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{9:23 LIBRARY
Michigan State
(pol University

 

 

 

This is to certify that the
dissertation entitled

CROSS-INHIBITION BETWEEN P2X AND NACHR LIGAND-
GATED ION CHANNELS IN THE ENTERIC NERVOUS
SYSTEM

presented by

Dima Alkawwas Decker

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Biochemistry

 

 

 

 

'.' Signature

  

MSU is an Affirmative Action/Equal Opportunity Employer

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5108 Kt/ProleccslPres/CIRC/DateDue Indd

 

CROSS-INHIBITION BETWEEN P2X AND NACHR LIGAND-GATED ION
CHANNELS IN THE ENTERIC NERVOUS SYSTEM

By

Dima Alkawwas Decker

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of

DOCTOR of PHILOSOPHY
Biochemistry and Molecular Biology

2009

ABSTRACT

CROSS-INHIBITION BETWEEN P2X AND NACHR LIGAND-GATED ION
CHANNELS IN THE ENTERIC NERVOUS SYSTEM

By
Dima Alkawwas Decker
The enteric nervous system (ENS) resides in the gut wall and contains
neurons that can control gastrointestinal function independently of the central nervous
system. The ENS contains two main types of ligand-gated ion channels that are
important mediators of fast excitatory synaptic transmission; P2X and the nicotinic
acetylcholine receptors (nAChRs). Although these channels are structurally distinct and
have been assumed to function independently, recent studies have shown that these

receptors have a mutually inhibitory interaction. Whole—cell patch clamp techniques
were used to assess the function of P2X; and (1334 nAChRs in cultured myenten'c neurons
and P2X and nAChRs expressed separately and together in human embryonic kidney

(HEK-293) cells, a heterologous system that permits molecular manipulations. These

molecular aspects included a detailed look at interactions between these receptors in three
different states; open, desensitized, and closed. This work provided evidence that P2X2

and nAChRs cross-inhibition occurs in all three of these states, including constitutive
inhibition between these receptors in the closed state. The specificity of this interaction

was investigated among the different subtypes of the P2X receptors. Cross-inhibition
occurred between (13134 nAChRs and the P2X3 and P2X4 receptor subtypes in open and

desensitized states.

 

More specific molecular manipulations included the determination of the direct
molecular mechanisms responsible for this interaction via the use of a truncated P2X2
receptor with a stop codon to shorten the C-terminal tail. Studies with this truncated

receptor provided evidence that the C-terminal tail of the P2X; receptor is important in
mediating cross inhibition of a3B4 nAChRs in the open and closed states. Furthermore,
with the use of a purified protein construct corresponding to the C-terminal tail of the
P2X2 receptor, competitive experiments were performed that provide evidence for a
physical interaction between the P2X2 receptor and the (ML; nAChR. A better

understanding of receptor interactions in enteric neurons would likely lead to novel drug
targets that would be usefiil in the treatment of common gastrointestinal motility

disorders such as irritable bowel syndrome.

 

To my mother, may she be proud of me.

iv

 

ACKNOWLEDGMENTS

This dissertation is culmination of 5 years of work, and would not have been
possible without the support of many people.

To my advisor, Jim Galligan, I thank you for your continued encouragement,
guidance, support and patience. The ideal environment that you provided was crucial in
my journey to becoming an independent investigator.

My committee members: Dr. John Lapres, Dr. Dave DeWitt, Dr. Eric Hegg, and
Dr. Tim Zhacharewski for your time and effort.

I would like to thank my colleagues and friends in the Galligan lab for their help
and for creating an amiable work environment.

I also need to thank my ‘biochem’ collegues. The Kodrofske lab for many
thought provoking discussions. Dr. John Wang for his advice and generosity. And Patty
Voss for her help with experiments, friendship and always finding the exact thing to say
to make me laugh my face off.

Without the support of my family, this would have not been possible. I want to

especially thank my husband Marc.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE OF CONTENTS
LIST OF TABLES viii
LIST OF FIGURES .. ix
Chapter 1: Introduction 1
1. The enteric nervous system 2
Overview ________________________________________________________ 2
Anatomy of the enteric nervous system __ _________ 3
Reflex circuitry of the enteric nervous system _____________________________________________________ 5
2. Synaptic transmission 11
Overview". ______________________________________________________________________________ 1 1
Slow synaptic transmission ___________________________________ 12
Fast synaptic transmission,“ ____________________________ 13
3. Purinergic signaling 16
ATP as an intercellular signaling molecule____ __ _____ 16
Purinergic receptors _________________ 16
P2X receptors 17
Multimerization 1 8
Electrophysiological and pharmacological characteristic of heterologously
expressed P2X receptors 18
P2X receptor subunit structure _ ___________________________________ 2 O
4. Cholinergic receptors 25
Muscarinic Cholinergic receptors _________________ 25
Nicotinic acetylcholine receptors (nAChRs) 26
Multimerization __ 27
Electrophysiological and pharmacological characteristic of heterologously
expressed nACRs _____ 28
nAChR subunit structure __________________________ 39
5. Cross-inhibition between nAChRs and P2X receptors 34
6. Research Goal 35
7. References 37
Chapter 2: Hypothesis and Specific Aims 42
Chapter 3: Cross-inhibtion between nicotinic acetylcholine receptors and P2X
receptors in myenteric neurons and HEK-293 cells 46
Abstract ______________________________________________________________________________________________ 4 7
Introduction _________________________________________________________________________________________________________________ 48
Materials and Methods ________________________________________________________________________________________________ 51
Results __________________________________________________________________________________________________________________________ 55
Discussion ____________________________________________________________________________________________________________________ 72
References 79

vi

“mum" . v'WJ'. um-

 

 

 

 

 

 

 

Chapter 4: Molecular mechanisms responsible for cross-inhibition 83
Abstract ______________________________________ .. 84
Introduction“, _______________________________________________________________________________ 86
Materials and Methods ________________________________________________________________________________________________ 88
Results ________________________________________________________________________________________________ 93
Discussion ______________________________________________________________________________________ 105
References __ ________________ 1 1 1

Chapter 5: Concluding remarks and future perspectives 115

 

vii

I nu blank}

 

F

LIST OF TABLES

Chapter 1.

Table 1.1: Neuron classification criteria

viii

'3 TI. ' .

LIST OF FIGURES

Images in this thesis/dissertation are presented in color.

Chapter 1:
Figure 1.1
Figure 1.2
Figure 1.3

Figure 1.4

Figure 1.5
Figure 1.6
Figure 1.7
Chapter 3:

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4
Figure 3.5

Figure 3.6

Figure 3.7

 

 

 

The anatomy of the enteric nervous system _______________________________________________________ 4
Reflex circuitry of the enteric nervous system ___________________________________________________ 8
Myenteric AH and S neurons ____________________________________________________________________________ 10
Fast excitatory synaptic transmission in myenteric neurons ,,,,,,,,,,,,,,,,,,,,,,,,,,, 15
Membrane topology of the P2X2 receptor _________________________________ 2 2
C-terminal tail length of P2X subtypes ________________________ “24
Membrane topology of nicotinic acetylcholine receptorsm 31
Cross- inhibition between P2X receptors and nAChRs in guinea pig ileum

cultured myenteric neurons 56

lmmunocytochemistry of HEK-293 cells _________________________________ 57

 

Comparison of pharmacological and functional properties of P2X2 receptors

and (13134 nAChRs expressed by cultured myenteric neurons and HEK-293
cells. __ 59

 

Cross-inhibition between P2X2 stable receptors and (13134 nAChRs expressed
by HEK-293 cells 61

Cross-inhibition between (13 [34 nAChRs stable and P2X; receptors expressed
by HEK-293 cells , 62

Cross—inhibition between P2X2 receptors and (13134 nAChRs expressed by
HEK-293 cells is maintained when receptors were activated by agonist

EC50 concentrations __ _ _ 64

ACh and ATP do not directly modulate P2X2 receptor or (13134 nAChR
function. 65

 

ix

Figure 3.8

Figure 3.9

Figure 3.10

Figure 3.11

Chapter 4:

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

Cartoon illustrating P2X subunit C-terminal tail amino acid length

 

 

 

 

 

 

 

 

 

 

 

differences among P2X2, P2X3 and P2X4 subunits ________ 6 6
P2X subunit C-terminal tail length does not alter cross-inhibition of 013134
nAChR function ___________________________________________________________________ “68
Cross inhibition between P2X; receptors and (13134 nAChRs is maintained
during receptor desensitization __ 69
Cross inhibition between P2X3 or P2X4 receptors and (13134 nAChRs is
maintained during receptor desensitization _______________________________ 71
Truncation of the P2X; C-terminal tail stops cross-inhibition between P2X2
and nAChRs 94
Purification of GST-fusion construct _____________________ 96
Cross-inhibition between nAChRs and P2X; receptors is mediated by the
P2X2 receptor C-terrninal tail _____ 98
Cross-inhibition between nAChRs and P2X2 receptors in myenteric neurons
is mediated by the P2X; receptor C-terminal tail 100
Constitutive inhibition of nAChRs ................................... 101
PPADS does not alter P2X; receptor mediated constitutive inhibition of
(13134 nAChRs 103
nAChRs and P2X2 receptors constitutively inhibit each other _____________________ 104

71'

LIST OF ABBREVIATIONS

ENS: Enteric nervous system

CNS: Central nervous system

ANS: Autonomic nervous system

GPCRs: G-protein coupled receptors

nAChRs: Nicotinic acetylcholine receptors

P2X: Purinergic 2X receptor

ACh: Acetylcholine

ATP: Adenosine triphosphate

fEPSPs: Fast excitatory post-synaptic potentials
sEPSPs: Slow excitatory post-synaptic potentials
HEK-293: Human embryonic kidney cells
DMEM: Dulbecco’s modified Eagle’s medium
ECso: Half-maximal effective concentration
FRET: Fluorescence Resonance Energy Transfer
5-HT: 5-hydroxy tryptamine

GABA: gamma-Aminobutyric acid

GST: glutatione S-transferase

xi

 

eri

CHAPTER 1

Introduction

 

Overview

The enteric nervous system (ENS) is a division of the autonomic nervous system
(ANS) that resides in the intestinal wall and controls gastrointestinal function (22). The
ANS is a system of neurons that control effectors that are not under voluntary direction
and include all neurally regulated tissues and organs (23). The ANS innervates visceral
organs of the thoracic, abdominal, and pelvic cavities including but not limited to the'
lungs, heart, digestive organs, kidneys, and the urinary bladder (23). Although
parasympathetic and sympathetic nerves supply the intestine, the ENS can still function if
these connections are severed. Therefore, the ENS can function independently of central
nervous system (CNS) control because it contains all the network elements needed for
reflex control of gastrointestinal function (29, 31). These elements include motor
neurons, inter-neurons, and sensory neurons that respond to mechanical and chemical
stimuli provided by intraluminal content (21).

The ANS is divided into three branches: the parasympathetic branch, the
sympathetic branch, and the enteric nervous system. Langley (1903, 1921) provided
three main features of the ENS branch. The ENS is a system of autonomic ganglia and
nerve fibers that reside within the walls of digestive organs. First, enteric neurons are
histologically different from other neurons of the ANS. Second, the autonomic pathways
from the CNS that make connections with enteric ganglia are different from those made
to other autonomic ganglion. Connections of the ANS are organized in a manner that

CNS connections are directly proportional to peripheral connections; one presynaptic

neuron connects to one postsynaptic neuron in the ganglia. However, ENS connections
differ in that CNS projections are not proportional to the connections in the ENS. More
specifically, there are many fewer extrinsic nerve fibers that make connections to the
ENS than there are enteric neurons. Lastly, there are complete reflex pathways that are
capable of operating without any connections to the CNS which reside within the enteric
division (38, 39). The ENS is the only substantial group of neurons that form circuits
capable of complete autonomous reflex activity outside of the CNS. This reflex arc

includes sensory, inter-neurons and motor neurons (21 , 22).

Anatomy of the enteric nervous system

The digestive organs referred to as the tubular digestive tract include the
esophagus, stomach, and the small and large intestines. The ENS in these organs is
composed of an interconnected network of neurons and glial cells. These networks of
nerve cells are found in two sets of ganglionated plexuses: the myenteric plexus and the
submucosal plexus. Axons from these plexuses project to and innervate the two muscle
layers of the ENS (longitudinal and circular) and the mucosa (22, 20).

The anatomic arrangement of these layers is in the following order from the
serosal to the lumen side: longitudinal smooth muscle, myenteric plexus, circular smooth
muscle, submucosal plexus, and mucosal layer (Figure 1.1). In the myenteric plexus,
individual ganglia contain up to 100 neurons. Interganglionic connections contain axons
which connect the ganglia together. Because the myenteric plexus is sandwiched

between the

Interganglionic

strand
Myenteric
Plexus

  
  

Circular
Muscle

  
 

Submucosa

   
 

Longitudinal
Muscle

Mucosa

Figure 1.1: The anatomy of the enteric nervous system.

A diagram adapted from Fumess and Costa 1987 illustrates the organization of
the enteric nervous system in whole mounts of the intestine. From top to bottom: the
serosal surface starts with the longitudinal muscle layer, followed by the myenteric
plexus, circular muscle, submucosal plexus and the mucosal layer.

muscle layers with axonal projections, there is an anatomical basis for the myenteric
plexus to have full control of both muscle layers. It is through its interganglionic
connective strands that the myenteric plexus is able to coordinate contractions and
relaxations along the gut for proper motility. In the other neuronal network, the
submucosal plexus, ganglia only contain up to eight neurons. It is possible for the
submucosal plexus to regulate secretory and absorptive functions of the mucosa] layer
due to its very close proximity to and axonal projections into the mucosa (22, 20). The
ENS contains more than 100 million neurons. These neurons can be organized via three

main criteria: functional classes, morphological classes and electrophysiological classes

(Table 1.1).

Reflex circuitry of the enteric nervous system and functional classifications

Myenteric neurons are organized into a functional network that regulates and
initiates the peristaltic reflex also known as motility. This reflex is activated via
distension. A bolus of food in the intestine causes distension and activates the peristaltic
reflex to relay information for coordinated contractions and relaxations. A bolus of food
can only be propagated along the intestine due to a distension-evoked contraction at the
oral side of the bolus followed by a relaxation at the anal side of the bolus. Repeated
cycles of this process are what mediate propulsion of food from the beginning of the
intestine down through the colon (22).

As mentioned earlier, the ENS can function without external nerve supply
because it contains three functional classes of neurons needed for a complete reflex

circuit: sensory neurons, interneurons, and motor neurons. When a bolus of food enters

Table 1.1: Neuron classification criteria

 

Electrophysiological

Functional definition Morphology Pro

 

The enteric nervous system contains over 100 million neurons. These neurons
are organized via three main criteria: functional classes, morphological classes, and
electrophysiological classes. The table lists the neuron types found in the guinea-pig
small intestine.

the intestine, there is a distention in the intestinal wall. This distension activates
mechanoreceptors that indirectly activate intrinsic primary afferent neurons (IPANS) at
their nerve terminal (they can also be activated by chemical stimuli). IPANS can
propagate the resulting action potential back to the cell body and relay the excitatory
cascade by releasing neurotransmitters onto a connecting intemeuron. It is from there
that this cascade propagates to more intemeurons, or activates motor neurons to cause
contractions or relaxations by releasing excitatory or inhibitory neurotransmitters,
respectively, onto the muscle (Figure 1.2; 22).

Morphological classifications

It is generally true that neurons in different species that have the same function
also have the same shape; this is also true for neurons in the ENS. By using morphology
as an indicator of equivalence of neurons of different species, ENS networks among
difference species can be studied and compared. It is important to note, however, that
neurons from different species do not have exactly the same shape according to function.
Neurons in larger animals have more elaborate dendritic trees compared to smaller
animals which contain less elaborate dendrites. This makes it less difficult to classify
neurons in smaller animals such as guinea-pigs compared to humans (22).

The first classification of enteric neurons by their morphology was studied by
Dogiel via methylene blue staining of neurons in the ENS. He provided a comprehensive
description of neurons of the myenteric and submucosal plexes of the intestine from
humans, guinea-pig, rabbit, rat, dog, and cat. These descriptions also included
illustrations of guinea-pig and human samples. Dogiel demonstrated two main groups of

enteric neurons based on soma shape, soma size and the number and distributions of

Ora] Anal

 

 

  
 
 

 

 

 

 

ACh 0 ATP . Longitudinal
Muscle
A I- , Myenteric
Q Plexus
1 Circular
P X nAChR Muscle
Action ‘ Q9 2 .
Potential
Mucosa

 

      

\ I
Bolus of food

seat}:

interneuron Motorneuron

Figure 1.2: Reflex circuitry of the enteric nervous system.

A diagram of the proposed model of the reflex circuitry of the enteric nervous
system responsible for mediating the peristaltic reflex. Intraluminal mechanical or
chemical stimulation indirectly activates intrinsic primary afferent neurons (IPANS , dark
grey) nerve terminals. This results in an antidromal action potential that results in the
release of neurotransmitters onto connecting intemeurons. Intemeurons (light grey)
activation results in the subsequent stimulation of motor neurons (black) to either
contract or relax the muscle. IPANS are AH neurons, while intemeurons and motor
neurons are S neurons.

dendrites. Therefore, the morphological classification scheme of enteric neurons is
named after him. Dogiel type I neurons are flat with stellate or angular shapes with cell
bodies ranging between 13-35 pm in length and 9-22 pm in width (Figure 1.3A; 58).
They have one axon (unipolar) and contain 4-20 short dendrites. These neurons have
been concluded to be motor and intemeurons of the ENS. Dogiel type II neurons are
angular, star or spindle shaped with multiple axonal (multiaxonal) projections to the
mucosa and adjoining neurons. With long axes of 22-47 um and short axes of 13-22 pm,

these neurons are recognized as sensory neurons (Figure 1.3B; 22, 58).

Electrophysiological classifications
Due to their electrophysiological characteristics, neurons in the reflex arc can be divided
into two types: AH neurons and S neurons. AH neurons are multi-axonal neurons (also

referred to as Dogiel type II neurons; Figure 1.33) in which a distinctive component of
the action potential is mediated by Ca2+. AH neuron action potentials are immediately

followed by a characteristic delayed and prolonged afierhyperpolarizing (AHP) potential
that lasts 1-20s (27, 58). As a result of the Ca2+ mediated shoulder, AH neuron action
potentials are only partially blocked by the sodium channel blocker tetrodotoxin (TI'X).
Although most AH neurons only receive slow synaptic input, some AH neurons receive
fast synaptic input (see synaptic transmission). Therefore, trains of stimuli elicit slow
excitatory potentials (sEPSPs) from all AH neurons while single electrical stimuli fail to
elicit a synaptic response in most AH neurons. All sensory neurons (IPANS) of the

reflex arc are AH neurons (22, 21, 7, 10, 27, 25).

Twai‘.

S neuron B AH neuron

‘i.

5

5 Cl trr-‘r

   

Dogiel type II

   

Ca2+ shoulder

10mV ms) (\ 10mVJfis/

Action potential Action potential

Figure 1.3: Myenteric AH and S neurons

A. Representative characteristics of S neuron morphology and
electrophysiological properties. S neurons are uniaxonal with multiple short
dendrites. Single stimuli elicit action potentials in S neurons that are tetrodotoxin
sensitive. B. Representative characteristics of AH neuron morphology and
electrophysiological properties. AH neurons are multiaxonal. Action potentials
in AH neurons have a calcium shoulder making them insensitive to tetrodotoxin,
and a long-lasting afierhyperpolarization.

S neurons (also referred to as Dogiel type I neurons; Figure 1.3A) are

intemeurons and motor neurons in this reflex arc. These neurons are uniaxonal, and lack
the Ca2+ and AHP components of the action potential, and they are therefore completely

blocked by TTX. Moreover, S neurons receive fast excitatory postsynaptic potentials
(fEPSPs) that are generally not observed in AH neurons. Single electrical stimuli elicit
tEPSPs in S neurons, while only some sEPSPs are elicited due to short trains of electrical
stimuli. Sensory processing of information in the intestine is mostly due to fast synaptic

transmission between these neurons (22, 21, 7, 10, 25, 27).
2. SYNAPTIC TRANSMISSION

Overview

Neurons communicate with each other through synaptic transmission, a process
mediated via specialized neuronal structures called synapses. A synapse is composed of a
presynaptic cell that is separated from a postsynaptic cell by a narrow cleft filled with
extracellular fluid preventing direct electrical communication between the cells (32, 34,
56,42)

Pre-synaptic actions. Cells communicate via neurotransmitters, these are small
hydrophilic molecules that are synthesized and stored in vesicles by neurons (51).
Release occurs due to the propagation of an action potential resulting in depolarization of
the nerve terminal. This activates calcium channels resulting in an influx of calcium, and
triggers the exocytosis of the content of neurotransmitter-containing vesicles clustered

near the nerve terminal membrane. The molecules of neurotransmitter diffuse across the

11

synaptic cleft and can bind to post-synaptic receptors. Some neurotransmitter molecules
are recovered via uptake through specialized transporters, while other molecules will be
degraded via enzymes located in the synaptic cleft (32, 34, 56, 42). The post-synaptic
actions of chemical synaptic transmission are classified into two main types: slow and

fast synaptic transmission.

Slow synaptic transmission

Slow synaptic transmission occurs via neurotransmitters acting at G-protein
coupled receptors (GPCRS). GPCR signaling is mediated by activating excitatory or
inhibitory interactions with intracellular heteromeric G-proteins (Bridges & Lindsley
2008). This activation alters the membrane potential of the post-synaptic cell. GPCR
family members are membrane proteins with an extracellular N-terminus, seven
transmembrane domains, and an intracellular C-terminus (35). The large third
intracellular loop of the receptor binds G-proteins which are trimeric containing a, [3, and
7 subunits. Agonist binding induces conformational changes which are thought to
involve at minimum the rearrangement of membrane helices 6 and 3. The activated
receptor now has a greater binding affinity for the G-protein and serves as a guanine-
nucleotide exchange factor to promote GDP dissociation, and GTP binding and
activation. The activated G-protein complex dissociates into Ga and 0137 subunits.

These subunits can now bind their respective downstream effectors including kinases,

phosphatases, small GTPases, and ion channels. Get-proteins include: GS coupled to
stimulation of adenylyl cyclase; Gi coupled to inhibition of adenylyl cyclase; Gq coupled

to the activation of phospholipase C; and G0 which activates KI and Ca2+ channels. GM

12

subunits can also directly activate channels and kinases. Hydrolysis of GTP to GDP by
the regulator of G-protein signaling proteins leads to the re-association of the
heterotrimer and termination of the activation cycle (13, 9, 50, 33).

GPCRs mediate slowly developing responses (latency 50 ms - 23) of long
duration (many seconds to minutes). GPCRs have latency in response because they
initiate a multi-step process that begins with agonist binding and depends on intracellular
signaling pathways. GPCRs mediate slow excitatory transmission, an important
signaling mechanism of the ENS (27). Some examples of slow synaptic transmission in

the ENS are acetylcholine acting at muscarinic M1 receptors, substance P acting at NK-3
receptors, S-HT acting at 5-HT1p receptors, and ATP acting at P2Y receptors. Activation

of these receptors results in a decrease in potassium conductance and an increase in

chloride conductance in myenteric neurons (25).

Fast synaptic transmission and ligand-gated ion channels in myenteric neurons

The second class of synaptic transmission in the ENS is ligand-gated ion channel
mediated fast synaptic transmission (25). Ligand-gated ion channels are membrane
proteins composed of multiple subunits in which the ligand binding site and the ion
channel are part of the same complex, forming an ionophore. Fast synaptic transmission
in the ENS is always excitatory. Since second messenger molecules are not needed, such
channels mediate a fast response (latency < 1 ms) with a short duration (< 100 ms).
Ligand-gated ion channels are localized to the somatodendritic region of the neuron
where they mediate fast excitatory synaptic transmission, and also to nerve endings

where they couple to the release of other excitatory neurotransmitters (25, 26, 27).

13

Several ligand-gated ion channels mediate fEPSPs in myenteric neurons,

including Cholinergic nicotinic acetylcholine receptors (nAChRs), purinergic P2X
receptors, and serotinergic 5-HT3 receptors (26). These channels are localized to S type

neurons and receive synaptic inputs from AH neurons and other S neurons for reflex
activation (see Figure 1.2). Moreover, the predominant mechanisms of excitatory fast
synaptic transmission of myenteric neurons of the myenteric plexus are mediated by
nAChRs and P2X receptors (22).

Studies performed by Galligan and Bertrand (24) using intracellular
electrophysiology methods to record from isolated preparations of guinea pig ileum
myenteric neurons maintained in vitro have shown that fast excitatory post-synaptic
potentials (fEPSPs) recorded from 25% of neurons are fully blocked by hexamethonium
(a nAChR antagonist). However, at 67% of neurons, hexamethonium only partially
blocked fEPSP responses, and complete inhibition was only established after the addition
of pyridoxal-phosphate-6-azophenyl-2',4'-disu]fonate (PPADS), a P2X receptor blocker.
This suggests that ATP and ACh are co-released by enteric neurons, and nAChR and

P2X are both located at the same synapse (24, 26; Figure 1.4).

14

Action Potential

    

Post-synaptic

at I

P2X Receptor Nicotinic acetylcholine Receptor

Pre-synaptic

Figure 1.4: Fast excitatory synaptic transmission in myenteric neurons.

A depiction of fast synaptic transmission synapse in the myenteric plexus. In
67% of myenteric neurons, fast excitatory post-synaptic potentials (fEPSPs) are
mediated by P2X and nicotinic acetylcholine receptors (nAChRs). This suggests that
these receptors are co—expressed by S type neurons of the myenteric plexus. In this type
of synaptic transmission, neurotransmitter would be released from the pre-synaptic cells
to act on their corresponding receptors of P2X and nAChR on the post-synaptic cell
membrane.

15

3. PURINERGIC SIGNALING

ATP as an intercellular signaling molecule

The first concept of ATP as a neurotransmitter, and not just as an energy carrier
for cellular metabolism, was proposed in 1972 by Burnstock after studying non-
adrenergic (norepinephrine) and non-Cholinergic (acetylcholine) nerve mediated
transmission in the gut and urinary bladder (30). There are many possible sources of
extracellular ATP. ATP can be released from injured cells as might occur under hypoxia
or inflammation (17, 6). ATP in millimolar concentrations is also stored inside vesicles
and granules of many neuronal and non-neuronal cells (62). These cells provide large
amounts of extracellular ATP from exocytotic release (16, 18). Once ATP is released
into the extracellular space, it is hydrolyzed into ADP by the membrane-bound enzyme
ecto-ATPase. Another enzyme ecto-diphosphohydrolase (ecto-apyrase) is capable of
hydrolyzing both ATP and ADP further down the cascade to AMP. AMP is then
hydrolyzed by 5’-nucleotidase to adenosine. Both AMP and adenosine are ligands for P1
receptors as mentioned above (62, 63). ATP acts at all P2 receptors; however, this
research will focus on ionotoropic P2X receptors as mediators of fast synaptic

transmission in the ENS.

Purinergic receptors
In 1978 purinergic receptors were divided into two pharmacological categories by
Burnstock (11, 12). P] for adenosine gated receptors and P2 for ATP/ADP gated

receptors (11, 1). All P1 adenosine receptors are metabotropic GPCRs that have been

divided into four cloned subtypes, A1, A2A, A23, and A3 (4). It was not until 1985 that

16

there was a proposal distinguishing two types of P2 receptors: P2X and P2Y. There have
been eight P2Y receptor subtypes identified to date, all of which belong to the GPCRs
family. The P2X receptor family are all ligand-gated ion channels of which seven

subtypes have been cloned so far (1 1, 1, l2).

P2X receptors

ATP-gated P2X receptors are a family of receptors that can mediate fast-synaptic
transmission in the nervous system. P2X receptors can be composed of seven (P2X1-
P2X7) different subunits activated by the neurotransmitter ATP (55, 18). The different
P2X subunits have physiologically distinct distributions. The rat P2X] receptor was
isolated by expression cloning of the vas deferens smooth muscle, with abundant
expression in smooth muscle preparations. P2X; receptors are widely expressed in the
nervous system, including but not limited to myenteric neurons, mouse and rat celiac
neurons, and dorsal horn neurons. P2X3 receptors have been observed in sensory
neurons and myenteric neurons, and they are believed to mediate pain sensations. P2X4
receptors are extensively found in the CNS. P2X5 receptors are present in some parts of
the brain, heart spinal cord, adrenal medulla, and lymphocytes. P2X6 receptors cannot
form homomers and are believed to exist as heteromers with other subtypes. P2X7
receptors are localized to immune cells and rat brain (37, 48). These subunits range in
size between 379 (P2X6) and 595 (P2X7) amino acids in length and present between 30 to

50% pair-wise identity in sequence (49, 37).

17

Multimerization

The stoichiometry of P2X receptors involves three subunits, thus the channel
could be a homo- or heterotrimer. Homomer association of the P2X subtypes lead, in
most of the receptors, to a functional ion channel and thus is the preferred state to study
the pharmacological properties of the receptors. For when the receptors form as

heteromers, some P2X subtypes dominate the pharmacological properties of the other
subtypes. All P2X subtypes can form homomers, except P2X6 which can only complex
with other subtypes to form a functional channel. Conversely, P2X7 receptors can only

form functional receptors as homomers, and do not mix with other subtypes (48, 49, 6).

Phannacologica] studies have provided evidence of heteromeric receptors
including: P2X2/3 receptors in sensory neurons and ganglion cells (49, 28), P2X4/6
receptors in CNS neurons (40), P2Xl/5 receptors, and P2X2/6 receptors (54, 49). P2X
subunits have different pharmacologic characteristics. For example, ATP activates all the
P2X subtypes, but a,B-methylene ATP is a more potent agonist at P2X] and P2X3

receptors. These pharmacological differences coupled with the different biophysical
properties of these channels are characteristics used to identify the composition of these

receptors in native and heterologous tissues (54, 6).

Electrophysiological and pharmacological characteristics of heterologously

expressed P2X receptors.

P2X receptors are non-selective cation channels with equal permeability for NaI,

KI, and even greater permeability for Ca2+. ATP binding activates P2X receptors within

18

a few milliseconds, with important kinetic differences among the different subtypes (48).
The continuous presence of agonist results in desensitization. The kinetics of

desensitization are contingent upon the time duration of agonist application.

Heterologously expressed P2X1 and P2X3 receptors desensitize in the presence of ATP

with time constants in the 100-300 ms range, whereas P2X2 and P2X4 receptors show
sustained currents with a very slow desensitization (time constant of seconds to tens of
seconds), occurring more rapidly in P2X4 subunits. P2X7 receptors show no

desensitization during application lasting for many seconds (48, 37, 49).

All P2X receptors are non-selective cation channels with equal permeability for

NaI, KI, and even greater permeability for Ca2+. However, different subtypes display
variable permeability properties. Homomeric P2X] and P2X4 receptors present a high
Ca2+ permeability (PCa/ PM, ~ 4), while permeability through P2X; receptors is lower

(PCa WM 2.5), and lowest at P2X3 receptors with PCa /PNa 1.2 (3 7, 49). At the whole cell

level, the currents induced by ATP show an inward rectification; this is due to a decrease
in the open probability of channels at more positive membrane potentials. This current-
voltage relationship also has a reversal potential of 0 mV, which is indicative of non-
selective cation permeability (49).

Agonist and antagonist pharmacology of P2X receptors. All cloned P2X

receptors are activated by ATP. Receptor subtype half maximal effective concentrations

(EC50) vary from 1 uM for P2X] and P2X3 to ~10 uM for P2X2, P2X4, P2X5, and P2X6.

The EC50 for P2X7 receptors is 100 uM (48).

19

‘3.

There are other P2X agonists that are ATP analogs, a,B-methylene-ATP ((1,0-

meATP), 2-methylthio-ATP (2MeSATP), and 2’,3’-(4-benzoy])-benzoyl ATP (BzATP).

a,[3-meATP is a selective agonist for P2X1 and P2X3 subunit containing receptors, while

BzATP is a selective agonist .for P2X7 receptors (37, 48, 49). The two main antagonists
for P2X receptors are suramin and PPADS. There are different sensitivities among the

subtypes to both antagonists. P2X], P2X2, P2X3, and P2X5 are blocked by suramin and

PPADS, while P2X4 and P2X7 are not (59, 37, 48, 49).

P2X receptor subunit structure

Each subunit of P2X receptors has two transmembrane domains (TMl and TM2)
with intracellular N and C-terrninal tails and a large extracellular loop (49). This
topology was determined by hydrophobicity plots and was confirmed by glycosylation
site determination and antibody identification towards epitope tags at the N- and C-

terminus that were only accessible due to permealization of the membrane (49, 37). Since

the main focus of this research is on myenteric neurons which mainly express P2X2

receptors, this work will mainly discuss the molecular physiology of P2X2 receptors

(Figure 1.5).
The amino-terminal tail. The N-terminal domains of P2X subunits are short
(ranging between 20 — 30 amino acids in length) relative to their C-terminal domains. A

consensus protein kinase C (PKC; Thr-X-Lys/Arg) site is present in the amino-terminus
of all P2X subunits. This site is especially important for P2X2 subunit function; mutation

of the Thr18 amino acid prevents phosphorylation by PKC and results in rapidly

desensitizing kinetics. This disrupts its typical slow desensitizing characteristics (as

20

explained above; 37, 8). Mutations of residues Asp15, Prol9, Val23, and Val24 in the
amino-terminal via cystiene-scanning mutagenesis indicated that methenethiosulfonates
binding to free cysteine recidues resulted in 60% inhibition of current, while mutations at
Tyr-16 resulted in a non-functional channel. These results indicate the importance of the

intracellular N-terminals in P2X channel conductance and function (49).

The extracellular loop. This domain is the bulk of P2X receptors at 280 amino
acids in length (amino acid 50-330 of the rat P2X; receptor), and it presents a high degree

of homology among the seven different mammalian P2X subtypes. It contains 10
conserved cysteines and 6 conserved lysines (two of which compose the binding site for

ATP see below). Also, depending on the P2X subtype, there are two to six putative N-
linked glycosylation sites (three sites for rat P2X2 at asparagines 182, 239 and 298) that

help channel function and proper insertion into the membrane (49, 37). The extracellular

loop domain is believed to contain the binding site for ATP and antagonists. The lysine
residue at position 246 in P2X2 receptors has been determined to be crucial for proper

binding of PPADS, while alanine scanning mutagenesis techniques indicate that lysine

residues at positions 69 and 71 are critical for ATP binding (3 7).

21

Outside

» J .-

that...

Inside

 

Figure 1.5: Membrane topology of the P2X; receptor.

Each subunit of P2X receptors has two transmembrane domains (TMl and
TM2) with intracellular N and C-terminal tails and a large extracellular loop. The N-
terminal domain contains a protein kinase C site at Thr18 ( . ) that mediates channel

kenetics. The extracellular loop contains 10 conserved cystiene residues (0) and lysine
residues (0) that mediate the binding site of ATP and the anagonist PPADS, along with
three glycosylation ( El ) sites. The C-terminal of the P2X2 receptor contains protein
kinase A (A) and protein kinase C ( I” ) sites that mediate channel kenetics. P2X
interactions with other proteins are believed to be mediated by the C-terminal tail.

22

The pore. The channel pore is composed of two transmembrane domains (TMl
and TM2) which span the plasma membrane in a-helix conformation as determined by
substituted cysteine accessibility. This method is where residues of the receptor are
mutated to cysteine residues individually and the accessibility of the side-chain in the
expressed channels are probed with modifying reagents to determine which residues line

the pore of the receptor. Studies have also shown that TM2 lines the pore and that in
P2X2 receptors it is an a-helix in the closed state that changes in the open state. The

narrowest part of the pore is at position 342 at a glycine residue about halfway through
the TM2 (37). Also, Val48 located in the outer edge of TMl together with residues

located in the out portion of TM2 (1328) play an important part in the gating movements

of P2X2 receptor channel opening (49).

The carboxy-termina] tail. The C-terminal tail is the most variable of the P2X
structure among different subtypes. It varies in amino acid composition and length with
the shortest for the P2X; receptor (30 amino acids) and the longest for the P2X7 receptor
(240 amino acids; F igurel .6). The C-terminal tail of P2X2 receptors contains a consensus
phosphorylation site for cyclic AMP (cAMP)-dependent protein kinase A (PKA, S431),
phosphorylation results in inhibition of current by increasing desensitization of the
channel. There is also another possible PKC site at T372 (3 7, 49).

These sites seem to play a stabilizing role on the slow phenotype of the P2X2
receptor (8). For truncations, deletions, mutations, and splicing of the C-terminal domain
greatly affect the kinetics, permeation and desensitization of the channel (37). In
addition, it seems that the C-terminal tail of the P2X; receptors is responsible for its

interactions with other proteins (3 7).

23

l a P2X4 [:1 28

P2X] :2 45

P2X, 53

 

 

P2X2 119
2x7 240

Figure 1.6: C-terminal tail length of P2X subtypes.

The C-terminal tail is the most variable of the P2X structure among
different subtypes. It varies in amino acid composition and length with the
shortest for P2X4 (30 amino acids) and the longest for the P2X7 receptor (240
amino acids).

24

4. CHOLINERGIC RECEPTORS

Cholinergic signaling is mediated by the neurotransmitter acetylcholine (ACh).
Acetylcholine is synthesized in nerve terminals from acetyl coenzyme A (acetyl CoA)
and choline in a reaction catalyzed by choline acetyltransferase (ChAT), an enzyme
which exist in soluble form in the nerve terminal cytoplasm (64, 51). Choline is
transported into cholinergic neurons by a high affinity transporter (this is the rate limiting
step of the reaction), while acetyl CoA is produced by mitochondria. After synthesis,
ACh is packaged into vesicles at the synapse via a vesicular ACh transporter (VAChT)
due to the proton gradient established by the proton pumping activity of a synaptic
vesicle-located vacuolar ATPase (64). Once released into the synapse, ACh is
hydrolyzed via acetylcholinesterase into acetate and choline; choline is then transported
back into the terminal for more ACh synthesis (51).

Adolf von Beayer was the first to discover choline, and he prepared ACh in 1867.
It was not until 1914 that ACh was discovered in ergot-containing plant material by Dale
and Ewins, and deduced to be a possible neurotransmitter. Many years later, in 1926,
Loewi provided strong evidence that ACh was the transmitter mediating parasympathetic
transmission in the frog heart (64). ACh acts at both muscarinic acetylcholine receptors
and nicotinic acetylcholine receptors (nAChRs). However, this work will focus on

nAChRs and their role in fast synaptic transmission.

Muscarinic cholinergic receptors
ACh produces its diverse physiological actions by binding and activating two

structurally and functionally distinct families of receptors. Muscarinic receptors

25

(mAChR) are GPCRs and encompass the metabotropic side of ACh mediated
transmission. Cloning studies have revealed five distinct mammalian subtypes Ml-Ms
(57). These five subtypes are divided into two major functional classes according to their
G-protein coupling affinities. M1, M3, and M5 receptors are selective for the Gq/G” G-
protein which couples to activation of phospholipase C (PLC) and generation of inositol
triphosphate and diacylglycerol (DAG). DAG activates PKC which can phosphorylate a
variety of intracellular targets, including ion channels. The M2 and M4 receptor subtypes
activate G; G-protiens which are coupled to inhibition of adenylate cyclase activity. Slow
synaptic excitatory transmission in the ENS is mediated by M1 receptors (57, 58, 25).
Fast excitatory synaptic transmission in the ENS is mediated by ACh acting at nicotinic

acetylcholine receptors (nAChRs; 25, 27,28).

Nicotinic acetylcholine receptors (nAChRs)

nAChRs do not only bind acetylcholine, but are also activated by nicotine (an
alkaloid extracted from tobacco plants, Nicotiana tabacum) and is antagonized by curate.
Curare is a mixture of plant toxins from Chondodendron tomentosum adopted by South
American Indians who used it as an arrowhead poison to immobilize their quarry (51).
Using these resources, it was Langley’s work in 1901 that first revealed the idea of the
nAChR. He reported that nicotine stimulated sympathetic nerve cells by ‘direct action
upon them’. In 1905 Langley reported that curare acts on the same ‘receptive substance’
as nicotine, and antagonizes its effects; what we now simply call a receptor (5). The
cloned subunit of the nAChR was the ACh binding a-subunit from the electric organ of

Torpedo, this electric ray produces and electric pulse to stun its prey. This electric organ

26

was a great source for receptor purification since nAChRs comprised 40% of the total
protein of this organ (3). This was the first primary structure of any vertebrate ion
channel protein to be purified (64).

nAChRs belong to the cys-loop superfamily of ligand-gated ion channels. This
family comprises both excitatory cation conducting (nAChR and 5-hydroxytryptamine
receptors 5-HT3) and inhibitory Cl' conducting channels (y-aminobutyric acid receptor
GABAA and the glycine receptor). Cys-loop family members are composed of five
subunits (pentameric), each of which has four transmembrane domains and extracellular
N and C-terminal tails (44). Nicotinic acetylcholine receptors can be subdivided into

muscle and neuronal types.

Multimerization

Muscle types of nAChRs consist of five subunits, two a; subunits, one 131 subunit,
one 7 subunit and either one 5 subunit, or one a subunit. Muscle receptors are usually
expressed postsynaptically (3, 64). This work will only focus on neuronal types of this

receptor. Neuronal nAChRs are heteromeric pentamers composed of two a and three B
subunits. There are eight different neuronal a (012- (19) and three different neuronal [3
([32— B4) subunits (43). In the brain, neuronal nAChR receptors have a presynaptic and

modulatory function by regulating the release of both ACh and other neurotransmitters as

autoreceptors via increasing the influx of Na+ and especially Ca2+ ions (16; 64). 'CNS
expressed subtypes include (1482, (16133, (17, a9, a5, and (12(14).
In the peripheral nervous system, including the ENS, the prominent subtype of

expression is (13134 and these receptors mediate fast excitatory transmission (64, 14). This

27

subtype will be the focus of this research. The electrophysiological and pharmacological

properties of nAChRs are determined by their specific subunit composition (64, 14, 3).

Electrophysiolocial and pharmacological characteristics of heterologously expressed

nAChRs

The predominant mechanism of excitatory neurotransmission in the ENS is via
nerve released ACh acting at nAChRs (25, 27, 28). In fEPSPs recorded from myenteric
plexus S neurons, 25% of neurons were purely cholinergic, and 67% of neurons were a
mix of purinergic and cholinergic signaling (26). ACh activates (1384 nAChRs with a very
short latency of 2 ms, with fastly desensitizing characteristics, within 4 seconds the
response is desensitized by 80% due to steady-state application of ACh (25, 61). ACh-
induced responses in myenteric neurons have an inwardly rectifying current-voltage
relationship with a reversal potential near 0 mV. This is a characteristic of non-selective
cation channels; nAChRs are permeable to Na+, KL, and Ca2+ with a PCa /PNa of 1 (61,
27, 19).

Data from immunocytochemical and pharmacological studies done in cultured
myenteric neurons suggest that S neurons do not express a7 subunits, but instead express
(13134 subunits. Although fEPSPs can be recorded from AH neurons, the amplitude of
nAChR mediated currents where smaller in amplitude from AH neurons compared to
current amplitudes recorded from S neurons. This suggests that AH neurons express
fewer nAChRs than S neurons (27, 26)

Agonist and antagonist pharmacology of nAChRs. All nAChRs are activated

by ACh application; however, there are other agonists for nAChR, such as nicotine,

28

DMPP, and cytisine. Cytisine is only a full agonist at nAChRs containing [34 subunits.
Concentration response curves in cultured myenteric neurons revealed. that DMPP is the
most potent at (13134 receptors with an ECSO of 44 uM, while ACh and nicotine where
equipotent at 200 uM, and the least potent agonist was cytisine at 400 uM. These
pharmacological studies provided evidence that myenteric neurons contain B4 subunits
(61).

Different nAChR subtypes also have different antagonist. a-bungarotoxin (a
southeast Asia snake Bungarus multicinctus venom that binds receptors in the
neuromuscular junction causing paralysis and death) only binds a7 subtypes. a-

bungarotoxin has no effect on nAChRs in the ENS, suggesting that (17 subunits are not

expressed there (27, 3). Other antagonists include hexamethonium, mecamylamine, and

DHBE, with mecamylamine inducing the most potent antagonism at [34 subunits, and

DHBE for [32 subunits. Half-maximal inhibition concentration values (IC50) of
antagonists in myenteric neurons were in the following rank order potency:
mecamylamine > hexamethonium > DHBE (27, 61). These data suggest that (13134
nAChRs are the most prominently expressed subtype in the ENS.
nAChR subunit structure

The membrane topology of nAChRs (a member of the cys-loop superfamily)
differs from that of P2X receptors. Cys-loop family members are composed of five
subunits (pentameric), each of which has four transmembrane domains and extracellular
N and C-terminal tails (44; Figure 1.7). They are referred to as cys-loop due to the

characteristic loop which contains a conserved sequence of 13 residues flanked by a

29

disulfide bond between two cysteine (Cys) residues that is important for complete
nAChR assembly (65, 15).

Since 2001, structural studies have described the cys-loop members in general
with an emphasis on nAChR structure. The first step was the elucidation of the X-ray
crystal structure of the mollusccm Lymnaea slagnalis ACh-binding protein AChBP at 2.7
A resolution. This protein aligns with the extracellular domains of nAChR at 24%, and
consists of a soluble homopentamer of 210 amino acid subunit. In the molluscan synaptic
cleft, AChBP is secreted from perisynaptic glial cells to the cleft under conditions of
active neurotransmission, and captures ACh molecules, therefore diminishing or
terminating synaptic transmission (65). The AChBP structure along with a subsequent 4
A resolution electron microscopy study of the Torpedo nAChR were used to create the
refined 4 A model of the whole receptor in its closed state. In this model, the receptor
was shown to have a length of 160 A, a diameter of 80 A, and a vestibule 20 A in
diameter (65, 15).

The extracellular domains. The amino terminal, an extracellular linker between
M2 and M3 (transmembrane domains) and the caboxy terminal of this receptor form
the extracellular domains. These extracellular domains of the receptor contain the ligand
binding site, glycosylation sites, and the 15 residue Cys-loop. The extracellular domain
is organized with 10 B-sheet structures connected via the Cys-loop and an amino a- helix.
This region contains many loop regions important for receptor function and assembly.
The ligand binding site is mainly mediated by the (1 subunits, but has been shown to be
formed at the interface between two subunits, with a “principle component” from (1

subunits, and a “complementary component” via a non-a subunits (2, 65, 41, 15). The

30

 

A - ., n: n C
Outside

Inside

Figure 1.7: Membrane topology of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors belong to the Cys-loop superfamily of receptors.
Cys-loop family members are composed of five subunits (pentameric), each of which has
four transmembrane domains and extracellular N- and C-terminal tails. They are referred
to as Cys-loop due to the characteristic loop with contains a conserved sequence of 13
residues flanked by a disulfide bond between two cysteine (Cys; 0) residues that are
important for complete nAChR assembly.

31

binding of agonist is mediated by five conserved aromatic side chains referred to as the
‘aromatic box’ that serves to capture the agonist (41).

Dose-response relationships for agonist activity have a Hill coefficient
approaching two, indicating that the channel is activated by two agonist molecules; one
for each a subunit (41, 15). Ligand binding causes a rotation of part of the extracellular
binding region around an axis that passes the cys-loop via a series of interactions. The
Cys-loop makes contact with a 01-132 loop and the M2-M3 transmembrane domain linker
that projects above the membrane. The [ll-[52 also interacts with residues near the M2
C-terminal domain. All these events result in the M2-M3 linker causing the M2 region to
move opening the channel (41 , 65).

The pore. Each subunit is formed by 4 highly hydrophobic a- helix segments
termed M1, M2, M3 and M4. The M2 transmembrane a-helix contributes to the pore,
while M1, M3 and M4 are in contact with the lipid membrane (2). The outer portion of
the vestibule that is exposed to the extracellular portion of the membrane is lined with
negatively charged residues on the a subunit (residues Glu13, Asp44, GluS 1 , Asp71) and
[3 subunits (residues Asp84, Asp 89, Asp 97, Glu 45, G1u82, Glu100) promoting a
cation-stabalizing environment and explains the vestibules selectivity for cations (65).
The pore is maximally constricted in the middle of the membrane (~6 A) due to side
chain interactions between hydrophobic residues of neighboring helices, forming a
hydrophobic girdle or gate with major contributions by Leu 251 and Val 255 of the M2
transmembrane domain (2, 65) .

Intracellular loop. This main hydrophilic portion of the receptor consists of a

large cytoplasmic loop between M3 and M4. This loop varies in length between subunits

32

(110-270 amino acids) and contains a curved u- helix. There are many phosphorylation
sites contained within this loop which modulate receptor function via desensitization, and
expression. Moreover, this portion of the receptor is involved in governing channel
kinetics and mediates protein-protein interactions (2, 65). These interactions include
binding with the cytoskeleton to induce receptor clustering. Also, it has been
demonstrated that residues in the large cytoplasmic loop near the intracellular end of M4

affect the assembly of subunits and electrophysiological properties of nAChRs (65).

33

5. CROSS-INHIBITION BETWEEN NICOTINIC ACETYLCHOLINE AND P2X
RECEPTORS.

P2X receptors and nAChRs are co-expressed by guinea-pig coeliac ganglion
neurons (53, 52), rat pheochromocytoma PC12 cells (45), myenteric neurons, (60) and rat
superior cervical ganglion neurons (46). P2X receptors and nAChRs are very structurally
distinct (Figure 1.5 and Figure 1.7), and they have therefore been assumed to function
independently. However, studies have shown a functional interaction between these
channels. Interactions between these receptors were first observed by Nakazawa et al in
1991. ATP activated currents and nicotine induced currents were studied in NGF
differentiated rat pheochromcytoma-l2 cells isolated from the adrenal medulla (45, 47).
Nakazawa observed that ATP and nicotine mediated currents were not additive. When
simultaneously activated, these receptors mediate an inhibitory interaction.
Simultaneous activation of both channels produces inward currents that are smaller than
the additive sum of individual currents (predicted current) that would occur if the
channels functioned independently.

P2X/nAChR interactions were later observed in guinea-pig celiac ganglion
neurons (52, 53), and superior cervical sympathetic neurons (46). These studies
suggested that P2X2-nAChR cross-inhibition is a result of “channel overlap” (45, 46).
This hypothesis is based on the idea that ATP and ACh activate a common channel with
different agonist binding sites (45, 52, 53). Another theory to explain this occlusion of
current was that P2X receptors have an allosteric inhibitory binding site for ACh, and that
nAChRs have a similar binding site for ATP (47, 52). These hypotheses are difficult to

test in these model systems where both P2X and nAChRs are endogenously expressed.

34

Further work included P2X and nAChRs co-expressed in cultured native
peripheral neurons, and cultured myenteric neurons that have shown this occlusion of
response in which the combined activation of receptors resulted in only 74% of the
predicted response (60). This non-additivity has also been reproduced in Xenopus
oocytes where P2X receptors and nAChRs were expressed heterologously (36). Unlike
G-coupled protein receptors, P2X receptors and nAChRs mediate quick responses
(latency, < 1 ms); hence, it is unclear how interactions take place in a short period of

time.

6. REASEARCH GOAL

The ENS is responsible for regulating motility, immune, and inflammatory
processes of the gastrointestinal tract. Disorders of this system result in motor, secretory
and circulatory dysfunctions (31). Details of the neuronal circuitry, receptor localization
and signaling responsible for regulating excitatory synaptic transmission in the ENS are
only partly understood. In particular, specific receptor sites responsible for regulation
would be excellent targets for drugs to facilitate neurotransmission and enhance or
decrease gut motility. Such drugs would help to better treat GI motility disorders such as
irritable bowel syndrome (29).

The goals of the present study were to investigate the interactions between
nAChRs and P2X receptors. This interesting interaction has not yet been studied
thoroughly. Since the ENS is the only part of the nervous system where ACh and ATP

have been shown to be co-transmitters at functionally defined synapses (26), it is an ideal

35

system to study this novel mechanism of synaptic integration. In this study, I will
investigate the interactions between P2X and nAChR during fast synaptic excitation.

A majority of previous work has been limited in its study of this unique ion
channel interaction because the systems utilized endogenously expressed both receptors.
This makes it difficult to verify such hypothesis as the “channel overlap” theory, or
allosteric binding sites. A heterologous system in which these receptors are not
endogenously expressed is a crucial tool in answering these scientific questions.
Although this interaction has been touched upon in Xenepos oocytes, our studies focus on
a mammalian system that has the same processing and trafficking of proteins as the

native environment of myenteric neurons, compared to an amphibian system.

36

REFERENCES

l. Abbracchiio MP, Burnstock G, Boeynaems JM, Barnard EA, Boyer JL,
Kennedy C, Knight G, Fumagalli M, Gachet C, Jacobson KA, Weisman GA.
International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled
Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to
Therapy. Pharmacol Rev, 58: 281—341, 2006.

2. Arias HR, Bhumireddy P, Bouzat C. Molecular mechanisms and binding site
locations for noncompetitive antagonists of nicotinic acetylcholine receptors. The

International Journal of Biochemistry & Cell Biology, 38: 1254—1276, 2006.

3. Albuquerque EX, Pereira EFR, Alkondon M, Rogers, SW. Mammalian nicotinic
acetylcholine receptors: from structure to function. Physiol Rev 89: 73—120, 2009.

4. Benarroch, EE. Adenosine and its receptors. Neurology, 70: 231-236, 2008.

5. Bennett MR. The concept of transmitter receptors: 100 years on.
Neuropharmacology 39: 523—546, 2000.

6. Bertrand PP. ATP and Sensory Transduction in the Enteric Nervous System.
Neuroscientist, 9(4): 243—260, 2003.

7. Bomstein JC, Costa M, Grider JR. Enteric motor and intemeuronal circuits
controlling motility. Neurogastroenterol. Motil. 16 (suppl 1): 34-38, 2004.

8. Boue—Grabot E, Archambault V, Seguela P. A protein kinase C site highly
conserved in P2X subunits controls the desensitization kinetics of P2X2 ATP-gated
channels. .1. Biol. Chem. 275(14): 10190-5, 2000.

9. Bridges TM, Lindsley CW. G-protein-coupled receptors: from classical modes of
modulation to allosteric mechanisms. ACS Chem. Biol. 3 (9): 530-541, 2008.

10. Brookes SJH. Classes of enteric nerve cells in the guinea-pig small intestine. The
anatomical record, 262: 58-70, 2001.

_ 11. Burnstock G. The journey to establish purinergic signalling in the gut.
Neurogastroenterol Motil, 20 (Suppl. 1): 8—19, 2008.

12. Burnstock G. Purinergic signalling: past, present and future. Braz J Med Biol Res
42(1): 2-8. 2009.

13. Clapham DE , Neer EJ. G-protein By subunits. Annu. Rev. Pharmacol. T oxicol
37:167—203, 1997.

37

14.

15.

16.

l7.

l8.

19.

20.

21.

22.

23.

24.

25.

26.

27.

Cordero—Erausquin M, Merubio LM, Klink R, Changeux JP. Nicotinic receptor
function: new perspectives from knockout mice. TiPS 21: 211-217, 2000.

Costa V, Nistri A, Cavalli A, Carloni P. A structural model of agonist binding to
the alpha3beta4 neuronal nicotinic receptor. Br J Pharmacol. 140 (5): 921-31, 2003.

Dajas-Bailador F, Wonnacott S. Nicotinic acetylcholine receptors and the
regulation of neuronal signalling. Trends Pharmacol Sci. 25(6):317-24, 2004.

Dubyak GR, el-Moatassim C. Signal transduction via P2-purinergic receptors for
extracellular ATP and other nucleotides. Am J Physiol-Cell. 265(3 Pt 1): C577-606,
1993.

Edwards FA, Gibb AJ. ATP- 3 fast neurotransmitter. FEBS 325: 86-89, 1993.
Fieber LA, Adams DJ. Acetylcholine-evoked currents in cultured neurons
dissociated from rat parasympathetic cardiac ganglia. J of Physiology 434, 215-237,
1991.

Fumess JB, Costa M. The enteric nervous system. pp 1-290, New York: Churchill
Livingstone. 1987.

Fumess JB, Sanger GJ. Intrinsic nerve circuits of the gastrointestinal tract;
identification of drug targets. Current opinion in Pharmacology, 2: 612- 622, 2002.

Fumess JB. The Enteric Nervous System. Blackwell Publishing, Massachusetts,
USA. p 1-274, 2006a.

Fumess JB. The organisation of the autonomic nervous system: peripheral
connections. Auton Neurosci. 30: 1-5. 2006b.

Gallgian and Bertrand PP. ATP mediates fast synaptic potentials in enteric neurons.
J Neurosci 14: 7563-7571,]994.

Galligan JJ. Mechanisms of excitatory synaptic transmissionin the enteric nervous
system. Tokia J Exp Clin Med. 23 (5): 129-136, 1998.

Gallligan JJ, LePard KJ, Schneider DA, Zhou X. Multiple mechanisms of fast
excitatory synaptic transmission in the enteric nervous system. J A uton Nerv Syst 81:

97-103, 2000.

Galligan JJ. Pharmacology of synaptic transmission in the enteric nervous system.
Current opinion in pharmacology, 2: 623-629, 2002.

38

28. Galligan JJ, North RA. Pharmacology and function of nicotinic acetylcholine and
P2X receptors in the enteric nervous system. Neurogastroenterol Motil, 16 (suppl 1)

64-70, 2004.

29. Gershon MD. Review article: serotonin receptros and transporters — roles in normal
and abnormal gastrointestinal motility. Ailment pharmacol T her. 20 (suppl. 7), 3-14,
2004.

30. Gourine AV, Wood JD, Burnstock G. Purinergic signalling in autonomic control.
Trends Neuro , April 7. [Epub ahead of print]

31. Goyal RK, Hirano I. The enteric nervous system. The New England Journal of
Medicine 334, 1106-1115,1996.

32. Hall ZW. Release of neurotransmitters and their interaction with receptors. Annu Rev
Biochem. 41:925-52, 1972.

33. Hamm HE. The many faces of G protein signaling. J of Biol Chem, 273(2): 669—672,
1998.

34. Hubbard JI. Mechanism of transmitter release. Prog Biophys Mol Biol. 21:33-124,
1970.

35. Ji TH, Grossmann M, Ji I. G protein-coupled receptors. J Biol Chem. 273: 17299-
302, 1998.

36. Khakh BS, Zhou X, Sydes J, Galligan JJ, Lester HA. State-dependent cross-
inhibition between transmitter-gated cation channels. Nature 406: 405-10, 2000.

37. Khakh BS. Molecular physiology of P2X receptors and. ATP signaling at synapses.
Nature Reviews 2: 165-174, 2001.

38. Langley JN. The autonomic nervous system. Brain 26: 1-26, 1903.
39. Langley JN. The autonomic nervous system. Heffer, Cambridge. p 80, 1921.

40. Le KT, Babinski K, Seguela P. Central P2X4 and P2X; channel subunits coassemble
into a novel heteromeric ATP receptor. J. Neurosci. 18 (18): 7152-9, 1998.

41. Lester HA, Dibas MI, Hahan DS, Leite JF, Dougherty DA. Cys-loop receptors:
new twists and turns. Trends Neurosci 27(6): 329-36, 2004.

42. Maio VD. Regulation of information passing by synaptic transmission: A short
review. Brain Research 1225: 26— 38, 2008.

39

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

McGehee DS, Role LW._Physiological diversity of nicotinic acetylcholine receptors
expressed by vertebrate neurons. Annu Rev Physiol. 57,521-46, 1995.

Miyazawa A, F ujiyoshi Y, Unwin N. Structure and gating mechanism of the
acetylcholine receptor pore. Nature 423: 949-955, 2003.

Nakazawa K, Fujimori K, Takanaka A, Inoue K. Comparison of adenosine

triphosphate- and nicotine-activated inward currents in rat phaeochromocytoma cells.
J Physiol 434: 647-60, 1991.

N akazawa K. ATP-activated current and its interaction with acetylcholine-activated
current in rat sympathetic neurons. J Neurosci 14:740-750, 1994.

Nakazawa K. Reduction of acetylcholine-activated current by low concentrations of
extracellular adenosine-triphosphate. Life Sciences 57: 351-356, 1995.

North RA , Surprenant A. Pharmacology of cloned P2X receptors. Annu Rev
Pharmacol T oxicol, 40: 563-8, 2000.

North RA. Molecular physiology of P2X receptors. Physiol Rev 82: 1013-1067,
2002.

Pierce KL, Permont RT, Leflcowitz RJ. Seven-transmemebrane receptors. Nature
Rev, molecular cell biology. 3: 639-650, 2002.

Purves D, Augustine G J, Fitzpatrick D, Katz LC, LaMantia AS, McNamara
JO, Williams SM. Neuroscience Second edition. Sunderland (MA): Sinauer
Associates, Inc., 2001.

Searl TJ, Redman RS, Silinsky EM. Mutual occlusion of P2X ATP receptors and
nicotinic receptors on sympathetic neurons of the guinea-pig. J Physiol 510: 783—791 ,
1998.

Silinsky EM, Gerzanich V. On the excitatory effects of ATP and its role as a
neurotransmitter in coeliac neurons of the guinea-pig. J Physiol 464: 197-212, 1993.

Torres G E, Egan TM. Voigt MM. Hetero-oligomeric assembly of P2X receptor
subunits. Specificities exist with regard to possible partners. J. Biol. Chem. 274(10):
6653-9, 1999.

Vial C, Roberts JA, Evans RJ. Molecular properties of ATP-gated P2X receptor
ion channels. TRENDS in Pharmacological Sciences. 25, 487-493, 2004.

Walmsley B, Alvarez FJ, F yffe RE. Diversity of structure and function at
mammalian central synapses. Trends Neurosci 21: 81-8, 1998.

40

57.

58.

59.

60.

61.

62.

63.

64.

65.

Wess J, Eglen RM, Gautam D. Muscarinic acetylcholine receptors: mutant mice

provide new insights for drug development. Nature reviews: drug discovery, 6: 72 ,
2007.

Wood JD, Kirchgessner A. Slow excitatory metabotropic signal transmission in the
enteric nervous system. Neurogastroenterol Motil. 16 (Suppl. 1):71—80, 2004.

Zhou X, Galligan JJ. P2X purinoceptors in cultured myenteric neurons of the
guinea-pig small intestine. J Physiol 496: 719-729, 1996.

Zhou X, Galligan JJ. Non-additive interaction between nicotinic cholinergic and
P2X purine receptors in guinea-pi g enteric neurons in culture. J Physiol 513: 685-
697, 1998.

Zhou X, Ren J, Brown E, Schneider D, CarabalIo-Lopez Y, Galligan JJ.
Pharmacological properties of nicotinic acetylcholine receptors expressed by guinea
pig small intestinal myenteric neurons. J Pharmacol Exp Ther 302: 889-897, 2002.

Zimmermann H. Signalling via ATP in the nervous system. TINS 17(10): 420-426,
1994.

Zimmerman H. Extracellular Purine metabolism. Drug Development Res. 39: 337-
352, 1996.

Zimmermann H. ATP and acetylcholine, equal brethren. Neurochemistry
International, 52 : 634—648, 2008.

Zouridakis M, Zisimopoulou P, Poulas K, Tzartos SJ. Recent advances in
understanding the structure of nicotinic acetylcholine receptors. I UBMB Life, 61(4):
407—423, 2009.

41

CHAPTER 2

Hypothesis and Specific Aims

42

OVERALL HYPOTHESIS

My hypothesis is that: nAChRs and P2X receptors form a heterodimer that

regulates fast synaptic excitation in the ENS. An alternative explanation for their fast

mutual inhibition would be that their interaction is charge-mediated. The first concern

regarding this interaction would be that it occurs as a result of Na+ or Ca2+ dependence,
especially since Ca2+ is a known mediator of downstream signaling events (both

receptors are Ca2+ permeable). However, studies in Ca2+ free solutions have not altered
the non-additive response (Zhou and Galligan 1998). These studies also provided
evidence, that this interaction is not Na+ dependent (Zhou and Galligan 1998). These

results rejected the idea that there are chemical interactions that can account for this
occlusion of response.

If these interactions are not ion dependent, further reflection would suggest that
this receptor-receptor interaction is associated with the formation of a heterodimer; a
hypothesis that is supported by the following evidence. First, the cross-inhibitory
response of these receptors is dependent on receptor density, that is a larger amplitude in
response to ATP or a greater number of receptors expressed correlate with a more
prominent occlusion. Second, this interaction is still observed in membrane patches,

suggesting no need for second messengers (Zhou and Galligan, 1998; Khakh et al.,

2000). Third, Fluorescence Resonance Energy Transfer (FRET) analysis of a (14132

nAChR and P2X2, which are subtypes expressed in the brain, revealed localization of

these receptors in a close proximity of ~80A apart (Khakh et al., 2005). This is evidence

43

of an Angstrom scale interaction between full-length P2X2 and a4B2 nAChR in the

plasma membrane of living HEK-293 cells. The FRET data imply that P2X2 interact
more strongly with the B subunits than with a subunit.

My hypothesis of heterodimer formation will be tested by two specific aims. The
goal of the first specific aim is to reproduce the inhibitory interaction between nAChR
and P2X in cultured myenteric neurons. The second will be to determine whether a
functional interaction occurs between nicotinic acetylcholine receptors (nAChRs) and
P2X receptors in a heterologous system expressing the specific receptor subtypes found

at enteric neuronal synapses. These receptor subtypes are the 0L3B4 nAChR subtype and

the P2X2 homotrimeric receptor. The rationale is to utilize the advantage of the human
embryonic kidney (HEK-293) heterologous expression system to determine the

specificities of this interaction.

Specific Aims:
Specific aim #1: Reproduce nAChR and P2X inhibitory interaction in cultured
myenteric neurons. The overall aim of this goal is to verify that this interaction can be

reproduced.

Specific aim #2: Express (13134 nAChR and P2X; in HEK-293 cells.

Specific aim 2a: Determine the function of a3B4 nAChR and P2X2 in this heterologous
system and how it compares to receptors in cultured myenteric neurons.

Specific aim 2b: Reproduce the functional interaction between P2X2 and a3B4 nAChR.

Specific aim 2c: Determine the specificity of the mutually inhibitory interaction between

nAChR and P2X.

44

Specific aim 2d: Determine if desensitization of individual receptors affect the mutually

inhibitory interaction.
Specific aim 2e: Determine the role of the C-terminal tail of the P2X2 receptor.

Specific aim 2f: Determine basal level of cross-inhibition between receptors.

45

CHAPTER 3

Cross-inhibition Between Nicotinic Acetylcholine Receptors and P2X

Receptors in Myenteric Neurons and HEK-293 cells1

 

I Data contained in this chapter have been published.
Decker, DA, Galligan, JJ. (April 2, 2009) American Journal of Physiology GI and Liver
[Epub ahead of print]

46

ABSTRACT

The enteric nervous system (ENS) controls gut function. P2X and nicotinic
acetylcholine receptors (nAChRs) are ligand-gated cation channels that mediate fast
synaptic excitation in the ENS. Close molecular coupling in enteric neuronal membranes
contributes to a mutually inhibitory interaction between these receptors; this effect is
called cross-inhibition. We studied the molecular mechanisms responsible for cross-
inhibition. Whole-cell patch clamp techniques were used to measure P2X- and nAChR-
mediated currents in cultured enteric neurons and HEK-293 cells. In cultured myenteric
neurons, ACh (3 mM) and ATP (1 mM) co-application evoked an inward current that
was only 57 :1: 6% (P < 0.05) of the predicted current that would have occurred if the two
populations of channels were activated independently. In HEK-293 cells co-expressing
a3B4 nAChR/P2X2 receptors, co-application of ATP and ACh caused a current that was '

58 i 7% of the predicted current (P < 0.05). To test the importance of P2X subunit C-

terminal tail length on cross-inhibition, P2X3 and P2X4 subunits, which have shorter C-
terminal tails were studied. Cross-inhibition with 0L3B4 nAChRs and P2X3 or P2X4

subunits was similar to that occurring with P2X2 subunits. P2X receptor or a3B4 nAChR
desensitization did not prevent receptor cross-inhibition. These data indicate that the
a3B4-P2X receptor interaction is not restricted to P2X2 subunits. In addition, active and

desensitized conformations of the P2X receptor inhibit nAChR function. These
molecular interactions may modulate the function of synapses that use ATP and ACh as

fast synaptic transmitters in the ENS.

47

INTRODUCTION

The enteric nervous system (ENS) is the division of the autonomic nervous
system (ANS) that controls gastrointestinal function (8). Although parasympathetic,
sympathetic and sensory nerves supply the intestine, the ENS can control gut function
independently from CNS control. This is possible because the ENS contains all the
neural elements needed for reflex control of gastrointestinal function (10,12). These
elements include motor neurons, inter-neurons and sensory neurons that respond to
mechanical and chemical stimuli provided by intraluminal content (9).

There are two classes of ligand-gated ion channel receptors that mediate fast

synaptic excitation of enteric neurons; the cys-loop receptors and P2X receptors. Cys-
loop receptors include nicotinic acetylcholine receptors (nAChRs), 5-HT3 receptors and
GABAA receptors. Cys-loop receptors are pentameric and each subunit has four
transmembrane domains and extracellular N and C-terminal tails (17,18). Neuronal
nAChRs are composed of two a and three B subunits; there are eight neuronal a (dz-a9)

and three neuronal B (Bz-B4) nAChR subunits (l 7). In the ENS, the predominant nAChR

subtype is composed of <13 and B4 subunits (39) and acetylcholine acting at nAChRs is

the primary fast excitatory neurotransmitter in the ENS (10). P2X receptors are trimeric
and each subunit has two transmembrane domains and intracellular N and C—terminal

tails. P2X receptors are homomeric or heteromeric receptors composed of one or more of

seven (P2X1-P2X7) subunits (7, 14, 26, 27, 31, 35). Enteric neurons express P2X2 and

P2X3 subunits which can form homomeric and heteromeric P2X2/P2X3 receptors (6, 29,

38). ATP acting at P2X receptors contributes to fast synaptic excitation in subsets of

48

enteric neurons and ATP and ACh are co-transmitters at many synapses in the ENS (10,
16,20,28,30)

P2X receptors and nAChRs are structurally different and it would be expected
that they would function independently. However, P2X receptors and nAChRs co-
expressed by rat pheochromcytoma—12 cells (23, 25) and superior cervical sympathetic
neurons (24) are functionally linked. Simultaneous activation of both channels produces
inward currents that are smaller than the additive sum of individual currents (predicted
current) that would occur if the channels functioned independently. The functional
interaction between nAChRs and P2X receptors was subsequently shown to occur in
guinea pig celiac ganglion neurons (32) and cultured myenteric neurons fiom the guinea-
pig ileum (3 8). This latter result is particularly important because ATP and ACh are co-
transmitters at functionally identified synapses in the guinea pig ileum myenteric plexus
(1, 16, 27) and ATP modulates the function of nAChRs activated by synaptically-released
ACh (l 3). The functional interaction between nAChRs and P2X receptors is proportional
to receptor density and the interaction occurs in cell-free patches obtained from cultured
myenteric neurons (38). These data suggest that intracellular signaling mechanisms are
not required for the functional interaction and that there may be direct molecular

interactions between the two types of receptors. A similar fimctional interaction occurs

between P2X receptors and other cys-loop receptors including 5-HT3 receptors in guinea
pig submucosal neurons (2) and GABAA receptors in dorsal root ganglion neurons (34).
Studies of heterologously expressed P2X2 and 5-HT3 receptors revealed that a portion of
the cytoplasmic C-terminal tail of the P2X2 receptor and a portion of the second

cytoplasmic loop of the 5-HT3A subunit are responsible for not only a functional

49

interaction but also may mediate a physical interaction between these receptors (3).
Similarly, the C-terminal tail is required for interaction of P2X3 receptors with GABAA

receptors expressed in Xenopus oocytes or dorsal root ganglion neurons (34). These
studies suggest that the P2X receptor C-terminal tail might contribute to interactions with
cys-loop receptors, a hypothesis that we test by studying interactions between nAChRs

and P2X subunits with varying C-terminal tail lengths.

Although a functional interaction between P2X2 receptors and a3B4 nAChRs
occurs when these receptors are expressed in Xenopus oocytes (13), the molecular
mechanism responsible for the interaction between P2X2 and (13 B4 nAChRs has not been
studied in detail. The present study was designed to test the hypothesis that the
functional interaction between P2X2 receptors and (13 B4 nAChRs could be reproduced in

a heterologous expression system (HEK-293 cells) and that this system could be used to

probe the molecular mechanisms responsible for interactions between these receptors.

50

MATERIALS AND METHODS

Tissue Culture

Cultured myenteric neurons. Neurons were cultured as described previously
(34,35). Two newborn guinea-pigs (<36 hrs old) were killed by severing the major neck
blood vessel following halothane anesthesia. The small intestine was removed from the

animals and placed in cold (4 °C) Krebs solution of the following composition
(millimolar): 117 NaCl; 4.7 KC]; 2.5 CaClz; 1.2 MgClz; 1.2 NaHzPO4; 25 NaHCO3 and

11 glucose. The longitudinal muscle myenteric plexus was stripped free using a cotton
swab and cut into 5 mm pieces. Tissues were digested with 1600 U of trypsin, followed
by trituration with a fire-polished Pasteur pipette. Afier incubation with 2000 U crab
hepatopancreas collagenase, the tissues were triturated again. The neurons were

resuspended in Eagle's minimum essential medium (MEM) containing 10% fetal bovine
serum, gentamicin (10 ug ml'l), penicillin (100 units ml'l) and streptomycin (100 units
ml”) and plated onto sterile, poly-L-lysine coated 35 mm plastic dishes and maintained

in an incubator at 37 °C in a 5% CO; atmosphere for up to 3 weeks.

Human embryonic kidney (HEK-293) cells. Cells were obtained from
American Type Culture Collection and grown in Dulbecco’s modified Eagle’s medium

(DMEM) F-12 containing 10% fetal bovine serum, 10% GluMax (Invitrogen, Carlsbad,
C A), and 100 units ml'l penicillin and streptomycin, except for the rat a3B4 nAChR stable
H‘EK-293 cell line which also contained 0.5 mg/ml geneticin (GIBCO, G418). Cells
were maintained at 37 °C in a 5% CO; atmosphere in a humidified incubator. Cells were

passaged once every 3 days when they reached 90% confluence. Transient transfection

51

of cells was accomplished using electroporation. A 0.4 cm Gene Pulser Cuvette (BIO-
RAD) was used with 2 ug of total plasmid protein along with 0.2 ug of GFP to help
identify transfected cells. The electroporation machine used was Gene Pulser XcellTM

Electroporation System (BIORAD). This resulted in 80% transfection efficiency.

Afterwards, cells were plated on 35 mm coverslips and maintained in the incubator for 24
hrs before use in electrophysiology experiments. A rat a3B4 nAChR stable HEK-293 cell
line was provided by Dr. Yingxian Xiao from Georgetown University (36). Plasmids
containing the coding sequences for rat P2X2, P2X3 and P2X4 receptor subunits were a
gift from Dr. Alan North (University of Manchester, Manchester, UK). Plasmids
containing the coding sequences for the murine <13 and murine B4 nAChR subunits were
provided by Dr. Jerry A. Stitzel (University of Colorado, Denver Colorado).

Whole cell recording: Whole-cell voltage-clamp measurements were obtained at
room temperature using standard methods. Coverslips containing cells were placed on a
stage of an inverted microscope (Nikon Diaphot, Mager Scientific, Inc. Dexter, MI) using
phase-contrast optics. Foridentifying co-transfected cells, a mercury bulb with excitation
wavelength of 495 nm was used with a filter for 520 nm emission. The pipette solution
contained (millimolar): 122.5 K-aspartate; 20 KC]; 1 MgClz; 10 EGTA; 5 HEPES; and 2
ATP. The pH was adjusted to 7.3 with KOH. The extracellular solution was a HEPES-
based buffer composed of (millimolar): 155 NaCl; 5 KCl; 2 CaClz; l MgClz; 10 HEPES;
and 12 glucose, pH was adjusted to 7.4 with NaOH. All recordings were made using an
Axopatch 2OOB amplifier (Molecular Devices, Inc. Sunnyvale, CA). Data was acquired
using pCLAMP 9.1 software (Molecular Devices). Whole-cell recordings were carried

out using patch pipettes with tip resistances of 3-5 M0; seal resistances were greater than

52

1 GS). Steady state current-voltage relationships for agonist-induced whole-cell currents
were obtained using step depolarization that changed the holding potential from an initial
value of -70 to 50 mV in 20 mV increments. There was a two minute interval between
all successive agonist applications at each holding potential, -70 mV for HEK-293 cells
and -60 mV for myenteric neurons.

Drug application. Agonists were applied onto individual neurons/cells by
gravity flow from linear array quartz tubes placed near the cell. The distance from the
mouth of the tubes to the cells was ~200 um with flow controlled manually using a
micromanipulator. Computer-controlled solenoid valves (General Valve, F airfield, NJ)
were used to gate solution flow through the tubes. Agonist-induced currents were
measured as the peak current amplitude. The difference between the predicted sum of the
individual peak currents was compared with the current amplitude caused by co-
application of two agonists.

Statistics. Data are expressed as the mean :1: S.E.M. and “n“ refers to the number
of cells from which the data were obtained. The predicted current amplitude that would
occur during agonist co-application was calculated by measuring the peak currents
caused by previous individual agonist applications in the same cell and then summing
these values. These are repeated measurements within the same cell; therefore, there is no
concern for error within a single measurement because it cancels itself out. These are
directly measured data that were used for statistical comparisons of actual and predicted
currents. Traces illustrating predicted currents were constructed using a simple math
function in Origin 7 (OriginLab Corp., Northharnpton, MA). Student’s t test or analysis

of variance were used to establish significant differences between control and treatment

53

groups. The significance level was P< 0.05. EC50 is the half-maximal effective

concentration and nH is the slope. Ymin and Ymax are the minimum and maximum

respectively. Agonist concentration-response curves were fit to the following logistic

function using Origin:
y = (_YJ_D_11_L:_Y_lfl_X.) + Ymax

1 + (x/ECSO) "H

lmmunocytochemistry (ICC): After transfection, cells were washed three
times with 0.1 M phosphate buffered solution (PBS) and then fixed for 20 minutes with
Zamboni’s fixative (2% formaldehyde and 0.2% picric acid in 0.1 M PBS), pH 7.0. The
cells were washed again with PBS and incubated with 0.1% Triton for 30 minutes prior to
addition of primary antibodies. Primary antibodies in PBS with 5% serum were applied
and incubated at 37°C for 2 hr. The cells were then washed with PBS, followed by
incubation with secondary antibody for 1hr at 37°C. Cells were washed again with PBS
before mounting on slides with anti-fade solution (Zhou et a]. 2002). Primary antibodies
were at 1:200 dilution: rabbit anti-P2X2 (Alomone, Jerusalem), goat anti-alpha 3 (SC-
1711, Santa Cruz Biotechnology), mouse anti-EB (Covance Research Products).
Secondary antibodies were F ITC at (1:40) and Cy3 (1:200) IgG (Jackson
Immunoresearch Laboratories). Cells were viewed and analyzed using a Leica

Microsystems confocal microscope and image acquisition and analysis software.

54

RESULTS

Inhibitory interaction between nAChRs and P2X receptors in cultured
myenteric neurons. ACh (3 mM) caused an inward current in 98% of the myenteric
neurons tested, while ATP (1 'mM) caused an inward current in 80% of the neurons
tested. The mean current amplitudes caused by individual application of ACh (3 mM)
and ATP (1 mM) were not statistically different (Fig. 3.1). However, when the two
agonists were applied simultaneously to the same neurons the peak inward current was
only 57 d: 6% of the predicted current that would have occurred had the two channels
functioned in an independent manner (n = 8, P < 0.05, Fig. 3.1); these data confirm
previously published work (3 5). In the remainder of the paper, we will call the inhibitory
interaction between nAChRs and P2X receptors “cross-inhibition”.

Properties of P2X2 receptors and a3B4 nAChRs expressed in HEK-293 cells.
We wanted to verify expression of these receptors in this heterologous system. First we
utilized a HEK-293 P2X2 stable cell line to visualize P2X2 receptors near the plasma
membrane (Fig. 3.2A). Wild-type HEK-293 cells were also transiently transfected with
a3B4 nAChR and GFP (Fig. 3.23). P2X2 stable HEK-293 cells transiently transfected
with a3B4 nAChR subunits displayed co-expression in the plasma membrane (Fig. 3.2C-
E). Therefore, we have succeeded in attaining HEK-293 cells that express both receptors,

singly and together. We verified that the pharmacological and functional properties of

heterologously expressed P2X2 receptors and a3 B4 nAChRs were similar to those seen in

cultured myenteric neurons. In HEK-293 cells stably expressing P2X2 receptors, ATP (1

mM) evoked a rapidly developing current that was largely sustained throughout the

55

Myenteric Neurons

 

 

 

 

 

A
ATP ACh ACh + ATP ATP ACh
E 0.2nA Actual
2 s ’
B 2.01 2
Predicted

I—l
(II
1

n= 6-8

Peak current (nA)
c —
in c
l l

 

 

9
c
I

IATP IACh Actual Predicted IATP IACh
L__Y_J
I ACh/ATP

Figure. 3.1: Cross- inhibition between P2X receptors and nAChRs in guinea pig
ileum cultured myenteric neurons.

A: Inward currents caused by ATP (1 mM), an agonist for P2X receptors and ACh (3
mM) an agonist for nAChRs in the same neuron. Co-application of ACh and ATP caused
an inward current that was smaller than the predicted current that would have occurred if
the two channels functioned independently (P < 0.05). Individual re-application of ATP
and ACh caused inward currents that were not statistically different from the original
responses (P > 0.05). B: Pooled data from experiments shown in “A”. “n” indicates the
number of neurons. *Indicates significantly different from the predicted current.

56

(1;; \ulumil

:\lll)llllll

50 pm

 

Figure 3.2: lmmunocytochemistry of HEK-293 cells.

A. In a P2X2 stable HEK-293 cell line, immunofluorescence for an antibody for the P2X2
receptor. B. In a3B4 nAChR HEK-293 cells, immunofluorescence for an antibody against
the (:3 subunit of the nAChR. C-E. In P2X2 stable HEK-293 cells transiently transfected
with a3B4 nAChR, immunofluorescence for P2X2 (C), (13 subunit of nAChR (D), and an
overlay (E) of C and D.

57

period of ATP application. This response was similar to that caused by ATP in cultured

myenteric neurons (Fig. 3.3A). ACh (3 mM) evoked a rapidly developing and
desensitizing current in HEK-293 cells stably expressing a3B4 nAChRs and this response
was similar to that measured in cultured myenteric neurons (Fig. 3.3B). Concentration-
response curves and current-voltage relationships were determined for ATP in HEK-293

cells stably expressing P2X2 receptors and for ACh in HEK-293 cells stably expressing
a3B4 nAChRs. ATP caused a concentration-dependent increase in current amplitude

with a half-maximal effective concentration (EC50) of 20 j: 10 uM; the maximum current

occurred at 300 uM (Fig. 3.3C). The Hill slope was 0.9 i 0.3 (n = 6). These values were
similar to those reported previously for ATP evoked inward currents in cultured
myenteric neurons (3 8).

ACh caused a concentration-dependent inward current in HEK-293 cells with an
EC50 of 130 3c 20 uM and a Hill slope of 2.0 :t 0.6 (Fig. 3.3C). These values were similar

to those reported previously for ACh-induced activation of nAChRs expressed by

cultured myenteric neurons (36). Current-voltage relationships for ACh- or ATP-
induced currents were studied in HEK-293 cells stably expressing a3B4 nAChRs or P2X2

receptors, respectively. ACh-induced currents exhibited an inwardly rectifying current
voltage relationship with a reversal potential of 8.0 :t 4.5 (n = 5, Fig. 3.3D). The slope

conductance at -50 was 33 :1: 6 n8 while at 50 mV this value was 7 i 4 n8. ATP-induced
currents in P2X2 stable cells also exhibited inward rectification and the reversal potential

was 15 :1: 6 (n = 4-5). At -50 mV the slope conductance was 48 :t 5 nS while at 50 mV

58

A

 

 

  

 

    
  
 

fl ACh
Myenteric . Myenteric
Neurons Neurons
. _J 0.2nA I
‘. , 28 0.2nA
-. /
v” Zs
B ATP ACh
P2X2 ' “3'34
Stable cell Stable cell -—I 0.2nA
line ‘ llne 25‘
‘ /
-/ _Jo.2 nA
Zs
1.0
C 1) Il
.. 100.1 I A1? I
t: O ACh -75 25 so 75
i8. 80' Voltage (mV)
8
g 60‘ 20 -I-AI‘P
. ' ' ‘ -e-ACh
g 40-
°\° 20. «3.01
ouw

 

 

4.0‘
.6 -5 .4 .3 Current (nA)
Log [Agonist]

Figure 3.3: Comparison of pharmacological and functional properties of P2X;

receptors and (13154 nAChRs expressed by cultured myenteric neurons and HEK-293
cells.

A: Inward currents caused by ATP (1 mM, left) and ACh (3 mM, right) in a cultured
myenteric neuron. The ATP response desensitized slowly while the ACh response
desensitized completely during ACh application. B: Inward currents caused by ATP (1
mM) in an HEK-293 cell line stably expressing P2X2 receptors (left) and by ACh (3 mM)
in an HEK-293 cell line stably expressing a3B4 nAChRs (right). Although larger in
amplitude, the properties of the currents in cultured myenteric neurons and HEK-293
cells were similar. C: Concentration-response curves for ATP and ACh in HEK-293
cells stably expressing P2X2 and a3B4 nAChRs. D: Current-voltage relationship for
ATP- (1 mM) and ACh- (3 mM) induced currents in HEK-293 cells stably expressing
P2X2 and a3B4 nAChRs.

59

this value was 6 A: 3 n8. These properties are very similar to those reported previously
for nAChRs and P2X receptors expressed by cultured myenteric neurons (37, 38, 39).
All subsequent experiments use saturating concentrations of ATP (1 mM) and ACh (3

mM) unless specified otherwise.
Cross-inhibition between P2X; receptors and a3B4 nAChRs in HEK-293 cells.

The data described above indicate that the functional and pharmacological properties of
heterologously expressed nAChRs and P2X receptors are similar to those of the receptors

expressed by cultured myenteric neurons. Therefore, we used HEK-293 cells that stably

expressed rat P2X2 receptors and that were also transiently transfected with murine a3

and B4 nAChR subunits to study cross-inhibition between these receptors. Due to

receptor overexpression, the mean current amplitudes caused by individual application of
ACh and ATP were larger than those recorded from cultured myenteric neurons (Fig.
3.4). However, as was observed in cultured myenteric neurons, simultaneous application
of maximum concentrations of ACh and ATP caused an inward current that was only 61
i 3 % (P < 0.05, n=9) of the predicted current that would have occurred had the
heterologously expressed P2X2 receptors a3B4 nAChRs fimctioned independently.
Responses caused by individual application of ACh and ATP obtained after agonist co-
application were not statistically different from the original current amplitudes (P > 0.05;
n = 9, Fig. 3.4). Furthermore, when the predicted current value was calculated using
individual agonist-evoked currents obtained after agonist co-application, the actual
current was significantly smaller than this predicted value (P <0.05; n=6). In the next

experiments, we used a cell line that stably expressed a3 B4 nAChRs and that was

60

P2X2 stable - a3B4 nAChR HEK-293 cells

 

 

A ATP ACh ACh + ATP ATP ACh
Actual
l1 nA
2 s
6.0- X Predicted

Peak cu rrent (nA)

%%

 

 

IATP IACh Actual Predicted [ATP IACh

H__J

I ACh/ATP

Figure 3.4: Cross-inhibition between P2X2 stable receptors and a3B4 nAChRs
expressed by HEK-293 cells.

A: ATP- (1 mM) and ACh- (3 mM) induced inward currents recorded from a HEK-293

cell stably expressing P2X2 receptors and transiently transfected with a3B4 nAChRs.

Currents caused by agonist co-application exhibited cross-inhibition. Individual re-

application of ATP and ACh caused inward currents that were not statistically different

from the original responses (P > 0.05). B: Pooled data from experiments shown in “A”.
'“n” indicates the number of cells. *Indicates significantly different from the predicted
current.

61

a3B4 nAChR stable - P2X2 HEK-293 cells

ACh ‘ ATP ACh + ATP

A ~—

u
”14‘

Actual

5 l0.5 nA
.. 2 s
B 3.0-

    

 

Predicted

% 2.0.

= T

0

l:

8

g 1.0-

a.

0.3 .

 

 

 

 

[ACh [ATP Actual Predicted

I ACh/ATP

Figure 3.5: Cross-inhibition between «3114 nAChRs stable and P2X; receptors
expressed by HEK-293 cells.

A: ATP- (1 mM) and ACh- (3 mM) induced inward currents recorded from a HEK-293
cell stably expressing a3B4 nAChRs and transiently transfected with P2X2 receptors.
Currents caused by agonist co-application exhibited cross-inhibition. B: Pooled data
from experiments shown in “A”. “n’ indicates the number of cells. *Indicates
significantly different from the predicted sum of currents (P < 0.05).

62

transiently transfected with P2X2 receptors. In these cells, currents caused by

simultaneous application of ACh and ATP were only 58 i 5% of the predicted current
(Fig. 3.5, P < 0.05, n = 13) that would have occurred if the receptors functioned
independently. Subsequent individual re-application of ACh and ATP caused currents
that were not statistically different from those elicited before agonist co-application (P >
0.05, n =13, data not shown). Furthermore, when the predicted current value was
calculated using individual agonist-evoked currents obtained after agonist co-application,
the actual current was significantly smaller than this predicted value (P<0.05, n = 13).

As mentioned above, receptor over-expression resulted in current amplitudes in
HEK-293 cells that were larger than those in cultured myenteric neurons. It is possible

that nAChR and P2X receptor activation caused by co-application of maximum agonist
concentrations produced local Na+ or Ca2+ depletion reducing the driving force required

for inward current. Another concern would be poor voltage control of large amplitude
currents evoked by co-application of maximum agonist concentrations. In order to rule
out these possibilities, EC50 concentrations of ACh (100 uM) and ATP (30 uM) were
used to activate nAChRs and P2X receptors, respectively. Because submaximal agonist
concentrations were used, neither receptor desensitized (Fig. 3.6). However, the inward
current caused by simultaneous activation of (13 B4 nAChRs and P2X2 receptors was only
58 :1: 7 % of predicted response (P < 0.05, n = 7).

ACh may interact directly with P2X receptors to modulate their function or ATP
may directly interact with nAChRs to modulate their function. Cultured myenteric

neurons cannot be used to evaluate this possibility as nearly all neurons express both

63

a3B4 nAChR stable - P2X2 HEK-293 cells
A 100 ttM 30 11M

 

    

 

 

 

 

 

Actual
411/
/ p l 1 nA
4'0 Predicted “-
B 2 s

2‘ 3.0-
5
E 2 0 -—1—
E
3
.a:

1.0-
E

0.3 .

IACh [ATP Actual Predicted
I ACh/ATP

Figure 3.6: Cross-inhibition between P2X; receptors and a3B4 nAChRs expressed

by EEK-293 cells is maintained when receptors were activated by agonist EC50
concentrations.

A: ATP- (30 uM) and ACh- (100 11M) induced inward currents recorded from a HEK-
293 cell stably expressing 0.3[34 nAChRs and transiently transfected with P2X2 receptors.
ACh current activated by the EC50 concentration does not desensitize. Currents caused by
agonist co-application exhibited cross-inhibition. B: Pooled data fiom experiments

shown in “A” “n’ indicates the number of cells. *Indicates significantly different from
the predicted current (P < 0.05).

64

P2X2 Stable EEK-293 cells a3B4 nAChR HEK-293 cells

A , ATP ACh ACh +ATP C ATP ACh ACh+ATP

 

l0.4 nA

 

 

    

. i I 0.5 nA 2 s
2 s
B 4.0 D
A n= 4
E 3.0 Q
:7 E.
E z 0 E n=6
3 a
J
i
2 1.0 A:
0.0
IATP IACh Ami“ IA" IACh Actual
L J

Y
[ACh/ATP IACIIATP

Figure 3.7: ACh and ATP do not directly modulate P2X2 receptor or a3B4 nAChR
function.

A: ATP (1 mM) caused an inward current in a HEK-293 cell stably expressing only the
P2X; receptor ACh (3 mM) did not cause an inward current in these cells. ATP and ACh
co-application caused an inward current that did not differ from the response caused by
ATP alone. B: Pooled data from experiments shown in “A”. In cells expressing only the
P2X2 receptor, co-application of ATP and ACh caused an inward current that is not
different from the response caused by application of ATP alone (P > 0.05). C: ACh (3
mM) caused an inward current in a HEK-293 cell stably expressing only the a3B4
nAChR. ATP (1 mM) did not cause an inward current in these cells. ATP and ACh co-
application caused an inward current that did not differ from the response caused by ACh
alone. C: Pooled data from experiments shown in “B”. In cells expressing only the a3B4
nAChR, co-application of ATP and ACh caused an inward current that was not different
from the response caused by application of ACh alone (P > 0.05).

65

 

30 P2X4

M 53 P2X3
119 P2X2

 

 

Figure 3.8: Cartoon illustrating P2X subunit C-terminal tail amino acid length
differences among P2X2, P2X3 and P2X4 subunits.

66

receptors. Therefore, we used HEK-293 cells expressing only P2X2 or 0L3B4 nAChRs to
test for the direct agonist modulation mechanism of cross inhibition. ACh (3 mM) did not
cause an inward current in HEK-293 cells expressing only P2X2 receptors and ACh did
not alter currents caused by ATP in these cells (P > 0.05, n = 4, Fig. 3.7A). Likewise,
ATP (1 mM) did not cause an inward current in cells expressing only a3B4 nAChRs and

ATP did not alter currents caused by ACh in these cells (P > 0.05, n= 7, Fig. 3.78).

P2X3 and P2X4 subunits interact with nAChRs. The specificity of the P2X

and nAChR receptor interaction was determined by transiently expressing P2X3 or P2X4
subunits in HEK-293 cells stably expressing a3B4 nAChRs. P2X3 and P2X4 subunits
differ from P2X2 subunits in the length of the intracellular C-terminal tail (Fig. 3.8). In

cells co-expressing a3B4 nAChR/P2X3 receptors co-application of ACh and ATP caused
an inward current that was only 53 d: 5% of the predicted current (Fig. 3.9A,B, n = 25, P
< 0.05). In cells co-expressing a3B4 nAChR/P2X4 receptors, agonist co-application

caused an inward current that was only 58 i 5% of the predicted current (P < 0.05, n =
21, Fig. 3.9C,D).

Receptor desensitization. We next investigated if the desensitized state of
nAChRs could maintain cross-inhibition of P2X2 receptors, individual activation currents
were 1.9 :t 0.3 nA and 2.5 i 0.3 nA for ACh and ATP respectively. ACh was
continuously applied toHEK-293 cells stably expressing a3B4 nAChRs and transiently
transfected with P2X; receptors. This treatment caused nAChR desensitization and then
ATP and ACh were co-applied. Because the nAChR was desensitized, the current should

only be due to activation of P2X2 receptors. The ATP-induced current was only 71 i 7%

67

u3B4 nAChR stable-P2X; HEK-293 cells “334 nAChR stable-P2X4 HEK-293 cells

A ACh ATP ACh+ATP B 401 ATP AC h+ \TP

AKc/tual Actual
_i 0.3 nA
2 s I
I l nA l l "A
2 3 Predicted 2 3 Predicted

2S

 

 

 

C 4.0 D
5 :5 3. 0
v =
E 3.0 T:
l:
t 2
‘5 2.0 g 2°
9 ca
-“ a:
3: 1.0 g 1.0
0.0
I“), I", Actual Predicted Im Actual Predicted
[Act/Arr I‘m“?

Figure 3.9: P2X subunit C-terminal tail length does not alter cross-inhibition of
(13454 nAChR function.

A: P2X2, P2X3 and P2X4 subunits differ mainly in their C-terminal tail amino acid
length. P2X4 subunits have the shortest C-terminal tail (30 amino acids). B: Inward
currents caused by ACh (3 mM) and ATP (1 mM) in HEK-293 cells stably expressing
a3B4 nAChRs and transiently transfected with P2X3 receptors. Inward currents carried by
P2X3 receptors desensitize rapidly. The inward currents caused by co-application of ACh
and ATP were smaller than the predicted current that would occur if the receptors
functioned independently. Histograms show pooled data from experiments shown in the
upper traces. C: Inward currents caused by ACh and ATP in HEK-293 cells stably
expressing a3B4 nAChRs and transiently transfected with P2X4 receptors. The inward
currents caused by co-application of ACh and ATP were smaller than the predicted
current that would occur if the receptors functioned independently. Histograms show
pooled data from experiments shown in the upper traces. *Indicates significantly
different from the predicted current (P < 0.05) in both sets of experiments.

68

(13134 nAChR stable-P2X2 HEK—293 cells a3B4 nAChR stable-P2X2 HEK-293 cells

 

 

 

   

 

 

 

 

 

 

 

 

 

ACh + ACh +
ACh ATP ACh E? ACh ATP ATP All)
A ‘ B * ,-
l ‘ ./"
i J ”A ‘l J 0.5 nA
I 2 s 2 s
.. It 'i
a:
3_o. 3.0'
n = 7 n = 11
A A
E 2.0- I E 7.0- l
E E
= E
.: L0" 3 1.0-
3 :2
9.; j n
I Ch [ATP I 0.“ i
A
ACh/A" IACII IATP I ACME,

Figure 3.10: Cross inhibition between P2X; receptors and (13134 nAChRs is
maintained during receptor desensitization.

A: Inward currents caused by ACh (3 mM) and ATP (1 mM) in HEK-293 cells stably
transfected with a3B4 nAChRs and transiently transfected with P2X2 receptors.
Prolonged ACh application desensitized the 0t3B4 nAChR but the inward current caused
by ATP in the continued presence of ACh was smaller than the current caused by ATP
prior to nAChR desensization. Histograms show pooled data from experiments shown in
the upper traces. B: Similar results were obtained in the same cells when the P2X2
receptor was first desensitized by prolonged application of ATP. The inward current
caused by ACh during P2X2 receptor desensitization was smaller than the ACh current
recorded before P2X2 receptor desensitization. Histograms show pooled data from
experiments shown in the upper traces. *Indicates significantly different from the current
recorded prior to (P < 0.05) in both sets of experiments.

69

of the initial current obtained prior to nAChR desensitization (P < 0.05, n = 7, Fig.
3.10A). The current recorded during simultaneous agonist application in the presence of
nAChR desensitization was 71% of the predicted current; this value is higher but not

statistically different (58 i 7%, n=13; P > 0.05) than that obtained during ATP and ACh

application when both the P2X2 and a3B4 nAChRs were fully function (i.e., not

desensitized). The same experiment was repeated, except this time the P2X2 receptors
were desensitized using prolonged ATP application immediately followed by co-
application of ACh and ATP. After P2X2 receptor desensitization, the ACh-induced

current was only 72 i 6 % (P < 0.05, n = 11, Fig. 3.10B) of the initial ACh induced

current. Again, this value was higher but not statistically different (P > 0.05, n=13) from
that measured during ATP and ACh co-application when the P2X2 and a3B4 nAChRs

were fully functional.

To determine the specificity of this interaction, similar experiments as above were
performed with the P2X3 and the P2X4 subtypes of the receptor. For nAChR/P2X3 cells,
ACh-induced currents after P2X3 receptor desensitization were 74 :h 9% of the initial
ACh current (11 = 9, P < 0.05, Fig. 3.11A). This value was significantly different from
that occurring during simultaneous P2X3 receptor and nAChR activation when these
receptors were fully functional (non-desensitized) (see above, 53 :1: 5%; P < 0.05). For
nAChR/PZX4 cells, ACh-induced currents after P2X4 receptor desensitization were only
75 :1: 6% of initial activation (P < 0.05, n = 9, Fig. 3.118). This value was significantly
different from that occurring during simultaneous P2X4 receptor and nAChR activation

when these receptors were fully functional (see above, 58 i 5%; P < 0.05).

70

a3B4 nAChR - P2X3 HEK-293 cells a3B4 nAChR - P2X4 HEK-293 cells
*
2.0-l

      

 

 

A B 9
n =
Q
5 A
E- E
5 E 1.0'
I: B
E 25;
//
0.0"
IACII [ATP 1 ACh/ATP Ixcn IATP I ACh/ATP

Figure 3.11: Cross inhibition between P2X3 or P2X4 receptors and (13134 nAChRs is
maintained during receptor desensitization.

A: Histograms showing the amplitude of ACh (3 mM) and ATP (1 mM) currents before
P2X3 receptor desensitization. Then ATP was applied continuously to desensitize the
P2X3 receptors and ACh was re-applied in the continued presence of ATP (see Figure 8
for protocol). The ACh current was significantly smaller than that recorded prior to P2X3
receptor desensitization. B: Histograms showing results of experiments similar to those
in “A” but using cells co-expressing P2X4 receptors and a3B4 nAChRs. The ACh current
recorded during P2X4 receptor desensitization was smaller than that recorded prior to the

desensitization protocol. *Indicates significantly different from the current recorded
prior to desensitization (P < 0.05) in both sets of experiments.

71

DISCUSSION

ACh and ATP are co-transmitters in the ENS (10, 28, 30) and interactions
between their target receptors has been shown in guinea-pig celiac ganglion neurons (32,
33), rat PC-12 cells (23, 25), superior cervical sympathetic neurons (24), cultured
myenteric neurons (38) and in heterologously expressed receptors in Xenopus oocytes

(13). In the present study, we have reproduced this receptor interaction in mammalian

cells expressing (x3B4 nAChRs and several P2X receptor subunits.
Functional and pharmacological properties of P2X; receptors and nAChRs

are retained in HEK-293 cells. We confirmed that P2X2 receptors and nAChRs are

functionally linked in guinea pig cultured myenteric neurons (38) and show that this
interaction can be reproduced in a heterologous expression system. Heterologous

expression of these receptors did not alter their localization and pharmacological
function. When the P2X2 and a3B4 nAChRs are heterologously expressed in HEK-293
cells individually, they are localized to the cell membrane. In cells that express both
P2X2 and (13 B4 nAChRs, both receptors are co-localized to the membrane as observed by
immunecytochemistry. This displays a spatial proximity of these receptors in the cell
membrane. P2X2 receptors desensitize slowly while responses mediated by enteric (13B4
nAChRs desensitize in less than 2 5; these properties were retained in HEK-293 cells
expressing a3B4 nAChRs or P2X; receptors. Concentration-response curves for ACh-
and ATP-induced inward currents were also similar in cultured myenteric neurons and

HEK-293 cells expressing a3B4 nAChRs or P2X; receptors. The current voltage
relationship for currents carried by P2X2 receptors a3B4 nAChRs exhibit inward

72

rectification (37, 38, 39) and this property was also retained in HEK-293 cells.
Therefore, HEK-293 cells are an appropriate model system to study the details of the
molecular interactions between P2X and nAChRs.

Cross- inhibition between nAChRs and P2X receptors expressed in HEK-

293 cells. We used HEK-293 cells that stably expressed P2X2 receptors and transiently
expressed 0L3B4 nAChRs or HEK-293 cells that stably expressed a3B4 nAChRs and

transiently expressed P2X2 receptors to study receptor cross inhibition. Similar data were
obtained with both types of receptor combinations. As occurs with cultured myenteric
neurons, when P2X2 receptors and nAChRs are co-activated in HEK-293 cells expressing

both receptors, the resulting inward current is substantially smaller than the current that
would have occurred if the two receptors functioned independently. If P2X receptors and
nAChRs function independently, co-activation of receptors would cause fully additive
currents. The response amplitude would be predicted by summing the currents evoked by
individual agonist application; this is the predicted current. The data obtained in HEK-
293 cells indicate that cross-inhibition is not a neuronal specific response and it does not
require neuron specific proteins or other molecules to maintain the receptor interaction.
We have also ruled out receptor rundown or desensitization as a mechanism responsible
for the proposed cross-inhibition as we were able to show that the currents caused by
individual application of ACh or ATP were stable in amplitude through the time course
of our studies.

It is possible that cross-inhibition occurs because P2X receptors have an allosteric
inhibitory binding site for ACh, or that nAChRs have a similar binding site for ATP (25,

31). Another possibility is that ACh activates muscarinic receptors or ATP activates P2Y

73

receptors which are both endogenously expressed by HEK-293 cells (22). These
receptors link to protein kinase C activation which could then alter the function of

nAChRs or P2X receptors. These hypotheses would be difficult to test in cultured
myenteric neurons, as nearly every neuron co-expresses P2X2 receptors and nAChRs (37,

38) and many myenteric neurons also express muscarinic and P2Y receptors (11, 21).

However, using HEK-293 cells it is possible to control receptor expression. We showed

that ACh does not activate P2X2 receptors, and more importantly, ACh does not modify
activation of the P2X2 receptor by ATP in the absence of nAChR expression. Similarly,
ATP does not modify nAChR function in the absence of P2X2 receptor expression.

These data indicate that ACh or ATP do not directly modulate the firnction of P2X;

receptors or nAChRs and do not indirectly modify their function via activation of
muscarinic- or P2Y-receptor linked signaling pathways.
We used saturating agonist concentrations in the studies of cross inhibition. It is

possible that cross inhibition is not due to direct receptor interaction but could be due to
local depletion of Na+ or Ca2+ ions near the channel during maximal activation.

Inadequate voltage control during maximal receptor activation could also account for

non-additivity of agonist responses. To address these concerns, we studied receptor cross
inhibition using EC50 concentrations of ACh and ATP. We found that even during

submaximal receptor activation, agonist co-application produced responses that were

smaller in amplitude than the predicted additive response. These data indicate non-
additivity between P2X2 and nAChRs is not attributable to local ion depletion or poor

voltage clamp of large amplitude currents.

74

Cross inhibition of a3B4 nAChR function is not specific for P2X; receptors.

We next determined if inhibition of (13B4 nAChR function was specific for P2X2 subunits.
P2X receptor subunits differ largely in the length and composition of their C-terminal

intracellular tail. The P2X2 subunit has a C-terminal tail length of 119 amino acids (26).
We focused on P2X3 subunits, with a C-terminal tail length of 53 amino acids, as these

are expressed by myenteric neurons (29) and the P2X4 subunit because it has the shortest

C-terminal tail at 30 amino acids (26). By narrowing down the region of the C-terminal
tail that might be responsible for this interaction, a better understanding of the sequence

regions or motif that mediate this interaction can be elucidated. We detected cross-
inhibition between or, B4 nAChRs and both P2X3 and P2X4 receptors suggesting that a3B4

nAChRs can interact with multiple P2X receptors subunits. Although P2X2 and P2X4 C-

terrninal tails do not have identical sequences (24), the 30 amino acid C-terminal tail

length of the P2X4 subunit is sufficient to produce the proposed allosteric linking to the

(13 B4nAChR. Others have shown that a segment of the C-terminal tail of the P2X2 subunit
(amino acids 22-119 from the end of the second transmembrane domain) interacts with
the cys-loop 5-HT3A and GABAA receptors (3, 4).

Desensitized states of P2X and nAChRs can mediate cross-inhibition.
Previous work (21) showed that cross inhibition between neuronal nAChRs and P2X

receptors requires the open state of the channels and little cross inhibition occurred

during desensitization of one of the receptors. However, we found cross-inhibition of

a3B4 nAChR function when P2X2, P2X3 or P2X4 receptors co-expressed by the same

cells were desensitized. Similarly, P2X2 receptor function could be inhibited by

75

desensitized a3B4 nAChRs in cells expressing both receptors. Although cross-inhibition

was maintained during receptor desensitization the amount of cross- inhibition was not

equivalent to that occurring when fully active, non-desensitized receptors were studied.

This difference was most prominent when nAChRs were co-expressed with P2X3 or
P2X4 subunits. This suggests that the desensitized state of one receptor (either the P2X

or 0L3B4 nAChR) is less efficient at impairing current flow through the linked receptor.

Therefore, differences in our data from those of Nakazawa (24) are based on the
magnitude of cross-inhibition caused by desensitized receptors rather than complete

inability of the desensitized receptor to cause cross inhibition. Khakh and co-workers

(13) studied the effects of receptor desensitization on interactions between P2X2
receptors and a3B4 nAChRs expressed in Xenopus oocytes. They used a mutant P2X2
receptor (T18A) which desensitizes more than 10-fold faster than wild type P2X2
receptors. These studies showed that currents carried by a3B4 nAChRs were inhibited
when the T18A P2X2 receptor was firlly activated but that nAChR currents partly
recovered as the T18A P2X2 receptor desensitized. These data are again consistent with
our data showing that the desensitized state of one receptor in the interacting pair (a3B4

nAChR-P2X2) inhibits current flow through the coupled receptor, but the inhibition is

reduced compared to that occurring when the channels are in the fully open state.
Conclusions. Cross-inhibition between Cys-loop type receptors and P2X

receptors maybe a mechanism of receptor modulation in the enteric nervous system (2,

38). This interaction does not require intracellular signaling mechanisms and occurs in

cell free patches of cultured neurons suggesting close coupling of the two receptors in the

76

neuronal membrane (38). Functional interaction between nAChRs and P2X2 receptors
also occurs when these receptors are expressed heterologously in HEK-293 cells

suggesting that the receptor interaction does not require nervous system specific proteins.
Conformational spread could be a mechanism for cross-inhibition between a3B4 nAChRs

and P2X receptors (13, 14) as may also occur with other receptors and channels (5).
Conformational spread would require that nAChRs and P2X receptors are so closely

associated in the membrane that opening of one channel impairs opening of the second

channel. Fluorescence resonance energy transfer analysis of a4B2 nAChRs (brain specific

receptors) and P2X2, receptors expressed in HEK-293 cells revealed that the receptors

were localized within 100 nm of each other in the plasma membrane. This close
association would make conformational spread during receptor activation possible
perhaps through heterodimer formation (15). Conformational spread would also be
consistent with our desensitization data. P2X receptors and nAChRs adopt different
conformations in the closed, open and desensitized states (12, 19). Conformation spread
from the open state readily impair current flow in the adjacent channel, while
conformation spread from the desensitized state of one receptor impairs current flow in

the adjacent channel less effectively.

Acknowledgements

We are grateful to Dr. Xiao (Georgetown University, Washington DC.) for

generously donating the a3B4 nAChR stable HEK-293 cell line, and Dr. Alan North

(University of Manchester, Manchester, UK) for the P2X2, P2X3 and P2X4 plasmids. We

77

would also like to thank Dr. Jerry A. Stitzel (University of Colorado, Boulder, CO) for

providing the mouse (13 and B4 plasmids.

78

10.

ll.

12.

REFERENCES

Bian X, Bertrand PP, Bornstein JC. Descending inhibitory reflexes involve
P2X receptor-mediated transmission from intemeurons to motor neurons in
guinea-pig ileum. J Physiol 528: 551-560, 2000.

Barajas-Lopez C, Montafio LM, Espinosa-Luna R. Inhibitory interactions
between 5-HT3 and P2X channels in submucosal neurons. Am J Physio] 283:
G1238-1248, 2002.

Boué-Grabot E, Barajas-Lopez C, Chakfe Y, Blais D, Bélanger D, Emerit
MB, Séguéla P. Intracellular cross talk and physical interaction between two
classes of neurotransmitter-gated channels. J Neurosci 23: 1246-1253, 2003.

Boué-Grabot E, Toulmé E, Emerit MB, Garret M. Subunit-specific coupling

between gamma-aminobutyric acid type A and P2X2 receptor channels. J Biol
Chem 279: 52517-52525, 2004.

Bray D, Duke T. Conformational spread: the propagation of allosteric states in
large multiprotein complexes. Annu Rev Biophys Biomo] Struct 33:53-73, 2004.

Castelucci P, Robbins HL, Poole DP, Furness JB. The distribution of purine

P2X2 receptors in the guinea-pig enteric nervous system. Histochem Cell Biol
117: 415-422, 2002.

Edwards FA, Gibb AJ. ATP - a fast neurotransmitter. FEBS 325: 86-89, 1993.

Furness, JB, Sanger GJ. Intrinsic nerve circuites of the gastrointestinal tract:
identification of drug targets. Curr Opin Pharmacol 2: 612-622, 2002.

Furness JB. The organisation of the autonomic nervous system: peripheral
connections. Auton Neurosci 30: 1-5, 2006.

Gallligan JJ, LePard KJ, Schneider DA, Zhou X. Multiple mechanisms of
fast excitatory synaptic transmission in the enteric nervous system. J A uton Nerv
Syst 81: 97-103, 2000.

Gwynne RM, Bornstein JC. Synaptic transmission at functionally identified
synapses in the enteric nervous system: roles for both ionotropic and
metabotropic receptors. Current Neuropharmacology 5: 1-17, 2005.

He ML, Koshimizu TA, Tomic’ M, Stojilkovic SS. Purinergic P2X2 receptor

desensitization depends on coupling between ectodomain and C-terminal
domain. Mol Pharmacol 62: 1187-1197, 2002.

79

13.

14.

15.

16.

l7.

l8.

19.

20.

21.

22.

23.

24.

Khakh BS, Zhou X, Sydes J, Galligan JJ, Lester HA. State-dependent cross-
inhibition between transmitter-gated cation channels. Nature 406: 405-10, 2000.

Khakh BS. Molecular physiology of P2X receptors and ATP signaling at
synapses. Nature Reviews 2:165-174, 2001.

Khakh BS, Fisher JA, Nashmi R, Bowser DN, Lester HA. An Angstrom scale
interaction between plasma membrane ATP-gated P2X2 and alpha4beta2
nicotinic channels measured with fluorescence resonance energy transfer and

total internal reflection fluorescence microscopy. J Neurosci 25: 6911-6920,
2005.

Lepard KJ, Galligan JJ. Analysis of fast synaptic pathways in myenteric
plexus of guinea pig ileum. Am J Physiol 276: G529-G538, 1999.

McGehee DS, Role LW. Physiological diversity of nicotinic acetylcholine
receptors expressed by vertebrate neurons. Ann Rev Physio] 57:521-546, 1995.

Miyazawa A, Fujiyoshi Y, Unwin N. Structure and gating mechanism of the
acetylcholine receptor pore. Nature 423: 949-955, 2003.

Mourot A, Rodrigo J, Kotzyba-Hibert F, Bertrand S, Bertrand D, Goeldner
M. Probing the reorganization of the nicotinic acetylcholine receptor during

desensitization by time-resolved covalent labeling using [3H]AC5, a
photoactivatable agonist. Mol Pharmacol 69: 452-461, 2006.

Monro RL, Bertrand PP, Bornstein JC. ATP participates in three excitatory

postsynaptic potentials in the submucous plexus of the guinea pig ileum. J
Physio] 556: 571-584, 2004.

Morno RL, Bornstein JC, Bertrand PP. Synaptic transmission from the
submucosal plexus to the myenteric plexus in Guinea-pig ileum.
Neurogastroenterol Motil 20: 1 165—1173, 2008.

Mundell SJ, Benovic JL. Selective regulation of endogenous G protein-coupled
receptors by arrestins in HEK293 cells. J Biol Chem 275 (17): 12900-12908,
2000.

Nakazawa K, Fujimori K, Takanaka A, Inoue K. Comparison of adenosine
triphosphate- and nicotine-activated inward currents in rat phaeochromocytoma

cells. J Physio] 434: 647-60, 1991.

Nakazawa K. ATP-activated current and its interaction with acetylcholine-
activated current in rat sympathetic neurons. J Neurosci 14:740-750, 1994.

80

25.

26.
27.

28.

29.

30.
31.

32.

33.

34.

35.

36.

37.

Nakazawa K. Reduction of acetylcholine-activated current by low
concentrations of extracellular adenosine-triphosphate. Life Sciences 57: 351-
356, 1995.

North RA, Surprenant A. Pharmacology of cloned P2X receptors. Ann Rev
Pharmacol T oxicol 40: 563-80, 2000.

North RA. Molecular physiology of P2X receptors. Physiol Rev 82: 1013-
1067, 2002.

Nurgali K, Furness JB ,Stebbing MJ. Analysis of purinergic and cholinergic
fast synaptic transmission to identified myenteric neurons. Neuroscience 116:
335-347, 2003.

Poole DP, Castelucci P, Robbins HL, Chiocchetti R, Furness JB. The
distribution of P2X3 purine receptor subunits in the guinea pig enteric nervous
system. Auton Neurosci 101: 39-47, 2002.

Ren J, Bertrand PP. Purinergic receptors and synaptic transmission in enteric
neurons. Purinergic Signal 4: 255-266, 2008.

Robertson SJ, Ennion SJ, Evans RJ, Edwards FA. Synaptic P2X receptors.
Curr Opin Neurobiol 11: 378-386, 2001.

Searl TJ, Redman RS, Silinsky EM. Mutual occlusion of P2X ATP receptors
and nicotinic receptors on sympathetic neurons of the guinea-pig. J Physiol 510:
783-791, 1998

Silinsky EM, Gerzanich V. On the excitatory effects of ATP and its role as a
neurotransmitter in coeliac neurons of the guinea-pig. J Physiol 464:197-212,
1993.

Toulmé E, Blais D, Léger C, Landry M, Garret M, Séguéla P, Boué-Grabot
E. An intracellular motif of P2X3 receptors is required for functional cross-talk
with GABAA receptors in nociceptive DRG neurons. J Neurochem 102: 1357-
1368, 2007.

Vial C, Roberts JA, Evans RJ. Molecular properties of ATP-gated P2X
receptor ion channels. Trends Pharmacol Sci 25: 487-493, 2004.

Xiao Y, Meyer EL, Thompson JM, Surin A, Wroblewski J, Kellar KJ. Rat
a3/B4 subtype of neuronal nicotinic acetylcholine receptor stably expressed in a

transfected cell line: pharmacology of ligand binding and fiinction. Mol
Pharmacol 54: 322-333, 1998.

Zhou X, Galligan JJ. P2X purinoceptors in cultured myenteric neurons of
guinea-pig small intestine. J Physiol 496: 719-729, 1996.

81

38.

39.

Zhou X, Galligan JJ. Non-additive interaction between nicotinic cholinergic

and P2X purine receptors in guinea-pig enteric neurons in culture. J Physiol 513:
685-697, 1998.

Zhou X, Ren J, Brown E, Schneider D, Caraballo-Lopez Y, Galligan JJ.
Pharmacological properties of nicotinic acetylcholine receptors expressed by

guinea pig small intestinal myenteric neurons. J Pharmacol Exp Ther 302: 889-
897, 2002.

82

CHAPTER 4
Molecular Mechanisms of Cross-inhibition Between Nicotinic

Acetylcholine Receptors and P2X Receptors in Myenteric Neurons and

HEK-293 cells

83

ABSTRACT
P2X2 and nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion

channels that mediate fast synaptic excitation in the enteric nervous system (ENS). P2X

receptors and nAChRs are functionally linked. This study examined the mechanisms
responsible for interactions between P2X2 and a3B4 subunit-containing nAChRs in HEK-

293 cells and cultured myenteric neurons from the guinea pig ileum. Whole-cell patch

clamp techniques were used to measure P2X and nAChR currents caused by
acetylcholine and ATP. In HEK-293 cells expressing a3B4 nAChRs and P2X2 receptors,

co-application of ATP and ACh caused an inward current that was 56 :1: 7% of the current
that should occur if these channels functioned independently (P < 0.05, n = 9); we call

this interaction cross-inhibition. Cross-inhibition did not occur in HEK-293 cells

expressing a3B4 nAChRs and transfected with a C-terminal tail truncated P2X2 receptor

(P2X2TR ; P >0.05, n = 8). Intracellular application of the C-terminal tail of the P2X2
receptor blocked nAChR-P2X receptor cross-inhibition in HEK-293 cells and myenteric

neurons. In the absence of ATP, P2X2 receptors constitutively inhibited nAChR
currents. Constitutive inhibition did not occur in cells expressing a3B4 nAChRs
transfected with P2X2TR. ATP-induced currents in cells stably expressing P2X2
receptors were unaffected by co-expression with a3B4 nAChRs in the absence of ACh.
These data indicate that a portion of the C-terminal tail of P2X2 receptors mediates cross-

inhibition between a3B4 nAChR-P2X2 receptors. The closed state of the P2X2 receptor

84

inhibits nAChR function. These molecular interactions may modulate transmission at

enteric synapses that use ATP and acetylcholine as neurotransmitters.

85

INTRODUCTION

Nicotinic acetylcholine receptors (nAChRs) and P2X receptors are ligand-gated
cation channels that mediate cholinergic and purinergic fast synaptic excitation of enteric
neurons (9, 10, 11). NAChRs are a member of the Cys-loop receptor family which also
includes 5-HT3 receptors and GABAA receptors. Cys-loop receptors are composed of 5
subunits and each subunit has four transmembrane domains and extracellular N and C-

terminal tails (21, 22). Neuronal nAChRs are composed of two or and three B subunits;

there are eight neuronal a (dz-(19) and three neuronal B (Bz-B4) nAChR subunits (21). In

the ENS, the predominant nAChR subtype is composed of a3 and B4 subunits, and

acetylcholine (ACh) acting at nAChRs is the primary fast excitatory neurotransmitter in
the ENS (9, 42). P2X receptors belong to a different family of ligand-gated cation
channels. P2X receptors are composed of three subunits; each subunit has two

transmembrane domains and intracellular N and C-terminal tails. P2X receptors are

homomeric or heteromeric receptors composed of one or combinations seven (P2X1-

P2X7) subunits (7, 16, 28, 29, 34, 38). Enteric neurons express P2X2 and P2X3 subunits

(5, 31, 41). ATP acting at P2X receptors contributes to fast synaptic excitation in subsets
of enteric neurons and ATP and ACh are co-transmitters at many synapses in the ENS (9,
19,30,33)

Because P2X receptors and nAChRs are so differently structured, they have been
assumed to function independently. However, studies have shown a functional
interaction between these channels. P2X and nAChR co-expressed in cultured myenteric

neurons and in human embryonic kidney cells (HEK-293), when simultaneously

86

activated, mediate a mutually inhibitory interaction. That is, activation of one channel
inhibits responses mediated by the other channel, resulting in fewer channels opening
than expected, also referred to as cross-inhibition (41, 15, 6). This functional interaction

has also been shown to occur between nAChRs and different subtypes of the P2X
receptor including P2X2, P2X3 and P2X4 (20). It seems that P2X receptors interact

promiscuously with different Cys-loop family members; studies have shown interactions
between P2X receptors and nAChRs, 5-HT3 and GABAA receptors (41, 15, 6, 3, 4).

More detailed studies have also observed that the functional interactions between these
receptors occur not only in the active, but also in the desensitized states of these receptors
in HEK-293 cells (6).

This study examines in more detail the molecular mechanisms that underlie the
functional relationship between these receptors and its dependence on a concurrent
physical association in HEK-293 cells and cultured myenteric neurons. Because HEK-
293 cells do no endogenously express any of these receptors, they are a unique
mammalian model to investigate the specific interactions that involve specific domains of

the receptors which may attribute to the functional coupling between them.

87

MATERIALS AND METHODS

Human embryonic kidney (HEK-293) cells

Cells were obtained from American Type Culture Collection and grown in

Dulbecco’s modified Eagle’s medium (DMEM) F-l2 containing 10% fetal bovine serum,

10% GluMax (Invitrogen. Carlsbad. CA), and 100 units ml'l penicillin and streptomycin,
except for the rat a3B4 nAChR stable HEK-293 cell line which also contained 0.5 mg/ml

geneticin (GIBCO, G418). Cells were maintained at 37 °C in a 5% C02 atmosphere in a

humidified incubator. Cells were passaged once every 3 days when they reached 90%
confluence. Transient transfection of cells was accomplished using electroporation. A
0.4 cm Gene Pulser Cuvette (BIO-RAD) was used with 2 ug of total plasmid protein

along with 0.2 pg of GFP to help identify transfected cells. The electroporation machine
used was Gene Pulser XcellTM Electroporation System (BIORAD). This resulted in 80%

transfection efficiency. Afterwards, cells were plated on 35 mm coverslips and

maintained in the incubator for 24 hrs before use in electrophysiology experiments. A rat

a3B4 nAChR stable HEK-293 cell line was provided by Dr. Yingxian Xiao from

Georgetown University (40). Plasmids containing the coding sequences for rat P2X2
receptor subunit was provided by Dr. Alan North (University of Manchester, Manchester,
UK). Plasmids containing the coding sequence for the rat P2X2TR where a generous gift
from Dr. Boué-Grabot (CNRS Université Victor Segalen Bordeaux, France). Plasmids
containing the coding sequences for the murine a3 and murine B4 nAChR subunits were

provided by Dr. Jerry A. Stitzel (University of Colorado, Denver Colorado).
88

Myenteric neurons

Neurons were cultured as described previously (41, 6). Two newbom guinea-pigs
(<36 hrs old) were killed by severing the major neck blood vessel following halothane

anesthesia. The small intestine was removed from the animals and placed in cold (4 °C)

Krebs solution of the following composition (millimolar): 117 NaCl; 4.7 KCl; 2.5 CaClz;

1.2 MgClz; 1.2 NaHzPO4; 25 NaHCO3 and 11 glucose. The longitudinal muscle

myenteric plexus was stripped free using a cotton swab and cut into 5 mm pieces. Tissues
were digested with 1600 U of trypsin, followed by trituration with a fire-polished Pasteur
pipette. After incubation with 2000 U crab hepatopancreas collagenase, the tissues were

triturated again. The neurons were resuspended in Eagle's minimum essential medium

(MEM) containing 10% fetal bovine serum, gentamicin (10 pg ml’l), penicillin (100 units
ml'l), and streptomycin (100 units ml']) and plated onto sterile, poly-L-lysine coated 35

mm plastic dishes and maintained in an incubator at 37 °C in a 5% CO; atmosphere for

up to 3 weeks.

Whole cell patch clamp recording

Whole-cell voltage-clamp measurements were obtained at room temperature
using standard methods. Coverslips containing cells were placed on a stage of an inverted
microscope (Nikon Diaphot, Mager Scientific, Inc. Dexter, MI) using phase-contrast
optics. For identifying co-transfected cells, a mercury bulb with excitation wavelength of

495 nm was used with a filter for 520 nm emission. The pipette solution contained

(millimolar): 122.5 K-aspartate; 20 KCl; 1 MgClz; 10 EGTA; 5 HEPES; and 2 ATP. The
89

pH was adjusted to 7.3 with KOH. The extracellular solution was a HEPES-based buffer
composed of (millimolar): 155 NaCl; 5 KCl; 2 CaClz; l MgClz; 10 HEPES; and 12

glucose, pH was adjusted to 7.4 with NaOH. All recordings were made using an
Axopatch 2008 amplifier (Molecular Devices, Inc. Sunnyvale, CA). Data was acquired
using pCLAMP 9.1 software (Molecular Devices). Whole-cell recordings were carried
out using patch pipettes with tip resistances of 3-5 MQ; seal resistances were greater than
1 GO. There was a two minute interval between all successive agonist applications at

each holding potential, -70 mV for HEK-293 cells and -60 mV for myenteric neurons.

Protein construct expression and purification
pGEX vectors with a GST gene Fusion System were generously provided by Dr.
Boué-Grabot from the CNRS Université Victor Segalen Bordeaux, France. We were

provided with A pGEX with GST (pGEX-GST) as a control vector along with another
vector that contains the C-terminal domain of the P2X2 receptor (P2X2-CT,

corresponding to amino acids 365-469). The vectors were heat-shock transformed into
BL21D3+ cells and grown on Cam (34ug/ml) and Amp (100ug/ml) LB plates.
Transformed clones were first grown in a 10 ml culture (LB media containing Cam and
Amp) at 30 °C to an A600 of 0.5 and then this was expanded into a 500 m1 culture
containing Cam/Amp and 20% Glucose. These bacterial cells where grown until A600 of
0.6 at which point 0.1 mM isoprople-D -thiogalactoside (IPTG) was added. Cultures
were induced for 3 hours at 30 °C and cells were finally collected by centrifugation, snap

frozen and stored at -80 °C.
90

Cell-free extracts (soluble protein fractions of cells disrupted by sonication) from
induced cultures were affinity purified on immobilized Glutathione agarose beads
(Pierce), 10 fractions were collected. Fractions were checked by SDS-Phage that was
silver stained. The two fractions that contained the most protein were pooled and
dialyzed overnight at 4 °C into the intracellular solution buffer. Bradford assay was
performed to determine protein concentration and proteins were also visualized with
SDS-Page silver-staining and Western blotting analysis.

Western blot analysis

Samples were run on a 12.5% SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose, and the membranes were blocked with 10% nonfat dried
milk in Tris-buffered saline containing Tween 20 (lOmM Tris, 0.5% Tween 20, 0.5M
NaCl, T-TBS) overnight at room temperature with gentle agitation. Membranes were
then incubated with the appropriate primary antibody diluted in 1% milk-T-TBS.

Primary antibodies are rabbit anti-GST (generously provided by Dr. John Wang’s lab
(39); Michigan State University) or rabbit anti-P2X2 (1:1,200) form Alamone Labs,

Jerusalem. This was incubated for 1 hour at room temperature. After washing four
times, 15 minutes each in T-TBS, the secondary HRP-conjugated Goat anti-Rabbit
(1:10,000; Jackson Laboratories) were added in 1%-milk-T-TBS for 1 h at room
temperature. Membranes were washed T-TBS as above, and proteins where visualized
using Western Lightning Chemiluminescence System (Perkin Elmer Life Sciences).
Drug application

Agonists were applied onto individual neurons/cells by gravity flow from linear

array quartz tubes placed near the cell. The distance from the mouth of the tubes to the
91

cells was ~200 pm with flow controlled manually using a micromanipulator. Computer-
controlled solenoid valves (General Valve, Fairfield, NJ) were used to gate solution flow
through the tubes. Agonist-induced currents were measured as the peak current
amplitude. The difference between the predicted sum of the individual peak currents was
compared with the current amplitude caused by co-application of two agonists.

Statistics. Data are expressed as the mean :t s.e.m. and “n“ refers to the number
of cells from which the data were obtained. The predicted current amplitude that would
occur during agonist co-application was calculated by measuring the peak currents
caused by previous individual agonist applications and then summing these values.
These are repeated measurements within the same cell; therefore, there is no concern for
error within a single measurement because it cancels itself out. These are directly
measured data that were used for statistical comparisons of actual and predicted currents.
Traces illustrating predicted currents were constructed using a simple math function in
Origin 8 (OriginLab Corp., Northhampton, MA). Student’s t test was used to establish

significant differences between control and treatment groups. The significance level was

P < 0.05. EC50 is the half-maximal effective concentration and m, is the slope. Ymin and

Ymax are the minimum and maximum respectively. Agonist concentration-response

curves were fit to the following logistic function using Origin:
y = firm—_Ylfli + Ymax

1 + (x/EC50) "”

92

RESULTS

Cross-inhibition between P2X2 receptors and a3B4 nAChRs in HEK-293 cells
We used HEK-293 cells stably expressed rat a3B4 nAChR receptors and

transiently transfected with rat P2X2 receptors to study cross-inhibition. Ach (3 mM) and

ATP (1 mM) were first applied individually and peak inward currents were measured.
Then the agonists were applied simultaneously and the resulting inward current was only
56 i 7 % (Fig. 4.1A and 4.18, P < 0.05, n = 9) of the predicted current that would have
occurred had the P2X2 receptors a3B4 nAChRs functioned independently. The predicted
current value was calculated by summing the amplitudes of the individual agonist-evoked
currents. Subsequent individual re-application of ACh and ATP caused currents that
were not statistically different from those elicited before agonist co-application (P > 0.05,

data not shown).

The P2X2 C-terminal tail mediates cross-inhibition

In the next experiments, we used HEK-293 cells that stably expressed a3B4
nAChRs and that were transiently transfected with a C-terminal tail truncated P2X2
receptor (P2X2TR). This receptor has a stop codon at amino acid 374, truncating a

majority of the P2X2 receptor cytoplasmic C-terminal domain. The truncated subunit can

assemble into a functional receptor (2). In these cells, currents caused by simultaneous
application of ACh and ATP caused an inward current that was not statistically different

from the predicted current value (82 :t 6%, P > 0.05, n = 8, Fig. 4.1C).

93

a3B4 nAChR stable - P2X2 HEK cells

A ACh ATP ATP+ ACh

1/

 
  

.‘1 K
'7 / Predicted

’1'

Actual

0.4nA

Outside

 
  

93

Peak current (nA)
N

 

rm, rm Actual Predicted 1m 1m Actual WW

I
I ACh/ATP “Ch/ATP

Figure 4.1: Truncation of the P2X; C-terminal tail stops cross-inhibition between
P2X2 and nAChRs.

A. Representative traces ATP- (1 mM) and ACh- (3 mM) induced inward currents
recorded from a3B4 nAChRs stable HEK-293 cell line transiently transfected with P2X2
receptors. B. Pooled data from experiments shown in “A”. In cells expressing only the
a3B4 nAChR and P2X2 receptors, co-application of ATP and ACh exhibited cross-
inhibition. C. a3B4 nAChRs stable HEK-293 cell line transiently with a truncated form
of the P2X2 receptor (P2X2TR), cross-inhibition was abolished. ‘n’ indicates the number
of cells. *Indicates significantly different from the predicted sum of currents (P < 0.05).

94

These data described above suggest that the C-terminal domain of the P2X2

receptor is a critical determinant of cross inhibition. To test this hypothesis further, we

used the C-terminal tail peptide as a competitive antagonist to block cross inhibition. The
peptide is a soluble form of the C-terminal tail of the P2X;;_ receptor (GST-P2X2-CT for
amino acids 365-469). A control peptide containing just the GST construct was also

tested. These constructs where expressed and purified via affinity chromatography on

glutathione beads. Proteins where eluted, pooled, and verified with silver staining and

blotted with anti-P2X2 antibody (with affinity for the P2X2 C-terminal tail amino acids
457-472) and anti-GST (Fig. 4.2). Using a rat a3B4 nAChR HEK-293 stable cell line
transiently transfected with rat P2X2 receptors, 50 pg of the GST-P2X2-CT peptide or the

control GST protein were added to the recording electrode. After the GST-P2X2-CT

peptide was allowed diffuse into the interior of the cell, individual application of ACh
(1.15 i 0.2 nA) and ATP (0.62 d: 0.2) were followed by co-application in which the

actual at 1.6 :t 0.3 nA was not statistically different from predicted at 1.8 i: 0.3 nA (Fig.
4.38, P > 0.05, n = 6 ). To verify that the interaction was in fact disrupted by the P2X2-

CT coding region, and not the GST-peptide itself, similar experiments with the GST-
control peptide were performed. After the GST-control peptide diffused into the cell, co-
application of agonists still resulted in cross-inhibition of receptors in which actual (0.9 d:

0.3 nA) was significantly less that predicted (1.5 d: 0.3 nA, Fig 4.3A, n = 6, P< 0.05).

95

Figure 4. 2: Purification of GST-fusion construct.

Lanes in A, B and C are in the following order from left to right (lanes 1-8 for GST-
P2X2-CT and lane 9 for GST): Lane , 1 lysate; lane 2, unbound fraction; lane 3, elution
fraction #2; lane 3, elution fraction #3; lane 5, elution fraction #4; lane 6, elution fraction
#5; lane 7, elution fraction #6; lane 8, pooled fractions #3 and #4; lane 9, pooled GST
fraction. A.Silver staining. B. Immunoblot with anti-GST. C. Immunoblot with anti-
P2X2.

96

A. Silver-stain .
104.-

97

m 1pc .-

-—- .ar

50— ..

P2X2-CT —->

37—
29—

312:1"

N l tititi
I

l

l

20— ,4

i

B. a-GST-Blotting
‘ 104 _
97 —

37‘ _ ...._... .
29 _ fi‘ - - ‘
R
20__ GST
1 2 3 4 5 6 7 8 9
C. a-P2X2-Blotting
104: 13"} fi’
97 . i

P2X2-CT -—;~‘ ~*?-L- -— _

 

37—~- u...
29- _.'
20—
1 2 3 4 5 6 7 8 9
. f JV
GST

97

a3B4 nAChR stable - P2X2 HEK cells

GST-control peptide GST-PZXZ-CT
ACh ATP ATP+ACh ACh ATP ATP+ ACh
A 7 W ., W * Actual B 4
. . _, 4 Actual
'. ,1 r" WNW” . , . ,/ w . i I;
‘_ Predicted '. /-_'.?
, _‘r/ . ,

.. , lsPredicted
'” I 0.5 nA
2 0.5 nA
s 2 s

 

 

 

 

 

 

 

 

 

2.0 n =6 2'5 n =6
A 2 2.0
E ‘5 . 5 T
.. E 1.5

2

E 10 l S
5 o 1.0
= i
,E 05 n. 0.5

0.: . 0.3 .

Actual Predicted Actual Predicted
[ACh/ATP lACh/ATP

Figure 4.3: Cross-inhibition between nAChRs and P2X; receptors is mediated by
the P2X; receptor C-terminal tail.

A. A GST-control peptide was added into the intracellular electrode and allowed to
diffuse into the cell. This did not alter cross-inhibition between nAChRs and P2X2
receptors. B. The addition of GST-P2X2-CT peptide construct corresponding to amino
acids 365-469 of the P2X2 C-terminal tail abolishes cross-inhibition. ‘n’ indicates the

number of cells. *Indicates significantly different from the predicted sum of currents (P <
0.05).

98

The P2X2 C-terminal tail mediates cross-inhibition in myenteric neurons

Whole cell recordings were obtained from myenteric neurons using pipette
solutions that contained the GST-control peptide. ACh (3 mM) and ATP (lmM) were
individually applied to myenteric neurons caused inward currents of 0.16 i 0.02 nA and
0.25 + 0.03 nA, respectively. Agonist co-application caused an actual response of 0.26 :1:

0.04 nA that was statistically different from the predicted (0.4 :t 0.05, Fig 4.4A, P < 0.05,
n = 6). After allowing sufficient time for the GST-P2X2-CT peptide to diffuse into the

neurons, cross-inhibition was abolished with no difference between actual and predicted

responses (Fig. 4.4B), 0.20 i 0.05 nA and 0.25 i 0.05 respectively (P > 0.05, n = 6).

Constitutive inhibition of nAChR function by P2X; receptors

We found that ACh currents in HEK-293 cells stably expressing a3B4 nAChRs
alone (2.7 i 0.2 nA, n = 10) were significantly larger than ACh current recorded from
HEK-293 cells stably expressing a3B4 nAChRs and transiently transfected with P2X2
receptors (Fig. 4.5A). ACh currents were significantly smaller 1.7 i 0.2 nA (Fig 4.5A, P
< 0.05, n = 10). Constitutive inhibition did not occur in cells stably expressing a3B4
nAChRs and transiently transfected with P2X2TR (Fig 4.5B, P > 0.05, n =16). In P2X2
stable cells, ATP currents were 1.9 i 0.3 nA; when these cells were transiently
transfected with a3B4 nAChRs, ATP currents (2.0 i 0.3 nA, Fig 4.5C, P > 0.05, n =12)

were not different from those measured in cells expressing only P2X2 receptors. These

data suggest that P2X2 receptors in the closed state inhibit a3B4 nAChRs. It is possible

99

Myenteric Neurons

 

 

 

 

   

 

 

 

 

 

 

A GST-control peptide B GST-PZXZ-CT
0.5' n =6 0_3. n :6
A 0.4- A
E * E |
*- :1 0.2.l
E 0.3-l T E
5 0 2- 5
i .2 0.1‘
fl 0.1- E
0-3 I 0.3 .
Actual Predicted Actual Predicted
I ACh/ATP I ACh/ATP

Figure 4.4: Cross-inhibition between nAChRs and P2X; receptors in myenteric
neurons is mediated by the P2X; receptor C-terminal tail.

A. Experiments were performed in cultured guinea pig ileum myenteric neurons. When
the GST-control peptide diffused into cell, cross-inhibition between nAChRs and P2X2
receptors was still present. B. Upon addition of the GST-P2X2-CT peptide construct to
the myenteric neurons, inhibitory interactions between the receptors where abolished. ‘n’

indicates the number of cells. *Indicates significantly different from the predicted sum of
currents (P < 0.05).

100

A nAChR stable - P2X2 HEK B nAChR stable - P2X2TR HEK

 

 

 

  

 

 

 

 

 

 

 

 

 

25. ACh (3 mM) 10. ACh (3mM)
A “:10 A n=16
E 2.0- * E
E 1.5- fi— E 2'0.
E 10. g
i I 3 1.0-
E 0.5- E

0.3 . 0.3 .

P2X2/nAChR nAChR PZXZTR/nAChR nAChR

C szzs‘ab'e'nAChR HEK D «3134 nAChR stable - P2X2 HEK cells

 

 

 

 

 

 

 

 

ATP(1 mM)
2.5 = 0.75-
] n 12 I ATP
g 2.0 a o 10 “M PPADS
T: If 0.5m
5 1.54 g
" t
‘5 l 0 5
Q . d O '
i i 0.25
C o
a 0.5 m
05“, ' 0.33 . . . _
P2X2/nAChR P2X2 '5 -4-5 -4 -3.5 -3

Log [ATP]

Figure 4.5: Constitutive inhibition of nAChRs.

A. ACh (3mM) induced currents in cells expressing 0L3|34 nAChRs alone, currents were
much larger than when the P2X2 receptor is co-express. B. When Q3134 nAChRs are co-
expressed with a truncated form of the P2X2 receptor (P2X2TR), ACh induced currents
are no longer suppressed compared to co-expression of a3B4 nAChRs with wild-type
P2X2 receptors. C. ATP (lmM) induced currents from P2X2 receptors alone are not
altered by co-expression with (1364 nAChRs. D. PPADS (lOuM) inhibits ATP induced

currents via the P2X2 receptor. ‘n’ indicates the number of cells. *Indicates statistically
different from control currents (P < 0.05).

101

that ATP released from surrounding cells might activate P2X2 receptors causing cross-
inhibition of nAChRs. Therefore recordings were obtained in the presence of the P2X
receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disu1fonate (PPADS 10 uM)

to block P2X2 receptors. At this concentration, PPADS blocked inward currents caused

by exogenous applied ATP (Fig. 4.5D). In HEK-293 cells only expressing 0:384 nAChR,
PPADS did not alter ACh induced currents (P > 0.05, n= 8, Fig. 4.6). However, when
these cells were transfected with P2X2 receptors, the ACh current was reduced (Fig. 4.6,

P<0.05, n=21). This effect was not altered by PPADS which blocks P2X2 activation (P <
0.05 compared to initial ACh application, n = 10, Fig. 4.6), suggesting that P2X2
receptors constitutively inhibit nAChRs.

To determine if there is allosteric inhibition of the nAChR by the P2X; receptor,

concentration response curves with ACh were performed in cells only expressing nAChR
where the EC50 was 1.2x 10'4 i 30 uM, and hill slope of 1.4 (n = 4-8). In cells with
nAChR/P2X2 co-expression the EC50 was 4.4x 10'4 i 90 uM and a hill slope of 0.6 (Fig

4.7A, n = 5-8). There is significant divergence of the nAChR/P2X2 curve from the
nAChR curve at 100 uM to 1 mM ACh concentrations (P < 0.05), and significant shift of

the curve to the right. When similar experiments are performed, but this time for the
ATP concentration response curve, here the EC50 is 3.4 x 10'5 i 7 uM, and a hill slope of
1.4 for only P2X2 receptors (11 = 5-6, and upon co-expression with nAChR the EC50 is 7.1

x 10'5 i 7 uM with a hill slope of 1.9 with statistical significance ATP concentrations of 3

— 30 pM and a significant rightward shift of the curve (Fig 4.78, n = 4).

102

 

ACh ACh + PPADS ACh ACh + PPADS

 

 

 

2.5-

2.0'I

1.0-l

0.5-

0.3
nAChR + + + +
P2X2 ' ' + +
PPADS - + - +

Peak current (nA)
‘1'

 

 

 

 

 

Figure 4.6: PPADS does not alter P2X2 receptor mediated constitutive inhibition of
0.354 nAChRs.

ACh (3mM) induced currents of a3 [34 nAChRs expressed individually are not altered by
PPADS. However, these currents are inhibited upon co-expression of (13 B4 nAChRs with
the P2X2 receptor. The addition of PPADS does not alter the constitutive inhibition. ‘n’

indicates the number of cells. *Indicates significantly different from control currents of
0L3B4 nAChRs expressed alone (P < 0.05).

103

a3|34 nAChR stable - P2X2 HEK szzstable - (1334 nAChR HEK

A B

      

       

 

 

"1" I nAChRn=4-8 1.1-
1.0- 1.0. _ 5 6
A 0.9. O nAChR w/P2X2 A 09- y pzx2 n— -
E “‘5‘“ < ' o P2X2w/nAChR
v 0.8. 5 0.8‘ “=4
E 0'7. E 0.7-
is 0.64 E 0.6,
3 05' 3 0.5-
% 0.4‘ I: 0.4-
“ 031 3 0.3-
a. 03‘ 9" 0.2+
0.1- 0.1.
W .
0.0- -5 _4 _3 0.0 -6 -5 .4 -3
LoglACh] Log I ATP]

Figure 4.7: nAChRs and P2X; receptors constitutively inhibit each other.

A. Concentration response curves to ACh in cells expressing a3B4 nAChRs alone, and in
cells co-expressing a3B4 nAChRs and P2X2 receptors. B. Concentration response curves
to ATP in cells expressing P2X2 receptors alone, and in cells co-expressing a3|34

nAChRs and P2X2 receptors. ‘n’ indicates the number of cells. *Indicates significantly
different between concentration response curves expressing individual receptors and both
receptors (P < 0.05).

104

DISCUSSION
I present evidence that two structurally distinct receptors, P2X2 and nAChR,

which mediate fast synaptic transmission in the ENS do not only interact functionally as
previously established, but in fact have a physical association. Many studies have looked
at the functional aspects of this interaction in guinea-pig celiac ganglion neurons (35, 36),
rat PC-12 cells (25,27), superior cervical sympathetic neurons (26), cultured myenteric
neurons (41) and in heterologously expressed receptors in Xenopus oocytes (15) and

human embryonic kidney (HEK-293) cells (6). This study establishes the physical
aspects of the relationship between P2X2 and nAChR and how it affects receptor
function.

The P2X2 C-terminal tail mediates cross-inhibition between receptors in

HEK-293 cells. Upon co-application of agonist to co-activate the receptors there is a
receptor cross-inhibition resulting in a mean current value that is significantly smaller

than the additive predicted current had the receptors functioned independently. Cross-

inhibition occurs in cells with low P2X2 receptor expression. However, when the C-
terminal tail of the P2X2 receptor is truncated from 119 to 21 amino acids, functional

interaction with nAChRs is eliminated. This suggests that the C-terminal tail of the P2X2

receptor contributes to cross-inhibition of nAChR function. Although similar experiments
have been previously performed in Xenopus oocytes (3), this is the first time that they

have been performed in a mammalian system.

The importance of the P2X2 receptor C-terminal tail in its interaction with other

cys-loop receptors such as 5HT3 (3) and GABAA (4) receptors has been established using
105

the P2X2TR. It seems that it is a general mechanism of regulation in which P2X2
receptors interact with cys-loop family members via their C-terminal tail. To better
understand the role of the P2X2 C-terminal tail, a GST-P2X2-CT construct

(corresponding to amino acids 365-469) was expressed in bacteria and purified. After

purity was verified, this peptide was utilized to compete for the binding sites between
P2X2 and nAChRs. The addition of this construct to cells expressing wild type P2X;

receptors and nAChRs resulted in a dominant negative effect in which cross-inhibition
between receptors was abolished. Loss of the functional interaction is not an artifact of

peptide addition into the recording electrode, as the GST-control peptide did not alter
cross-inhibition. These data demonstrate the role of the cytoplasmic sequence of P2X2

receptors in its functional and physical interaction with nAChRs in the heterologous

expression system of HEK-293 cells and in native cultured myenteric neurons. The GST-

P2X2-CT construct has also previously been utilized to abolish interactions between
P2X2 and other cys-loop members (5HT3 and GABAA 3, 4). And although the GST-

P2X2-CT has been used previously to disrupt the interaction between P2X2 and nAChRs

in Xenopus oocytes (3), it has never before been attempted in the mammalian systems of

HEK-293 cells and cultured myenteric neurons as this study has shown.

The direct correlation of loss of cross-inhibition due to P2X2 receptor truncation
or competitive binding with GST-P2X2-CT minimizes the possibility that cross-inhibition
is due to second messenger molecules generated by endogenous G-protein-coupled

receptors such as P2Y or muscarinic receptors that are expressed by both HEK-293 cells

106

and myenteric neurons (24, 13, 23). This suggest that although it has been shown that

metabotropic receptors like dopamine (D5) receptors can modulate ion channels such
GABAA receptors, that is not the case here (20,1).

P2X; receptors in the closed state produce constitutive inhibition of nAChRs.

Our data suggests a physical association between these receptors that mediates a cross-
inhibition upon activation. Moreover, that there is a constitutive inhibition present in the

closed states of the receptors as ACh currents mediated by nAChRs are reduced when co-

expressed with P2X2 receptors. This constitutive inhibition of nAChR function is lost
when the P2X2 truncated receptor is co-expressed. It seems that even in its closed state,

the P2X; receptor can lead to inhibition of nAChRs via its C-terminal tail. To exclude
the possibility that ATP is being release from surrounding HEK-293 cells to activate the
P2X2 receptor, PPADS was used to block P2X2 receptors. Even in the presence of

maximum PPADS concentrations constitutive inhibition of nAChRs persisted. This was

not due to PPADS altering nAChR function. To better test constitutive inhibition,
concentration response curves (CRC) of ACh with nAChRs alone and nAChRs/P2X2
receptors were constructed. Upon co-expression of receptors, there was a shift to the

right of the ACh CRC suggesting that there is allosteric inhibition of nAChRs by P2X2

receptors. These data suggest that P2X2 receptors result in a shifi of agonist sensitivity

for nAChRs and that functional cross-inhibition does not only occur when both receptors
are activated. Previous studies performed by Ren et al. (32) support this hypothesis.

ACh and ATP mediate fast synaptic responses in murine myenteric plexus as it occurs in

107

guinea pig intestine, suggesting a role for both P2X2 receptors and nAChRs (8, 18, 14).
In studies by Ren et a1, P2X2 subunit contribution to fast synaptic excitation in mouse
myenteric neurons was studied using wild type and P2X2 knockout mice. Ren et a].
where able to show that fast excitatory post-synaptic potentials (fEPSPs) in wild type
mice were the same in amplitude, rise time and time to half-decay as knockout P2X2
mice. If P2X2 receptors contributed to fEPSPs in wild type mice (as shown by a decrease
in amplitude of fEPSPs by the addition of PPADS), how can P2X2 knockout mice show
the same amplitude fEPSPs? The answer is that in the presence of P2X2 receptors, there

is a constitutive inhibition of nAChRs. When the P2X; receptors are deleted, this

constitutive inhibition is lifted, allowing full activation of the nAChRs. This results in

fEPSPs that are the same in amplitude as in wild type mice. These data support our
hypothesis that P2X2 receptors can constitutively inhibit nAChRs even in the closed

state.

However, the same was not true for the reverse scenario; experiments showed
that nAChRs did not constitutively inhibit P2X2 receptors, at least not as effectively as
the inhibition of P2X2 receptors. At maximal concentrations, ATP (lmM) mediated
current was not altered by co-expression with nAChRs. In the ATP CRC there is a small
change at lower concentrations of P2X2 receptor mediated currents upon co-expression
with nAChRs. Our studies indicate that there is constitutive inhibition between these

receptors that is not equal in intensity, that P2X2 receptors more potently inhibit nAChRs

than nAChR inhibition of P2X2 receptors. In Xenopus oocytes, nAChR occlusion by
108

P2X2 receptors was stronger than the occlusion of P2X2 receptors by nAChRs (15). A

similar disproportion of inhibition between P2X and GABAA receptors in dorsal root

ganglion neurons has also been previously reported, in which one receptor is more
effective at inhibiting than the other (37).

Conclusions. ACh and ATP are co-transmitters in the ENS (9, 30, 33). The
interactions between P2X2 and nAChR could play a regulatory role in excitatory synaptic

transmission. This cross-inhibition may regulate neuronal excitability and plasticity to
limit the level of calcium influx and depolarization of neurons, a mechanism that might
be missing in pathological conditions of hyperactivity such as irritable bowel syndrome
(IBS). IBS is a functional bowel disorder in which there is a change in motility and in

the function of sensory innervations (12). Fluorescence resonance energy transfer
analysis of (14132 nAChRs (brain specific receptors) and P2X2, receptors expressed in
HEK-293 cells revealed that the receptors were localized within 100 nm of each other in

the plasma membrane (1 7). This supports our finding that P2X2 and nAChRs physically

associated via the P2X2 C-terminal tail, and that this physical association mediates cross-

inhibition of these receptors. We also conclude that cross-inhibition is not only mediated

in the active states, but also in the closed states of the receptors. A constitutive inhibition
that is not proportional with P2X2 receptors more effectively inhibiting nAChRs. An
inhibition state that was supported by experiments in myenteric neuron fEPSPs in P2X2

knockout mice (32). This receptor-receptor mediated interaction which is isolated to the

intracellular portion of the membrane is a very direct method of regulation that might be

109

more prominent in the nervous system than our current knowledge base can attest to;

something that more exploration of receptor mediated interactions would elucidate.

Acknowledgements

We are grateful to Dr. Boue—Garabot (CNRS Université Victor Segalen Bordeaux,

France) for providing the P2X2TR plasmid along with the GST-control peptide and GST-
P2X2-CT constructs. Dr. Xiao (Georgetown University, Washington DC.) for donating
the (13134 nAChR stable HEK-293 cell line, and Dr. Alan North (University of

Manchester, Manchester, UK) for the P2X; plasmids. And most importantly, we are very

grateful for the Wang lab for its guidance and aid in expressing and purifying the GST

peptide constructs.

110

10.

ll.

12.

REFERENCES

Agnati LF, Ferre S , Lluis C, Franco R, Fuxe K. Molecular mechanisms and
therapeutical implications of intramembrane receptor/receptor interactions among
heptahelical receptors with examples from the striatopallidal GABA neurons.
Pharmacol Rev 2003; 55:509-550.

Boue-Grabot E, Archambault V, Seguela P. A protein kinase C site highly
conserved in P2X subunits controls the desensitization kinetics of P2X2 ATP-gated
channels. J. Biol. Chem. 2000; 275(14): 10190-5.

Boué—Grabot E, Barajas-Lopez C, Chakfe Y, Blais D,.Bélanger D, Emerit MB,
Séguéla P. Intracellular cross talk and physical interaction between two classes of
neurotransmitter-gated channels. J Neurosci 2003; 23: 1246-1253.

Boué-Grabot E, Toulmé E, Emerit MB, Garret M. Subunit-specific coupling

between gamma-aminobutyric acid type A and P2X2 receptor channels. J Biol Chem
2004; 279: 52517-52525.

Castelucci P, Robbins HL, Poole DP, Furness JB. The distribution of purine P2X2
receptors in the guinea-pig enteric nervous system. Histochem Cell Biol 2002; 117:
415-422.

Decker DA, Galligan JJ. Cross-inhibition Between Nicotinic Acetylcholine
Receptors and P2X Receptors in Myenteric Neurons and HEK-293 cells. Am J
Physiol Gastrointest Liver Physiol. 2009; Apr 2. [Epub ahead of print].

Edwards FA, Gibb AJ. ATP - a fast neurotransmitter. FEBS 1993; 325: 86-89.

Galligan JJ, Bertrand PP. ATP mediates fast synaptic transmission in enteric
nerves. J Neurosci 1994; 14: 7563-7571.

Gallligan JJ, LePard KJ, Schneider DA, Zhou X. Multiple mechanisms of fast
excitatory synaptic transmission in the enteric nervous system. J A uton Nerv Syst
2000; 81: 97-103.

Galligan JJ. Pharmacology of synaptic transmission in the enteric nervous system.
Current opinion in pharmacology 2002; 2: 623-629.

Galligan JJ, North RA. Pharmacology and function of nicotinic acetylcholine and
P2X receptors in the enteric nervous system. Neurogastroenterol Motil, 2004; 16
(suppl 1) 64-70.

Galligan JJ. Enteric P2X receptors as potential targets for drug treatment of the
irritable bowel syndrome. Br J of Pharm 2004; 141: 1294-1302.

111

13.

l. 4.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Gwynne RM, Bornstein JC. Synaptic transmission at functionally identified
synapses in the enteric nervous system: roles for both ionotropic and metabotropic
receptors. Current Neuropharmacology 2005; 5: 1-17.

Johnson PJ, Shum OR, Thornton PD, Bornstein JC. Evidence that inhibitory
motor neurons of the guinea pig small intestine exhibit fast excitatory postsynaptic
potentials mediated via P2X receptors. Neurosci Lett 1999; 266: 169-172.

Khakh BS, Zhou X, Sydes J, Galligan JJ and Lester HA. State-dependent cross-
inhibition between transmitter-gated cation channels. Nature 2000; 406:405-10.

Khakh BS. Molecular physiology of P2X receptors and ATP signaling at synapses.
Nature Reviews 2001; 2:165-174.

Khakh BS, Fisher JA, Nashmi R, Bowser DN, Lester HA. An Angstrom scale
interaction between plasma membrane ATP-gated P2X2 and alpha4beta2 nicotinic
channels measured with fluorescence resonance energy transfer and total internal
reflection fluorescence microscopy. J Neurosci 2005; 25: 6911-6920.

LePard KJ, Messori E, Galligan JJ. Purinergic (P2X) fast excitatory postsynaptic
potentials in myenteric neurons of guinea pig; distribution and pharmacology.
Gastroenterology 1997; 113: 1522-1534.

Lepard KJ, Galligan JJ. Analysis of fast synaptic pathways in myenteric plexus of
guinea pig ileum. Am J Physiol 1999; 276: G529-G538.

Liu F, Wan Q, Pristupa ZB, Yu X-M, Wang YT, Niznik HB. Direct protein -
protein coupling enables cross-talk between dopamine D5 and g-aminobutyric acid A
receptors. Nature 2000; 403: 274 — 280.

McGehee DS, Role LW. Physiological diversity of nicotinic acetylcholine receptors
expressed by vertebrate neurons. Ann Rev Physiol 1995; 57:521-546.

Miyazawa A, Fujiyoshi Y, Unwin N. Structure and gating mechanism of the
acetylcholine receptor pore. Nature 2003; 423: 949-955.

Momo RL, Bornstein JC, Bertrand PP. Synaptic transmission from the
submucosal plexus to the myenteric plexus in Guinea-pig ileum. Neurogastroenterol

Motil 2008; 20: 1165—1173.

Mundell SJ, Benovic JL. Selective regulation of endogenous G protein-coupled
receptors by arrestins in HEK293 cells. J Biol Chem 2000; 275(17): 12900-12908.

112

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

Nakazawa K, Fujimori K, Takanaka A, Inoue K. Comparison of adenosine

triphosphate- and nicotine-activated inward currents in rat phaeochromocytoma cells.
J Physiol 1991; 434: 647-60.

Nakazawa K. ATP-activated current and its interaction with acetylcholine-activated
current in rat sympathetic neurons. J Neurosci 1994; 14:740-750.

Nakazawa K. Reduction of acetylcholine-activated current by low concentrations of
extracellular adenosine-triphosphate. Life Sciences 1995; 57: 351-356.

North RA, Surprenant A. Pharmacology of cloned P2X receptors. Ann Rev
Pharmacol Toxicol 2000; 40: 563-80.

North RA. Molecular physiology of P2X receptors. Physio] Rev 2002; 82: 1013-
1067.

Nurgali K, Furness JB ,Stebbing MJ. Analysis of purinergic and cholinergic fast
synaptic transmission to identified myenteric neurons. Neuroscience 2003; 116: 335-
347.

Poole DP, Castelucci P, Robbins HL, Chiocchetti R, F urness JB. The distribution
of P2X3 purine receptor subunits in the guinea pig enteric nervous system. Auton
Neurosci 2002; 101: 39-47.

Ren J, Bian X, DeVries M, Schnegelsberg B, Cockayne, DA, Ford AP, and
Galligan JJ. P2X2 subunits contribute to fast synaptic excitation in myenteric
neurons of the mouse small intestine. J Physiol 2003; 522.3: 809-821.

Ren J, Bertrand PP. Purinergic receptors and synaptic transmission in enteric
neurons. Purinergic Signal 4: 2008;255-266.

Robertson SJ, Ennion SJ, Evans RJ, Edwards FA. Synaptic P2X receptors. Curr
Opin Neurobiol 2001; 11: 378-386.

Searl TJ, Redman RS, Silinsky EM. Mutual occlusion of P2X ATP receptors and
nicotinic receptors on sympathetic neurons of the guinea-pig. J Physio] 1998; 510:
783-791.

Silinsky EM, Gerzanich V. On the excitatory effects of ATP and its role as a
neurotransmitter in coeliac neurons of the guinea-pig. J Physiol 1993; 464:197-212.

Sokolova E, Nistri A, Giniatullin R. Negative cross talk between anionic GABAA

and cationic P2X ionotropic receptors of rat dorsal root ganglion neurons. J Neurosci
2001; 21(14): 4958-4968.

113

38.

39.

40.

41.

42.

Vial C, Roberts JA, Evans RJ. Molecular properties of ATP-gated P2X receptor
ion channels. Trends Pharmacol Sci 2004; 25: 487-493.

Voss PG, Gray RM, Dickey SW, Wang W, Park JW, Kasai KI, Hirabayashi J,
Patterson RJ, Wang JL. Dissociation of the carbohydrate-binding and splicing
activities of galectin-1. Arch of Biochem and Biophys 2008; 478(1): 18—25.

Xiao Y, Meyer EL, Thompson JM, Surin A, Wroblewski J, Kellar KJ. Rat org/B4
subtype of neuronal nicotinic acetylcholine receptor stably expressed in a transfected

cell line: pharmacology of ligand binding and fiinction. Mol Pharmacol 1998; 54:
322-333.

Zhou X, Galligan JJ. Non-additive interaction between nicotinic cholinergic and
P2X purine receptors in guinea-pig enteric neurons in culture. J Physiol 1998; 513:
685-697.

Zhou X, Ren J, Brown E, Schneider D, Caraballo-Lopez Y, Galligan JJ.

Pharmacological properties of nicotinic acetylcholine receptors expressed by guinea
pig small intestinal myenteric neurons. J Pharmacol Exp T her 2002; 302: 889-897.

114

CHAPTER 5
Concluding remarks and future perspectives

Previous work to investigate the interactions between P2X2 and nAChRs in model
systems such as guinea-pig coeliac ganglion neurons, rat pheochromocytoma PC12 cells,
myenteric neurons and rat superior cervical ganglion neurons has provided a barrier for

detailed probing into this interaction. For all of these systems express both P2X2 and

nAChRs, a draw back that my work has circumvented.

My studies have utilized the mammalian heterologous system of HEK-293 cells;
these cells do not endogenously express P2X2 and nAChRs, making it possible to test

previous hypotheses such as the “channel overlap” theory, and the presence of agonist
allosteric binding sites on these receptors. Another major concern in previous work has

been that other receptors (muscarinic for ACh, P2Y for ATP) whose activation can lead

to desensitization of P2X2 and nAChRs results in a cross-inhibition artifact. Being able to

express P2X2 and nAChRs individually, as in our system, has provided a powerful tool to

test hypotheses that have been difficult to test in previous model systems that express
both receptors. In cells that express only nAChRs, ACh induced currents were not
altered by co-application of ATP. The same was true for P2X receptors expressed
individually. Our studies have provided evidence that the ‘channel overlap’ theory, the
presence of allosteric binding sites, and a role for desensitization of P2X and nAChR by

muscarinic or P2Y receptors mediated hypotheses are not accurate.

The work presented in this thesis furthers our understanding of interactions

between P2X and nAChRs in three significant ways: this is the first comprehensive study

115

of interactions between these receptors in the open, desensitized and closed states.

Second, these studies have provided evidence that interactions of the nAChR with the
P2X receptor are not specific to the P2X2 subtype, but also occur with the P2X3 and
P2X4 subtypes in the open and desensitized states. And third, the molecular
mechanisms responsible for these interactions were determined to be mediated by a
physical interaction of the P2X2 C-terminal tail with the (13134 nAChR in HEK-293 cells

and myenteric neurons. This verifies results suggesting that a physical interaction

between these receptors are not an artifact of expression in a heterologous system, but in

fact due to the specific interaction between the P2X2 receptor and the (1304 nAChR.

Future studies to investigate the specificity of this physical interaction would be
fruitful. A more direct physical association between these receptors can be studied with a

co-immunoprecipitation of these receptors. Preliminary data was collected from

experiments in which an antibody against the (1304 nAChR was cross-linked to protein A
beads. I observed that when incubated with an (1304 nAChR stable cell line transfected
with P2X2 receptors, the (13134 nAChR can be immunoprecipitated and blotted, but you
cannot blot of the P2X2 receptor. If the converse cell line was incubated with the same
cross-linked beads (a P2X2 stable cellline transiently transfected with (13134 nAChR) the
P2X2 receptor can be blotted for (the opposite of protein of the antibody) while there
would be no signal for the immunoprecipitation or blot of the (1304 nAChR. Similar

experiments were repeated with beads that where cross-linked to the P2X2 receptor

antibody. It seems that for a proper signal to be detected on the western, the protein has
116

to be stably expressed, for if it is not, it seems that there is an insufficient amount of

protein to be detected.

These preliminary results are positive, because it shows that even if you
immunoprecipitate for the (13134 nAChR, you can still blot for the P2X; receptor, and the

reciprocal was also true. However, it is disconcerting how the signal for the transfected
proteins on the immunoblot cannot be detected. It does not make sense that there are
enough proteins there to co-immunoprecipitate the opposite protein, and that they can be
activated via whole cell voltage clamp, but not enough for the immunoblot. One concern
would be that the stably expressed proteins are being randomly enriched by the beads,
and that is the signal that was observed. However, I performed experiments with a

‘dummy’ antibody, and there were no such result.

To solve these problems in the following attempts, the transfected protein
concentration can be increased, or the total amount of protein (1 added 1mg) added to the
beads can be increased to a higher concentration. Also, this might be due to not enough
antibody population to pick up all of the stably transfected protein, so more antibody can
be cross-linked to beads (I used 2 pg, it can be increased to 4 pg or 6 pg). Furthermore,
there are other controls that have to be performed to verify these previous results.

Controls in which: cells are only stably expressing each of the individual proteins, and

show that the converse protein cannot be immunoblotted for both the (1384 nAChR and

the P2X2 receptor. Controls of ‘pre-immune’ antibody, or of that is not available, a

sample of rabbit antibodies that have been affinity purified in the same manner as the

117

antibodies used in the previous experiments against a protein that is known to not interact

with either the (1304 nAChR or the P2X2 receptor also have to be performed.

Another method to determine direct physical interactions is with site direct
mutagenesis which can be implemented to determine more specifically which amino acid

residues or sequence motifs might mediate these protein-protein interactions. There is

evidence in the literature that P2X2 receptors also interact with other cys-loop family
members such as the GABAA receptor and the 5-HT3 receptor. Work by Boue-Garabot
et al. has shown that interactions between the P2X2 receptor and the 5-HT3 receptor are
mediated via the P2X2 C-terminal tail and the second large intracellular loop of the 5-HT3
receptor. Sequence homology for the second large intracellular loop of the 5HT3 receptor
and the nAChR is 26% for the (:3 subunit and 30% for the B4 subunit. I believe that
interactions between the P2X2 receptor and the (13134 nAChR occur in a similar manner.
A peptide constructs of the large intracellular loop for the (13 subunit and B4 subunit of the

nAChR could be utilized in a similar manner as the GST-P2X2-CT to determine if the
large intracellular loop of the nAChR is in fact responsible for interactions between the

P2X2 and the (1304 nAChR, and more specifically, if both (13 and B4 subunits mediate

interactions with the P2X; receptor, or if only one of them does.

Future work to investigate the trafficking of these receptors would also be of
interest. This study has provided evidence of cross-inhibition in the desensitized states of

the receptors. It would be interesting to determine if even upon desensitization and

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internalization of the receptors co-localize would still be observed. A stable association

would be suggested if desensitization and internalization of nAChR results in subsequent

internalization of the P2X2 receptor. The same would be true for the opposite, if
desensitization and internalization of the P2X2 receptor also intemalizes the nAChR.

Ratios of the P2X2 receptor and nAChR in the endocytosed vesicles can be determined

using confocal microscopy.

The foundation for this unique protein-protein interaction between P2X receptors
and nAChRs has been laid by this thesis. In the future, the detailed physical mechanism
between these receptors can be investigated. For this unique mechanism of regulation
during fast excitatory synaptic transmission plays an important role in gastrointestinal
motility. Gastrointestinal propulsion of food down the intestine is based on neuron
activity. If myenteric plexus neurons in the reflex are are under-stimulated then there are
fewer contractions and relaxations of the muscle. This results in a low level of motility in
the intestine leading to constipation. If the same neurons are over-stimulated, there is an
increase in propulsion resulting in diarrhea. The level and relay of neuronal stimulation
is regulated via fast synaptic transmission. The accurate amount of stimulation results in

proper propulsion.

P2X; receptors and nAChRs are located on descending neurons in the reflex arc

of the ENS; more specifically on inter—neurons and motor neurons that release nitric
oxide onto the muscle. The descending pathway of neuronal stimulation mediates
relaxation of the muscle. Relaxation of the muscle is a crucial response for motility, for

without it food could not be propelled down the intestine. I believe that P2X2 receptors
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and nAChRs are co-localized in the descending pathway as a safety mechanism. Even
though both receptors are cation permeable, both are present as a guarantee that there will
be at least one functional receptor to mediate relaxation. Even if one receptor

desensitizes or no longer functions, there is a backup receptor to pick up the slack. If

both receptors are present and fully functional, cross-inhibition occurs between the P2X2
and nAChR receptors to prevent a large infux of Ca2+ into the cell (high intracellular

Ca2+ can by cytotoxic).

In most gastrointestinal motility disorders, there is an imbalance of neuronal
communication that results in hypo-contractility (constipation) or hyper-contractility
(diarrhea). By better understanding the mechanisms that regulate the relay of information
between neurons in the myenteric plexus (synaptic transmission), we can better
understand what goes wrong in disease states. It is a possibility that in disease states of
hyper-contractility; this unique mechanism of regulation between P2X receptors and
nAChRs is no longer present. That lack of cross-inhibition between these receptors
results in an increase in synaptic transmission compared to non-diseased states. By better
understanding this protein complex, new drugs can be discovered and implemented to
treat many gastrointestinal motility disorders in which this mechanism of regulation

might be lacking.

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