MECHANISMS OF SOUTHERN GRASSHOPPER MOUSE (ONYCHOMYS 

TORRIDUS) MUSCLE RESISTANCE TO THE PARALYTIC AND LETHAL TOXINS IN 

ARIZONA BARK SCORPION (CENTRUROIDES SCULPTURATUS) VENOM 

 
By 

Abhijna A. Parigi 

A DISSERTATION 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Submitted to  

Michigan State University  

in partial fulfillment of the requirements  

Integrative Biology—Doctor of Philosophy 

Ecology, Evolutionary Biology and Behavior—Dual Major  

for the degree of 

 

 

 

2018 

MECHANISMS OF SOUTHERN GRASSHOPPER MOUSE (ONYCHOMYS 

TORRIDUS) MUSCLE RESISTANCE TO THE PARALYTIC AND LETHAL TOXINS IN 

ARIZONA BARK SCORPION (CENTRUROIDES SCULPTURATUS) VENOM 

ABSTRACT 

 
By 

Abhijna A. Parigi 

Voltage-gated sodium ion channels (Nav) are transmembrane proteins responsible for 

initiating electrical signals in excitable cells. Because Nav channels play a crucial role in 

neuromuscular coordination, they are targeted by a diverse array of neurotoxins 

produced across the animal kingdom. Arizona bark scorpions (Centruroides 

sculpturatus) produce toxins that disrupt Nav channel function, causing pain, muscle 

paralysis and respiratory failure. Southern grasshopper mice (Onychomys torridus) hunt 

bark scorpions. In response to selection by scorpion venom, grasshopper mice have 

evolved physiological resistance to toxins that cause pain and death. Although previous 

work identified modifications in one grasshopper mouse Nav channel (Nav1.8) that 

provide resistance to venom pain, mechanisms underlying resistance to muscle 

paralysis remain unknown. In skeletal muscle, Nav1.4 channels regulate muscle 

contraction. Previous studies showed that toxins in C. elegans and C. vittatus venoms 

disrupt Nav1.4 gating mechanisms. Thus, I tested the hypothesis that C. sculpturatus 

venom contains toxins that target Nav1.4, and that grasshopper mice are resistant to the 

effects of these toxins via molecular changes to their Nav1.4.  Using molecular and 

electrophysiological analyses, I compared the structural and functional properties of 

grasshopper mice Nav1.4 channels to those of house mice and rats (rodents that are 

both sensitive to scorpion venom) and found that grasshopper mice Nav1.4 has evolved 

	

reduced sensitivity to C. sculpturatus venom. Further, I identified amino acid changes in 

the grasshopper mice Nav1.4 protein that contribute to reduced toxin sensitivity. Finally, 

I show that the beta subunits (accessory proteins that modulate Nav1.4 channel gating 

kinetics) of grasshopper mice do not have species-specific effects on channel function 

in the presence or absence of venom. My results demonstrate that highly conserved 

proteins can be evolutionarily modified with minimal effects to their baseline functional 

properties. 

	

ACKNOWLEDGEMENTS 

 

I am immensely grateful to have worked with some of the greatest minds in 

evolutionary biology. Each member of my dissertation committee has provided me with 

tremendous support and encouragement. In particular, I want to thank the chair of my 

dissertation committee, Dr. Ashlee Rowe, for her constant and unending support of my 

scientific interests. In the four years that I have spent in her lab, Ashlee has taught me a 

great deal about academic research and professionalism.  

The advice I received from colleagues at Michigan State University (MSU) has 

been invaluable for my success. A special thanks to the members of Dr. Ke Dong’s lab 

for sharing their expertise and equipment as I learned electrophysiology. I also want to 

thank the team of undergraduate research assistants whose tireless efforts in the lab 

enabled me to accomplish this work within a reasonable timeframe. Working with these 

curious and enthusiastic young scientists has been the highlight of my PhD.  

  

This final note of thanks is to family and friends. My parents have been a 

constant source of encouragement, strength and comfort from afar. I could not have 

undertaken this 6-year endeavor without their full support. Most importantly, I want to 

say a big thank you to my partner, Danny. He has stuck with me through the good times 

and bad, and continues to add adventure to all my days.  

This work would not have been possible without the financial support provided by 

the National Science Foundation and the Department of Integrative Biology at MSU.  

 

iv 

TABLE OF CONTENTS 

 

LIST OF TABLES .......................................................................................................... viii 

LIST OF FIGURES ......................................................................................................... ix 

KEY OF ABBREVIATIONS .............................................................................................. x 

INTRODUCTION .............................................................................................................1 
REFERENCES ............................................................................................................6 

 
CHAPTER 1: Molecular mechanisms of resistance to paralytic and lethal scorpion 
(Arizona bark scorpion, Centruroides sculpturatus) toxins in a scorpion predator 
(southern grasshopper mice, Onychomys torridus) .......................................................11 
INTRODUCTION .......................................................................................................11 
MATERIALS AND METHODS ...................................................................................16 
RNA isolation and cDNA synthesis .....................................................................16 
PCR and cloning for sequencing .........................................................................16 
Models of molecular evolution .............................................................................17 
Electrophysiology ................................................................................................18 
PCR and cloning for expression in Xenopus laevis oocytes ................................18 
Site directed mutagenesis ...................................................................................19 
Expressing Scn4a in Xenopus laevis oocytes .....................................................20 
Recording ............................................................................................................21 
C. sculpturatus venom hydration and dilution ......................................................21 
Protocols, data extraction and analysis ...............................................................22 
1) Nav channel activation ...............................................................................22 
2) Na+ current decay constant (tau) ...............................................................23 
3) Na+ current decay slope ............................................................................23 
4) Voltage dependence of steady-state inactivation .......................................24 
5) Recovery from fast-inactivation..................................................................24 
RESULTS ..................................................................................................................25 

Grasshopper mouse Scn4a encodes amino acid substitutions at conserved sites
 ............................................................................................................................25 
Models of molecular evolution to find amino acids under positive selection ........27 
Grasshopper mouse Nav1.4 channels are less sensitive to Centruroides toxins .27 
Effect of C. sculpturatus venom on activation ................................................27 
b toxin effect #1 (hyperpolarized shift in voltage dependence of activation)
 .................................................................................................................27 
b toxin effect #2 (decrease in peak Na+ current).......................................27 
Effect of C. sculpturatus venom on inactivation kinetics ................................31 
Effect of a toxin on tau ..............................................................................31 
Effect of a toxin on Na+ current decay slope ............................................34 
Steady-state inactivation and recovery ..........................................................36 

 

v 

Role of unique amino acid substitutions in conferring resistance to C. 
sculpturatus venom .............................................................................................36 

b toxin effects # 1 (hyperpolarizing shift in voltage dependence of activation) 
on triple mutant channels ...............................................................................37 
b toxin effects # 2 (reduction in peak Na+ current amplitude) on triple mutant 
channels ........................................................................................................38 
Effect of a toxin on tau ...................................................................................40 
Effect of a toxin on Na+-current decay slope ..................................................40 

Relative contributions of DI, DIII and C-terminus insert towards reduced C. 
sculpturatus venom sensitivity .............................................................................41 
Effect of DI mutations on sensitivity to C. sculpturatus venom .......................43 
Effect of DIII mutations on sensitivity to C. sculpturatus venom .....................44 
Effect of C-terminal insert on sensitivity to C. sculpturatus venom .................44 
DISCUSSION .............................................................................................................45 

Grasshopper mouse Nav1.4 channels are less sensitive to the a- and b-toxin 
effects of C. sculpturatus venom .........................................................................45 
Three distinct regions of the Nav1.4 channel contribute to reduced venom 
sensitivity .............................................................................................................49 
Effect of structural modifications on resistance to a and b toxins may be additive
 ............................................................................................................................51 
Proposed mechanism of resistance: role of amino acids in DI, DIII and C-
terminus ..............................................................................................................52 
Physiological costs associated with modifications that reduce sensitivity to C. 
sculpturatus venom .............................................................................................53 
Summary and significance ..................................................................................54 
REFERENCES ..........................................................................................................56 

 
CHAPTER 2: Role of grasshopper mouse beta subunits in modulating the function of 
skeletal-muscle voltage-gated ion channels (Nav1.4) ....................................................64 
INTRODUCTION .......................................................................................................64 
MATERIALS AND METHODS ...................................................................................67 
Molecular biology ................................................................................................67 
Electrophysiology ................................................................................................68 
Expressing Scn1b and Scn4b in Xenopus laevis oocytes ..............................68 
Recording ......................................................................................................69 
Data extraction and analysis ..........................................................................69 
RESULTS ..................................................................................................................70 

Sequence of genes encoding grasshopper mouse beta 1 and beta 4 subunits 
from skeletal muscle............................................................................................70 
Electrophysiological properties of GaGb, GaMb and GaRb channels .................71 
Voltage dependence of activation .......................................................................71 
Effect of C. sculpturatus venom on voltage dependence of activation ...........72 
Effect of C. sculpturatus venom on peak current ...........................................74 
Fast inactivation kinetics .....................................................................................75 
Voltage dependence of steady-state inactivation and recovery from fast 
inactivation ............................................................................................................78 

	

DISCUSSION.............................................................................................................78 
REFERENCES ..........................................................................................................83 

 
CONCLUDING REMARKS ............................................................................................88 

	

LIST OF TABLES 

 

Table 1: Boltzmann parameters for normalized conductance vs. voltage relationships of 
wildtype grasshopper mouse, house mouse and rat Nav1.4 channels ...........................29 
 
Table 2: Boltzmann parameters for normalized conductance vs. voltage relationships of 
wildtype grasshopper mouse and triple mutant Nav1.4 channels ...................................37 
 
Table 3: Boltzmann parameters comparing normalized conductance vs. voltage 
relationships for wildtype grasshopper mouse, triple mutant and single mutant (Di, Diii, 
NoC) Nav1.4 channels ...................................................................................................43 
 
Table 4:  Boltzmann parameters for normalized conductance vs. voltage relationships of 
GaGb, GaMb and GaRb channels .................................................................................74 
 

 

viii 

LIST OF FIGURES 

 

 

Figure 1: Nav channel alpha subunit structure and sequence ........................................26 
 
Figure 2: Effect of C. sculpturatus venom on the voltage dependence of activation of 
wildtype Nav1.4 channels expressed in Xenopus laevis oocytes ...................................28 
 
Figure 3: Effect of C. sculpturatus venom on voltage-current relationships for wildtype 
Nav1.4 channels expressed in Xenopus laevis oocytes .................................................30 
 
Figure 4: Effects of C. sculpturatus venom on inactivation kinetics of Nav1.4 channels 
expressed in Xenopus laevis oocytes ............................................................................33 
 
Figure 5: Effects of C. sculpturatus venom on the decay slope of Nav1.4 current ..........35 
 
Figure 6: Effect of C. sculpturatus venom on the activation of triple mutant Nav1.4 
channels expressed in Xenopus laevis oocytes. ............................................................39 
 
Figure 7: Effect of C. sculpturatus venom on the inactivation kinetics of triple mutant 
Nav1.4 channels to measure a-toxin effects ..................................................................40 
 
Figure 8: Effect of C. sculpturatus toxins on the voltage dependence of activation of Di, 
Diii and NoC mutant channels ........................................................................................42 
 
Figure 9: A partial multi-species amino acid sequence alignment of the beta 4 protein .71 
 
Figure 10: Influence of grasshopper mouse beta subunits on the voltage dependence of 
activation of grasshopper mouse Nav1.4 channels ........................................................73 
 
Figure 11: Effects of species- specific beta subunits on modulating Nav1.4 activation 
and peak current in the presence of C. sculpturatus venom ..........................................75 
 
Figure 12: Effects of species-specific beta subunits on modulating Nav1.4 alpha subunit 
baseline inactivation kinetics ..........................................................................................76 
 
Figure 13: Effects of species-specific beta subunits on modulating Nav1.4 alpha 
subunit’s inactivation kinetics in the presence of C. sculpturatus venom .......................78 
 

 

ix 

KEY TO ABBREVIATIONS 

 

ANOVA  

Analysis of Variance 

BSA 

 

cDNA  

CIP 

 

cRNA  

D 

 

GaGb 

 
GaRb 

 
GaRb 

Kd  

Na+ 

Nav 

 

 

 

OCB 

PAML 

PCR 

RNA 

S 

TTX 

Bovine Serum Albumin  

Complementary Deoxyribonucleic Acid 

Calf Intestinal Phosphate 

Capped Ribonucleic Acid 

Domain 

Grasshopper mouse Alpha subunits co-expressed with Grasshopper 
mouse Beta subunits 

Grasshopper mouse Alpha subunits co-expressed with House mouse 
Beta subunits 

Grasshopper mouse Alpha subunits co-expressed with Rat Beta subunits 

Dissociation Constant  

Sodium 

Voltage-Gated Sodium Ion Channel 

Open Channel Blocker 

Phylogenetic Analysis using Maximum Likelihood  

Polymerase Chain Reaction 

Ribonucleic Acid 

Segment 

Tetrodotoxin

 

x 

INTRODUCTION 

A central goal in evolutionary biology is to understand the molecular mechanisms 

underlying adaptive traits. Given that predator-prey interactions over evolutionary time 

have resulted in some of the most specialized adaptations (Brodie & Brodie 1990; 

Miyatake et al. 2004; Gosline & Rodd 2007; Stuart-Fox et al. 2008; Voss & Jansa 2012; 

McCabe et al. 2016), predators and their prey can serve as ideal models for 

understanding adaptive traits and the molecular processes that produce them. My 

research uses defensive neurotoxic venoms and their ion channel targets in 

predators as a model to study the molecular and physiological bases of 

neuromuscular adaptations. 

The survival of both predators and prey in the wild depends on their ability to 

detect and respond to environmental cues. In animals, the neuromuscular system 

coordinates stimulus detection and response via specialized proteins called voltage-

gated sodium (Nav) ion channels (Hodgkin & Huxley 1952; Marban et al. 1998; Yu & 

Catterall 2003). Given the importance of these channels in mediating all vital activities 

(e.g. obtaining food, navigation, detecting mates), neuromuscular systems in general, 

and Nav channels in particular are conserved across diverse taxa (Goldin 2002; Meisler 

2005). Consequently, some of the most effective offensive and defensive traits are 

those that target and manipulate Nav channels, ultimately disrupting neuromuscular 

activity (Possani et al. 1999; Al-Sabi et al. 2006; Wingerd et al. 2017).

	

1 

 An excellent example of a Nav channel-manipulating trait is neurotoxic venom 

produced by species of scorpion in the genus Centruroides (Kirsch et al. 1989; Gordon 

et al. 1996; Cestèle et al. 1999; Possani et al. 1999; Campos et al. 2008; Zhang et al. 

2015). Commonly known as “bark scorpions”, these small arthropods (approximately 

0.5 –1.0 gram) defend themselves from potential predators by inflicting toxic stings that 

can cause pain, paralysis and death (Patterson 1960; Rimsza et al. 1980; LoVecchio & 

McBride 2003). While we would predict most predators to simply avoid hunting bark 

scorpions, one predatory rodent, the grasshopper mouse (Onychomys spp.) is known to 

hunt and consume bark scorpions in the wild. Previous research in our lab has shown 

that grasshopper mice have evolved physiological resistance to venom (Rowe & Rowe 

2008) through modifications to at least one Nav channel (Rowe et al. 2013). Such an 

evolutionary solution is astounding, as even a few amino acid changes to highly 

conserved proteins can cause devastating diseases in humans (Hoffman et al. 1995; 

Vicart et al. 2005; Drenth & Waxman 2007; Struyk et al. 2008; Webb 2009; Jurkat-Rott 

et al. 2010; Dib-Hajj et al. 2012; Groome et al. 2014). In this study, my aim was to 

understand the molecular mechanisms underlying venom resistance in grasshopper 

mice, and the potential physiological costs that might arise from modifications to 

important proteins. 

Voltage-gated Nav channels, transmembrane protein complexes expressed in 

nerve and muscle cells, mediate electrical signals to and from the brain by regulating 

the flow of charged Na+ ions (McCleskey & Gold 1999; Goldin 2001; Wood & Baker 

2001; Wood et al. 2004). Each Nav channel is made of one large pore forming alpha 

subunit (~270kD) and two accessory proteins called beta subunits (32-35 kD); one beta 

	

2 

subunit binds covalently with the Nav alpha subunit, while the other associates non-

covalently (Catterall 2000; Winters & Isom 2016). Mammals express 9 different types of 

Nav alpha subunits and at least 4 types of beta subunits (beta 1- beta 4), all of which are 

expressed with distinct tissue specificity (Isom et al. 1992; Isom et al. 1995; Yu & 

Catterall 2003; Patino & Isom 2010; Brackenbury & Isom 2011; Isom 2016; Kruger & 

Isom 2016). Previous studies showed that toxin peptides in the venom of some species 

of Centruroides bind to specific regions on the alpha subunit of Nav channels, causing 

the channels to activate prematurely (near resting membrane potential), and delay fast 

inactivation (Kirsch et al. 1989; Gordon et al. 1996; Cestèle et al. 1998; Mantegazza & 

Cestèle 2005; Campos et al. 2008). Some peptides target Nav channels of pain-sensing 

neurons, while others bind to Nav channels in muscle and cause paralysis (Watt 1990; 

LoVecchio & McBride 2003). When stung by the Arizona bark scorpion, small mammals 

(and human infants) often die from suffocation due to paralysis of their diaphragm 

(Rimsza et al. 1980; LoVecchio & McBride 2003).  

Three species of grasshopper mice, O. torridus, O. arenicola, O. leucogaster, are 

carnivorous, desert-dwelling rodents of Southwestern United States and Mexico (Riddle 

& Honeycutt 1990) that regularly hunt bark scorpions (Rowe et al. 2006; Rowe et al. 

2008). Grasshopper mice are undeterred by bark scorpion stings, having evolved 

physiological resistance to the venom peptides (Rowe & Rowe 2008). Previous work in 

the Rowe lab showed that one Nav channel isoform expressed in the pain pathway of 

grasshopper mice (Nav1.8) has amino acid substitutions that allow the channel to bind 

venom peptides and block pain signals - put simply, grasshopper mice use venom as an 

anesthetic (Rowe et al. 2013). However, we still do not understand how grasshopper 

	

3	

mice can tolerate peptides that cause paralysis and death. Preliminary data obtained 

from whole muscle and nerve preparations suggested that the muscles of grasshopper 

mice are resistant to C. sculpturatus venom. Whereas the muscles of house mice 

exhibited a temporary venom-induced increase in the force of muscle contraction 

followed by a block of contraction, grasshopper mice muscles exhibited the venom-

induced increase in force of contraction only at high concentrations of venom. 

Moreover, muscle contraction was never blocked in grasshopper mice, even when the 

muscle was exposed to high concentrations of venom for prolonged periods of time (> 8 

hours, A. Rowe data unpublished). Additionally, previous work (Rowe et al. 2011; 

Vandendriessche et al. 2010) showed that isolated peptides from the venoms of C. 

vittatus and C. elegans (respectively) target Nav1.4 channels expressed in heterologous 

systems. My hypothesis is that the C. sculpturatus venom contains peptides that 

alter the gating function of Nav1.4, and that the grasshopper mouse Nav1.4 

channel complex (alpha and beta subunits) has evolved structural and functional 

modifications that impart resistance to these peptides.  

In the first chapter of my dissertation, I demonstrate that C. sculpturatus venom 

prematurely activates and then delays inactivation in Nav1.4 channels from Mus 

musculus (house mice) and Rattus norvegicus (rats) (control species known to die from 

scorpion envenomation). In comparison, grasshopper mouse Nav1.4 was less sensitive 

to the effects of C. sculpturatus venom. Using models of molecular evolution and 

mutagenesis studies, I identified the specific amino acid modifications involved in 

conferring reduced sensitivity to venom.  

	

4	

In Chapter 2, I examined the role of grasshopper mouse accessory beta subunits 

in modulating Nav1.4 baseline functional properties and response to C. sculpturatus 

venom. My results show that the grasshopper mouse beta subunits do not have any 

special modulatory effects on their alpha subunits either in the presence or absence of 

venom. 

	

5	

REFERENCES

	

6 

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10	

CHAPTER 1: Molecular mechanisms of resistance to paralytic and lethal scorpion 

(Arizona bark scorpion, Centruroides sculpturatus) toxins in a scorpion predator 

(southern grasshopper mice, Onychomys torridus) 

 

INTRODUCTION 

Voltage-gated sodium (Nav) ion channels are complex transmembrane proteins 

that regulate the activity of excitable cells, such as neurons and myocytes. Nav channels 

underlie the rising phase of the action potential, and, thus, are essential for neuronal 

signal transmission and muscle contraction (Hodgkin & Huxley 1952; Denac et al. 

2000). Because these Nav

 channels are critical for survival, their structure and function 

is highly conserved across diverse animal taxa (Goldin 2002; Kruger & Isom 2016). 

Consequently, Nav channels are ideal targets of naturally occurring predatory and anti-

predatory chemical weapons that aim to disrupt neuromuscular activity.  

Offensive and defensive neurotoxic weapons specific to Nav channels have 

arisen independently in multiple taxa across the phylogeny of life (Mebs 2001; Al-Sabi 

et al. 2006; Clement et al. 2007; Vandendriessche et al. 2008; Watt & Simard 2008; 

Klint et al. 2012; Yang et al. 2013; Han et al. 2017; Prashanth et al. 2017; Drukewitz et 

al. 2018; Prentis et al. 2018). Among species that produce chemical weapons, 

scorpions are particularly intriguing. Compared to species like puffer fishes (Indumathi & 

Khora 2017), newts (Hanifin et al. 1999) and blue ringed octopus (Sheumack et al. 

1978; Williams et al. 2011), which harbor the symbiotic bacteria that produce a single 

neurotoxic compound (tetrodotoxin, TTX), or poison frogs that assimilate toxic 

chemicals from their diet (Saporito et al. 2011) – scorpions evolved venom glands, 

	

11 

which express a family of genes that encode complex mixtures of toxins (low-molecular-

weight peptides, small molecules) selective for both predator and prey. Moreover, 

scorpions have evolved a specialized delivery system, hypodermic needle-like stingers 

that inject toxins into potential enemies (predators, prey, competitors) (Gwee et al. 

2002; Watt & Simard 2008).  

Most species of scorpions that produce lethal neurotoxins belong to the family 

Buthidea (~500 species) (Possani et al. 1999). One desert-dwelling Buthid species, 

Centruroides sculpturatus (Arizona bark scorpion), produces painful and lethal 

neurotoxic venom as a defense against potential mammalian predators (van der 

Meijden et al. 2017). C. sculpturatus venom is a cocktail of peptides that selectively 

disrupt the function of Nav channels (Possani et al. 1999; Possani et al. 2000; Bosmans 

& Tytgat 2007; Escalona & Possani 2013;), causing intense pain, paralysis of the 

diaphragm muscles, and fatal asphyxiation in small mammals (including human infants) 

(Patterson 1960; Cahalan 1975; Ismail 1995; LoVecchio & McBride 2003; Valdez-Cruz 

et al. 2004; Webber & Graham 2013).  

Despite being chemically protected, these deadly scorpions are regularly preyed 

upon by carnivorous desert rodents called grasshopper mice (Onychomys torridus) 

(Rowe & Rowe 2006). Staged behavioral trials between grasshopper mice and C. 

sculpturatus show that the mice get stung numerous times during a hunt, but 

experience little discomfort, and are seldom deterred by the scorpion (unlike venom-

sensitive house mice, Mus musculus) (Rowe & Rowe 2008). This apparent 

physiological resistance to pain and muscle paralysis seen in grasshopper mice is 

generally understood to be an adaptation to foraging in habitats where C. sculpturatus 

	

12	

scorpions are an abundant resource that are unavailable to venom-susceptible 

competitors. Prior work has shown that one Nav channel isoform involved in the pain 

pathway, Nav1.8, has acquired modifications in grasshopper mice that make the 

channel bind venom peptides and block pain signals (Rowe et al. 2013). While this work 

revealed the molecular mechanism underlying resistance to pain-inducing toxins in bark 

scorpion venom, how these carnivorous mice avoid muscle paralysis and death is not 

yet understood. To understand the molecular basis of resistance to the lethal 

components of C. sculpturatus venom, I investigated the role of Nav1.4. I chose to study 

Nav1.4 because it is the only Nav channel paralog expressed in myocytes (muscle cells) 

(Goldin et al. 2000), and it is a known target of other Centruroides venoms in sensitive 

mammals (namely rats and humans). 

Centruroides toxins manipulate the gating kinetics of sensitive Nav channels 

(Catterall et al. 2007). The gating mechanism and function of a Nav channel (Figure 1) is 

closely coupled to its structure. A typical mammalian Nav channel is made of a large 

pore forming alpha subunit (~260 kDa) and two smaller accessory beta subunits (32-36 

kDa) (Namadurai et al. 2015, Isom L. 2014). The Nav alpha subunit has 4 homologous 

domains (DI – DIV) and 6 transmembrane segments (S1-S6) per domain (Figure 1) 

(Marban et al. 1998). In the three-dimensional structure, the extracellular, reentrant 

loops that connect S5 to S6 of each domain come together to line the channel pore 

(Guy & Seetharamulu 1986). The S4 segments of DI- DIV are called the voltage 

sensors because they contain positively charged amino acid residues that move 

outward in response to changes in membrane potential (Catterall 1986). This upward 

and outward movement of the voltage sensors causes a conformational change that 

	

13	

opens the Nav channel pore (Catterall 2000; Catterall 2010). Activated channels remain 

selectively conductive to Na+ ions for a few milliseconds, after which the hinged lid 

mechanism of the inactivation gate closes to block further influx of ions – the channel is 

inactivated. The intracellular loop connecting DIII to DIV forms the hinged inactivation lid 

of the channel, and its movement is linked to the voltage sensor of DIV, such that 

complete outward movement of the DIV voltage sensor segment pulls the hinged lid of 

the inactivation gate, blocking the pore on the intracellular end (Armstrong 1981; 

Stühmer et al. 1989; Goldin 2003). According to some studies, the sequential 

movement of DI-DIII voltage sensors may be sufficient for channel activation, while the 

primary function of the DIV voltage sensor is likely related to the movement of the 

inactivation gate (Campos et al. 2004; Chanda & Bezanilla 2002; Horn et al. 2000; Silva 

& Goldstein 2013; Bosmans et al. 2008).  

C. sculpturatus toxins that target Nav channels are peptides, typically ranging 

from 60 to 75 amino acids long (Possani et al. 1999; Gordon et al. 1998). The peptides 

are classified as either a or b toxins (designated a and b to distinguish from alpha and 

beta Nav channel subunits) based on their binding sites and effects on the channels 

(Couraud et al.1982; Gordon et al. 1998). The a scorpion toxins in the venom of some 

Centruroides species primarily bind to the extracellular loop connecting S3-S4 

segments in DIV (Tejedor & Catterall 1988; Thomsen & Catterall 1989), preventing the 

movement of the DIV voltage sensors and, consequently, the inactivation gate (see 

structure-function relationship above) (Kirsch et al. 1989; Rogers et al. 1996; Campos et 

al. 2008).  Therefore, a-toxin bound channels activate normally, but experience delayed 

inactivation. On the other hand, b toxins bind to the extracellular loop connecting S3-S4 

	

14	

segments of the channel’s DII, and open the channel prematurely (i.e. at more negative 

membrane potentials than physiologically normal) (Possani et al. 1999; Cestèle & 

Catterall 2000; Mantegazza & Cestèle 2005; Escalona & Possani 2013). Overall, 

scorpion-venom bound Nav channels activate prematurely and remain active longer 

than necessary for normal physiological processes. In the pain pathway, these effects 

lead to anomalous and prolonged pain signals. However, in muscles, scorpion toxins 

induce unwarranted muscle contractions and twitches that can paralyze the diaphragm, 

causing respiratory failure (Rimsza et al. 1980). 

In this study, I investigated the effects of venom from C. sculpturatus on native 

grasshopper mouse muscle Nav1.4 channels expressed in Xenopus oocytes. My study 

is the first to demonstrate the effects of whole Centruroides venom on mammalian 

Nav1.4 channels expressed in heterologous cells. I show that grasshopper mouse 

Nav1.4 channels are less sensitive to C. sculpturatus venom than those of rats and 

house mice (control animals that die from C. sculpturatus envenomation) Further, I 

identify the specific structural modifications in the grasshopper mouse Nav1.4 protein 

that are responsible for reducing the channel’s sensitivity to certain effects of C. 

sculpturatus toxins. Remarkably, Nav1.4 channels employ a mechanism of resistance 

that is significantly different from that seen in the grasshopper mouse Nav1.8 channels 

of the pain pathway. My results suggest that these functionally conserved Nav channels 

can evolve several structural modifications in response to multifarious selection 

imposed by venom, without significant adverse effects to overall physiology.  

 

 

	

15	

MATERIALS AND METHODS 

In mammals, the Nav1.4 protein is encoded by the Scn4a gene. To investigate 

the role of Nav1.4 channels in muscle resistance to C. sculpturatus venom, I sequenced 

the complete coding region of the grasshopper mouse Scn4a. First, I extracted mRNA 

from the muscle tissue of three different grasshopper mouse species, and then used 

PCR and cloning to amplify the Scn4a gene for sequencing.  

 

RNA isolation and cDNA synthesis 

For RNA extraction from skeletal muscle of Onychomys torridus (3 animals), O. 

arenicola (2 animals), O. leucogaster (1 animal), and CD-1 Mus musculus, I used 

TRIzol (Ambion) and followed the manufacturer’s protocol. I converted mRNA to cDNA 

(complementary DNA) using SuperScript III First Strand Synthesis Reverse 

Transcriptase PCR kit (Invitrogen). A gene specific reverse transcription primer 

(6327RT), along with Oligo d(T)20 was used to prime the reverse transcription reaction 

for cDNA synthesis.  

 

PCR and cloning for sequencing 

I downloaded the coding region of Scn4a from multiple closely related species 

from the NCBI database and aligned them using the MUSCLE algorithm in Geneious 

(version 7.1.5). Based on the alignment, I designed degenerate primers to amplify the 

grasshopper mouse and house mouse Scn4a gene in two overlapping pieces using 

New England Biolabs’ (NEB) Q5 DNA polymerase. Primer design and PCR conditions 

were determined based on manufacturer’s guidelines. I then ran the amplified fragments 

	

16	

on a 0.8% w/v agarose gel, excised the appropriate sized bands, and purified the DNA 

using Wizard SV Gel and PCR Clean-Up System (Promega). These purified PCR 

products were sequenced at GENEWIZ.  

 

Models of molecular evolution 

To identify amino acids under positive selection in the Onychomys lineage, I 

used likelihood based models of molecular evolution in Codeml of PAML. I selected the 

branch-site model of Codeml because I was looking for episodic adaptive evolution on a 

few amino acids in a specific lineage (Zhang et al. 2005). I tested for an association 

between venom resistance in the Onychomys lineage and signatures of selection acting 

on the Scn4a gene. CODEML calculates a ratio of non-synonymous to synonymous 

substitutions, omega (w), as a proxy for selection. When a protein is under negative or 

purifying selection, w < 1, whereas w > 1 indicates positive selection for 

structural/functional changes to the protein. When w = 1, neither substitution type is 

favored, and the protein is likely evolving under neutral pressures.  

Based on species relationships from published studies (Fabre et al. 2012; 

Nyakatura & Bininda-Emonds 2012; Springer et al. 2012), I created a species 

phylogenetic tree, and manually converted it to Newick format. I added the Scn4a 

sequences of all Onychomys species to the previously generated multi-species 

alignment of Scn4a orthologous sequences (see PCR and cloning section) and 

exported the alignment to PHYLIP format supported by PAML. I set the Onychomys 

lineage as the “foreground” branch in which w values could be estimated at > 1, 

whereas all other branches were “background” in which w ranged from 0-1. Using the 

	

17	

likelihood ratio test, I compared this model to a null model that did not allow for 

positively selected sites and had a fixed w of 1 in the “foreground” lineages.  

 

Electrophysiology 

Do the structural modifications in grasshopper mouse Nav1.4 channels impart 

resistance to C. sculpturatus venom? To answer this question, I expressed the 

grasshopper mouse Nav1.4 protein in Xenopus laevis oocytes, and then used the two-

electrode voltage-clamp technique to evaluate the effects of C. sculpturatus venom on 

the voltage and time-dependent properties of channels. During these experiments, the 

Nav1.4 channels of two sensitive mammals, rat (Rattus norvegicus) and house mouse 

(Mus musculus), served as controls. I limited comparisons to the Nav1.4 alpha subunit 

by co-expressing the alpha subunit of all species with rat beta 1 and beta 2 accessory 

subunits. 

 

PCR and cloning for expression in Xenopus laevis oocytes 

For cloning of the Scn4a gene, I designed new primers (specific to grasshopper 

mouse and house mouse Scn4a coding regions) to amplify the full-length gene in one 

piece. Then I sub-cloned the purified PCR products into the p-GEM-t-easy vector 

following the manufacturer’s protocol. Clones were checked by sequencing 

(GENEWIZ), and clones with the correct insert sequence were selected for further 

processing. Since neither the grasshopper mouse nor house mouse Scn4a sequence 

contained EcoRI restriction sites, I used this enzyme to excise the full gene from the p-

GEM-t-easy vector for cloning into pcDNA3.1+ expression vector (contains the T7 

	

18	

promoter for in vitro RNA synthesis). I similarly digested pcDNA3.1+ with EcoRI and calf 

intestinal phosphatase (CIP), and ligated the sticky ends overnight using NEB’s DNA 

ligase (manufacturer’s protocol with 3:1 ratio of insert: vector). I transformed the ligated 

plasmid constructs into MAX Efficiency™ Stbl2™ competent cells (Invitrogen) and 

modified the manufacturer’s protocol for sodium channel cloning according to Feldman 

& Lossin (2014). At each stage, I extracted plasmid DNA using either PureLink™ 

HiPure Plasmid Miniprep Kit or PureLink™ HiPure Plasmid Maxiprep Kit (ThermoFisher 

Scientific) depending on desired amount of DNA. I preserved all colony cultures (prior to 

plasmid preparation) in 50% glycerol and stored them at -80oC for future use. Once 

cloned, I re-sequenced the entire coding region of Scn4a gene to confirm orientation 

and base identity.  

 

Site directed mutagenesis 

I created four different mutagenic constructs from the wildtype grasshopper 

mouse Scn4a clone: a) Triple mutant in which the isoleucine at position 333 (I333) and 

glutamic acid at position 334 (E334) were changed to glutamic acid E333 and glycine 

G334; similarly, tyrosine Y1182, lysine K1133 were changed to serine S1182, glutamic 

acid E1183, and the C-terminal insert, unique to grasshopper mice, was removed. b) Di 

mutant in which only isoleucine I333, glutamic acid E134 were changed to glutamic acid 

E333, glycine G134. c) Diii in which only tyrosine Y1182, lysine K1133 were changed to 

serine S1182, glutamic acid E1183. e) NoC mutant in which the C-terminal insert was 

removed from the wildtype grasshopper mouse construct. I used the NEB Q5® Site-

Directed Mutagenesis Kit for all mutagenesis reactions and selected the primers and 

	

19	

cycle conditions according to manufacturer’s guidelines 

(http://nebasechanger.neb.com/). All mutagenized constructs were completely re-

sequenced at GENEWIZ to ensure the lack of unwanted mutations.  

 

Expressing Scn4a in Xenopus laevis oocytes 

I purchased Xenopus laevis oocytes from Xenopus1 (MI), and manually removed 

the follicles surrounding the oocytes using forceps. For incubating oocytes overnight, I 

used ND-96 culture media containing 1.8 mM calcium chloride (CaCl2), 2 mM 

potassium chloride (KCl), 1mM magnesium chloride (MgCl2), 5 mM 4-(2-hydroxyethyl)-

1-piperazineethanesulfonic acid (HEPES), 2.5 mM sodium pyruvate (C3H3NaO3) and 

0.5 mM theophylline. I also supplemented the culture solution with 100 mg/L gentamicin 

and adjusted to a pH of 7.5 with 2N NaOH.  

To synthesize capped mRNA (cRNA) from all plasmid constructs, I used the 

mMESSAGE mMACHINE™ T7 Transcription Kit (Thermo-Fisher Scientific) following 

NotI linearization of the plasmid at the 3’ end of the gene. Post-linearization, I used 

phase separation with phenol-chloroform to clean DNA of RNases before cRNA 

synthesis, and then quantified the synthesized cRNA with a nanodrop 

spectrophotometer (Thermo-Fisher Scientific). Because beta subunits are important 

modulators of alpha subunit activity, prior to injection of cRNA into Xenopus oocytes for 

expression, I mixed the alpha subunit cRNA of Scn4a genes from house mouse, 

grasshopper mouse and rat (gift from Dr. G.K.Wang) with rat Scn1b and rat Scn2b 

cRNA (coding for beta1 and beta2 accessory proteins, gift from Dr. G.K.Wang) in a 

1:1:1 ratio. Injected oocytes remained at 16oC until recording, for a maximum of 4 days.  

	

20	

Recording 

Borosilicate glass electrodes (heat = 729, Pull = 100, Vel = 50, Time = 50, Sutter 

Instruments) filled with 3M KCl served as the current (resistance < 1 MW) and voltage 

electrodes (resistance < 2 MW). I placed oocytes in 450 µl of filter sterilized recording 

solution (ND96 supplemented with 2 mM KCl, 1.8mM CaCal2, 1mM MgCl2 and 10mM 

HEPES and adjusted pH to 7.5 with 2N NaOH). I custom built the recording chambers 

from disposable petri dishes with hot-glue damns connected by salt bridges. Each 

chamber was used only once. My recording amplifier and digitizer for oocyte clamp 

were OC725C (Warner Instruments) and Digidata 1200A (Axon Instruments) with 

pCLAMP10.6 software (Axon instruments). Leak currents were subtracted using P/4 

technique.  

 

C. sculpturatus venom hydration and dilution 

I hydrated lyophilized crude C. sculpturatus venom (Rowe et al. 2013) to 40 µg/µl 

using sterile water, and stored aliquots at -80oC. I pre-washed venom tubes with 1.5 

µg/µl of bovine serum albumin (BSA) to prevent venom from sticking to tube walls. To 

maintain osmolality of recording solution, I diluted venom in recording solution and BSA 

to a concentration of 1.5 µg/µl prior to use. Then I pipetted 50 µl of 1 µg/µl venom (final 

concentration of 0.15 µg/µl) directly into the recording chambers (final volume of 500 µl) 

to test the effects of venom on voltage and time dependent properties of Nav channels.  

 

 

 

	

21	

Protocols, data extraction and analysis 

I used Axon™ pCLAMP™ 10 Electrophysiology Data Acquisition & Analysis 

Software (Axon Instruments) to visualized current traces, Clampfit to extract data from 

current traces and R Studio for processing and statistical analysis. The 

electrophysiology protocols and statistical analyses are as follows: 

1) Nav channel activation: b toxins in Centruroides venom bind to the S3-S4 

extracellular loop of DII causing two different effects: 1) a hyperpolarized shift in the 

voltage dependence of channel activation, and 2) an overall decrease in peak Na+ 

current amplitude. To test these b effects of C. sculpturatus venom on Xenopus oocytes 

expressing the Nav1.4 channels, I measured the voltage dependence of activation. I 

depolarized Xenopus oocytes expressing either rat, house mouse or grasshopper 

mouse Nav1.4 channels to +10 mV from a holding potential of -100 mV in a series of 5 

mV steps. Before each test potential, the oocytes were given a pre-pulse of +10 mV to 

induce brief channel activation for b toxin binding. At each potential I extracted peak 

current using Clampfit software (Axon Instruments) and created a csv file for further 

analysis in R Studio. To calculate normalized conductance from current-voltage curves, 

I used the following equation GNa = Imax/V-VNa, where Imax is the peak current, V is the 

depolarized conditioning potential and VNa is the reversal potential. VNa was estimated 

for individual oocytes using the equation I = [1+ exp(-0.03937 * z* (V-V50))]-1 * g* (V – 

VNa), where z is the gating charge, g is a factor related to the number of channels 

contributing to macroscopic current, V is the voltage and V50 is the voltage of half-

maximal activation (as in Smith & Goldin 1998). The numeric value of conductance is 

directly related to the number of “active” Nav1.4 channels. I fit all conductance-voltage 

	

22	

plots to a single or sum of two Boltzmann equations: P/(1 + exp((V50a-V)/k1)) + ((1-P)/(1 

+ exp((V50a-V)/k2), where P is the proportion of channels, and k1 and k2 are slopes. For 

single Boltzmann fits, P = 1. To statistically compare conductance values between 

different species or channel types at one voltage step (-20mV), I used a two-way 

repeated measures ANOVA design with species, venom and their interaction as factors. 

To compare reduction in peak Na+ current post venom application, I plotted peak 

current across the range of membrane potentials and used a three-way repeated 

measures ANOVA in the nlme function of R Studio software package (factors: voltage, 

venom, species) to test for significant differences.  

2) Na+ current decay constant (tau): a toxins in Centruroides venom bind to the 

S3-S4 segment of DIV and hold the inactivation gate longer than normal. This a toxin-

induced impairment of the inactivation mechanism can be measured as a change in the 

Na+ current decay constant across a range of voltage steps. Using the voltage 

dependence of activation protocol described above, I fit the decay phase of each 

current trace to a single exponential equation and extracted the decay time constant 

parameter (tau). To ensure the capture of current from only “inactivating” channels, I 

used the distal part of the current trace (> 4 ms), and compared data obtained from 

different channels using a three-way repeated measures ANOVA (factors: species, 

venom, voltage).  

3) Na+ current decay slope: As another measure of inactivation kinetics, I 

obtained the slope parameters for current traces using the “statistics” function of the 

Clampfit software (https://www.moleculardevices.com/en/assets/tutorials-videos). In 

theory, the decay slope parameter yields the same information as tau, however, there 

	

23	

are two major differences. First, while single exponential equations are fit to the distal 

part of the decay trace (> 6 ms after depolarization), decay slopes are calculated based 

on peak Na+ current. Second, decay slope values are standardized by peak Na+ current 

for each given trace and are therefore not influenced by the amplitude of the current. I 

used a two-way repeated measure ANOVA (species, voltage) as factors to assess 

statistical significance for pre-venom data. To determine the extent to which venom 

alters the Na+ current decay slope, I measured the proportional change in decay slope 

after venom treatment. The proportion was calculated as: [(slope of Na+ current before 

venom – slope of Na+ current after venom)]/ slope of Na+ current before venom. Then, I 

used a two-way repeated measures ANOVA to assess statistical significance between 

species at different voltage steps.  

4) Voltage dependence of steady-state inactivation: I used a standard two-

pulse protocol to measure the voltage dependence of steady-state inactivation. I 

depolarized oocytes expressing either rat, house mouse or grasshopper mouse Nav1.4 

channels to +10 mV for 50 ms in 5 mV steps, from a holding potential of -100 mV. A test 

pulse to +10 mV enabled me to measure the fraction of non-fast-inactivated current over 

the range of voltages. I plotted normalized currents across voltage steps, and then fit 

those data to a single Boltzmann equation: 1/(1+ exp((V-V50)/k)).  

5) Recovery from fast-inactivation: To measure Nav1.4 recovery from fast 

inactivation, I applied a conditioning pulse to oocytes (+10 mV for 100 ms) to induce fast 

inactivation in channels, and then varied the recovery time periods (0-20 ms in 0.5 ms 

incremental steps). Current from the fraction of recovered channels could be measured 

with a test pulse to +10 mV. I did not fit the data to any standard equations because 

	

24	

there were no visible differences between channel types or pre and post venom 

treatments (overlapping ± 1SE).  

 

RESULTS 

Grasshopper mouse Scn4a encodes amino acid substitutions at conserved sites 

Preliminary data showed that skeletal muscle from grasshopper mice was less 

sensitive to C. sculpturatus venom (A. Rowe, unpublished). The gene Scn4a encodes 

the alpha subunit Nav1.4 expressed in muscle.  A multi-species alignment comparing 

Scn4a sequences from three species of grasshopper mice (Onychomys spp.) with other 

rodents and non-rodent mammalian species revealed that grasshopper mouse Scn4a 

encodes amino acid substitutions and an insert at highly conserved sites in the channel 

protein (Figure 1A). Moreover, the results showed variation in Scn4a among the three 

grasshopper mouse species. A partial species alignment showed a negatively charged 

glutamate (E at position 333 in the protein) was shifted to an adjacent site in the DI pore 

module (Figure 1B). In the DIII pore module, a negatively charged glutamate at position 

1183 was substituted with a positively charged lysine (K) in O. torridus, while the 

adjacent serine (S at position 1182) in all three species is substituted with tyrosine (Y) 

(Figure 1B). Finally, the C-terminus carried an insert that was unique to grasshopper 

mice. The insert in the three grasshopper mice species (Figure 1C) comprised 

seemingly random combinations of serines (S), alanines (A), prolines (P), valines (V) 

and leucines (L). O. torridus had the longest (48 amino acids) insert. 

 

	

25	

A

Extracellular

Intracellular

B
O. torridus 
O. arenicola
O. leucogaster
Deer mouse 
Chinese hamster
Prairie vole 
House mouse 
Rat
Human
C
O. torridus 
O. torridus 
O. arenicola
O. arenicola
O. leucogaster
Deer mouse
O. leucogaster
Deer mouse
Chinese hamster
Chinese hamster
House mouse
House mouse
Rat
Rat
Human
Human

DI

DII

DIII

DIV

+
+
4
+

1 2 3

5

6

1

2 3

+
+
4
+

5

6

1 2 3

+
+
4
+

5

6

1 2 3

+
+
4
+

5

6

Inactivation gate

DIII

C-terminus

DI

YYCVNTTTYKRFDISVVNNKSE
YYRVNTTTYERFDISVVNNKSE
YYCVNTTTYERFDISVVNNKSE
YYCVNTTTSERFDISVVNNKSE
YYCVNTTTSERFDVSVVNNKSE
YYCVNTTTSERFDISVVNNKSE
YYCINTTTSERFDISVVNNKSE
YYCVNTTTSERFDISVVNNKSE
YYCINTTTSERFDISEVNNKSE

NYTFDWDAYINDIENFYFLEGANDALLCGNSSDAG
O. torridus 
NYTFDWDAYINDVENFYFLKGANDALLCGNSSDAG
O. arenicola
NYTFDWDAYINDIDNFYFLEGANDALLCGNSSDAG
O. leucogaster
NYTFDWDAYINDEGNFYFLEGSNDALLCGNSSDAG
Deer mouse 
NYTFDWDAYINDEGNFYFLEGSNDALLCGNSSDAG
Chinese hamster 
NYTFDWDAYINDEGNFYFLEGSNDALLCGNSSDAG
Prairie vole 
NSTFDWEAYINDEGNFYFLEGSNDALLCGNSSDAG
House mouse 
NSTFDWEAYINDEGNFYFLEGSNDALLCGNSSDAG
Rat 
NDTFDWDAYISDEGNFYFLEGSNDALLCGNSSDAG
Human 
LDTAQTPPPPPSAPLPSSVSLPPSAPLPPSAPPPSVSLPSSAPSPSSAPSPSSAPLPPSAPPPPSPPPPS
LDTAQTPPPPPSAPLPSSVSLPPSAPLPPSAPPPSVSLPSSAPSPSSAPSPSSAPLPPSAPPPPSPPPPS
LDTAQTPPPPPSAPLPSSVSLPPSAP-PPSVSLPSSAPSPS-----------------SAPPPPSPPPPS
LDTAQTPPPPPSAPLPSSVSLPPSAP-PPSVSLPSSAPSPS-----------------SAPPPPSPPPPS
LDTAQTPPPPPSAPLPSSVSSPPSAPLPPSVSLPP-----------------------SAPPPPSPPPPP
LDTAQTPPPPPSAPLPSSVSSPPSAPLPPSVSLPP-----------------------SAPPPPSPPPPP
LDTAQTPPPPP-----------------------------------------------SASPPPSASPQI
LDTAQTPPPPP-----------------------------------------------SASPPPSASPQI
LDAALTTPPPP-------------------------------------------------S---PPLPSS
LDAALTTPPPP-------------------------------------------------S---PPLPSS
SDTALTPPPPS-----------------------------------------------------PPPPSS
SDTALTPPPPS-----------------------------------------------------PPPPSS
SDTALTPSPPP-----------------------------------------------------LPPSSS
SDTALTPSPPP-----------------------------------------------------LPPSSS
SDTAWPPAPPP-----------------------------------------------------------
SDTAWPPAPPP-----------------------------------------------------------

C-terminus

 

Figure 1: Nav channel alpha subunit structure and sequence. A) Primary structure 

of a typical Nav) channel alpha subunit. Blue diamonds represent location of unique 

substitutions seen in grasshopper mice, and red stars represent known Centruroides 

toxin binding sites. B) Partial multi-species amino acid sequence alignment of Nav1.4. 

Amino acids of interest are shown in color. Dots below alignment indicate the amino 

acids under positive selection. Amino acid corresponding to the black dot in DI was 

under 97% positive selection in the Onychomys lineage, whereas the black dot in DIII 

shows amino acid under 67% positive selection. C) Insert in C-terminus unique to 

grasshopper mice is shown in color. Insert length varies among Onychomys species. 

 

 

	

26	

Models of molecular evolution to find amino acids under positive selection 

Given that the Scn4a gene is highly conserved across taxa, I expected to see w 

< 1. However, many conserved proteins often experience episodic selection at specific 

active sites that may have subtle but significant effects on protein function (e.g. Jansa & 

Voss 2011). As expected, the branch-site model of CODEML found that the 

grasshopper mouse Scn4a gene had an w << 1, yet, seven amino acids (codons) in the 

Onychomys lineage were under positive selection (P = 0.012). Of these amino acids, 

E333I (transmembrane segment 5 of DI, 97% probability of positive selection) and 

S1182Y (transmembrane segment 5 of DIII, 67% probability of positive selection) are in 

extracellular loops physically close to known a and b toxin binding sites on the Nav 

channel (Figure 1B).  

 

Grasshopper mouse Nav1.4 channels are less sensitive to Centruroides toxins  

Effect of C. sculpturatus venom on activation 

b toxin effect #1 (hyperpolarized shift in voltage dependence of activation) : The 

effect of b toxins on the voltage dependence of activation is demonstrated by the 

relationship between normalized conductance and voltage (Figure 2A-C). The voltage of 

half maximal activation (V50) parameters derived from fitting the conductance-voltage 

relationship to a Boltzmann equation are shown in Table1. V50a is the voltage of half 

maximal activation for channels that are unaffected by venom, whereas V50b 

corresponds to the population of channels affected by venom. In the absence of venom 

there was a small but statistically significant difference between the V50a of grasshopper 

mice vs. the control species (Figure 2A, Table 1). The V50a of grasshopper mice (6.75 

	

27	

mV ± 0.15) was shifted 0.76 mV and 1.96 mV in the positive direction compared to the 

V50a of house mice (z = -4.25; P << 0.01) and rat (z = -7.44; P << 0.01), respectively.  

In the presence of venom, all species experienced premature, post-venom 

increases in conductance at voltages ranging from -55 to -10 mV (Figure 2B-C, Table 

1). However, a greater number of rat channels were activated at hyperpolarized 

potentials compared to those of house mice or grasshopper mice. To make statistical 

comparisons, I chose one voltage step, -20 mV, at which the post-venom conductance 

was visually most different from pre-venom conductance. In the presence of venom, the 

normalized conductance in grasshopper mouse channels was significantly lower than in 

rat channels (t = 3.653; df = 2, 38; P << 0.01), but not house mouse channels (t = 0.68; 

df = 2, 38; P = 0.50). These data suggest that b toxins negatively shift the voltage 

dependence of activation of a larger number of rat channels compared to house mouse 

or grasshopper mouse channels. 

.
1

0 Gmouse
Mouse
Rat

8
.
0

A

6
.
0

4
.
0

)

A
N
g
(
 
e
c
n
a
t
c
u
d
n
o
C
 
d
e
z

a
m
r
o
N

i
l

2

.

0

0

.

0

B

8
.
0

6
.
0

4
.
0

)

A
N
g
(
 
e
c
n
a
t
c
u
d
n
o
C
 
d
e
z

a
m
r
o
N

i
l

2

.

0

0

.

0

C

8
.
0

6
.
0

4
.
0

)

A
N
g
(
 
e
c
n
a
t
c
u
d
n
o
C
 
d
e
z

a
m
r
o
N

i
l

2

.

0

0

.

0

.
1

0 Gmouse

Gmouse + Venom
Rat
Rat + Venom

.
1

0 Gmouse

Gmouse + Venom
Mouse
Mouse + Venom

−75

−55

−35

−15

0

Voltage (mV)

 
Figure 2: Effect of C. sculpturatus venom on the voltage dependence of activation 

−75

−55

−35

−15

0

Voltage (mV)

−75

−55

−35

−15

0

Voltage (mV)

of wildtype Nav1.4 channels expressed in Xenopus laevis oocytes. A) Normalized 

conductance plotted as a function of membrane potential for grasshopper mouse 

(black), house mouse (purple) and rat (green) channels. B) Conductance-voltage 

	

28	

relationship pre (solid symbols) and post venom (open symbols) for grasshopper mouse 

(black) compared to rat Nav1.4 (green). C) Conductance-voltage relationship in 

grasshopper mouse (black) Nav1.4 channel compared to house mouse Nav1.4 (purple) 

channels. All curves are fit the to a single (pre-venom) or double (post-venom) 

Boltzmann equation.  

 

 

Table 1: Boltzmann parameters for normalized conductance vs. voltage 

relationships of wildtype grasshopper mouse, house mouse and rat Nav1.4 

channels. V50a and V50b are the voltages of half maximal activation for the proportion of 

channels affected and not affected by C. sculpturatus venom, respectively. K1 and K2 

are the slope parameters, P is the proportion of channels unaffected by venom, and n is 

the number of oocytes recorded.  

 

b toxin effect #2 (decrease in peak Na+ current): To evaluate the effect of b toxins 

on the peak Na+ current, I plotted normalized current as a function of voltage (Figure 

3A-B). C. sculpturatus venom caused a significant reduction in peak Na+ currents in all 

species (P < 0.01; df = 2, 76; t = 2.721) at voltages ranging from -5 mV to +55 mV. 

However, a three-way repeated measures ANOVA found post-venom peak currents 

were larger for grasshopper mice compared to rat channels (t = 2.086; df = 2, 76; P = 

	

29	

0.04), but there was no difference between grasshopper mice and house mice (t = 1.64; 

df = 2, 76; P = 0.105). Additionally, the difference in peak current (before vs. after 

venom) at +5 mV shows the greatest change occurred in rat Nav1.4, whereas the 

smallest change occurred in grasshopper mice (Figure 3C). The data in Figure 3 

illustrate that rat channels are most sensitive to the b-toxin effects of C. sculpturatus 

venom, house mice display intermediate sensitivity and grasshopper mice are the least 

sensitive. 

A

0
0

.

B

0
0

.

.

4
0
−

t

n
e
r
r
u
C
d
e
z

 

.

4
0
−

t

n
e
r
r
u
C
d
e
z

 

.

8
0
−

i
l

a
m
r
o
N

.

2
1
−

C

V
m
5
@

 

 
k
a
e
p

 

n

i
 

e
c
n
e
r
e

f
f
i

D

6

.

0

0

.

0

Rat
Gmouse
Rat + Venom
Gmouse + Venom
30
Voltage (mV)

−40

0

−80

.

8
0
−

i
l

a
m
r
o
N

.

2
1
−

60

90

D

Mouse
Gmouse
Mouse + Venom
Gmouse + Venom
30
Voltage (mV)

−40

0

−80

60

90

Gmouse Mouse

Rat

△5mV

50ms

10mV

10ms

-100mV

10ms

-100mV

 

Figure 3: Effect of C. sculpturatus venom on voltage-current relationships for 

wildtype Nav1.4 channels expressed in Xenopus laevis oocytes. All peak currents 

were normalized by pre-venom peak currents at each voltage step. A) Voltage-current 

	

30	

curves for rat (green) and grasshopper mouse (black) Nav1.4 channels before (solid 

symbols) and after (open symbols) the application of 0.15 µg/µl of C. sculpturatus 

venom. B) Voltage-current curves for house mouse (purple solid) and grasshopper 

mouse (black solid) Nav1.4 channels before and after (open symbols) the application 

venom. C) Difference between pre and post venom peak current observed at +5 mV for 

grasshopper mouse (grey), house mouse (purple) and rat (green). D) Illustration of 

activation protocol used to generate voltage current curves. All data represent mean ± 

1SE. 

 

Effect of C. sculpturatus venom on inactivation kinetics: To determine the a-

toxin effect of C. sculpturatus venom on Nav1.4, I applied a single depolarizing potential 

of 0 mV for 50 ms to oocytes expressing either grasshopper mouse or control Nav1.4 

channels, and then overlaid the current traces produced before and after the application 

of venom. In the absence of venom (solid lines), representative current traces from all 

three species demonstrated rapid inactivation of Na+ currents (Figure 4A). In contrast, 

the application of C. sculpturatus venom (dashed lines) slowed the decay of Na+ current 

in grasshopper mice, house mice and rats, suggesting a-toxin inhibition of the fast 

inactivation mechanism in all three species (Figure 4A).  

I quantified a toxin effects on Na+ current inactivation by measuring two different 

parameters: 1) time constant of Na+ current decay (tau); and 2) slope of the Na+ current 

decay. 

Effect of a toxin on tau: When averaged across voltages ranging from -25 mV to 

+30 mV, there were no statistically significant baseline differences in the tau values of 

	

31	

grasshopper mice, house mice and rats (Figure 4B; F-value = 2.0739; df = 2, 38; P = 

0.140). However, at certain voltages there were significant differences in the baseline 

tau values of the three species (F-value = 2.409; df = 22, 416; P << 0.01). For example, 

at -20 mV, the tau of grasshopper mouse channels was significantly higher than that of 

rat (t-value = -2.391; df = 416; P = 0.017), and marginally higher than house mice (t- 

value = -1.804; df = 416; P = 0.072). At -25 mV, the tau value for grasshopper mouse 

channels was significantly higher than that of house mouse channels (t-value = -4.230, 

df = 416; P < 0.01) but not rat channels (t-value = -0.527; df = 416; P = 0.598).  

The application of venom significantly increased the Na+ current tau values in all 

three species (Figure 4C and 4D; df = 1, 454; F-value = 58.364; P << 0.01). At -25 mV, 

the tau of grasshopper mouse channels was significantly higher than house mice 

channels (t-value = 3.866; df = 454; P << 0.01) but not rat channels (t- value = -0.421; df 

= 454; P = 0.6740). At all other voltages, venom had similar effects on the tau of 

grasshopper mice, house mice and rats.  

	

32	

A

Rat

Gmouse

   

Mouse

   

   

5 ms

5 ms

5 ms

B

8
4

.

0
4

.

2

.

3

)
s
m

(
 

u
a
T

4

.

2

6

−25

.

1

8

.

0

0

.

0

8

.
4

0
.
4

D

2
.
3

)
s
m

(
 

u
a
T

4

.

2

6

Mouse
Rat
Gmouse

0

10

−10
Voltage (mV)

20

30

Mouse
Mouse+Venom
Gmouse
Gmouse+Venom

C

8

.
4

0
.
4

2
.
3

4

.

2

)
s
m

(
 

u
a
T

6

.

1

8

.

0

0
.
0

−25

Rat
Rat+Venom
Gmouse
Gmouse+Venom

.

1

8

.

0

0
.
0

−25

20

30

0

10
−10
Voltage(mV)

0

10

−10
Voltage (mV)

20

30

 
Figure 4: Effects of C. sculpturatus venom on inactivation kinetics of Nav1.4 

channels expressed in Xenopus laevis oocytes. A) Na+ current traces generated 

from a single 0mV depolarizing pulse. Solid lines show pre-venom current for rat (green 

circles), grasshopper mouse (black diamonds), and house mouse (purple squares) 

channels, whereas dashed lines are post venom current traces. B) Time constant of Na+ 

current inactivation (tau) in absence of venom for grasshopper mice (black), house mice 

(purple) and rats (green). Effect of venom on the time constant of inactivation of C) rat 

channels (green open circles) compared to grasshopper mouse (black open diamonds), 

and D) house mouse channels (purple open squares) compared to grasshopper mouse 

(black open diamonds).  

	

33	

Effect of a toxin on Na+ current decay slope: I measured the slope of Na+ current 

decay over a range of membrane potentials, before and after venom (Figure 4). In the 

absence of venom there were species-specific differences in current decay slopes at 

membrane potentials ranging from -15 to +10 mV (Figure 4A). A two-way repeated 

measures ANOVA revealed that the decay slope for grasshopper mouse channels was 

significantly shallower than that of rats (df = 2, 37; t = 2.49; P = 0.017), but not house 

mice (t = 1.34; df = 2, 37; P = 0.19) (Figure 4D). Shallower Na+ current decay slopes 

and higher tau values produced by grasshopper mouse Nav1.4 suggests that these 

channels experience delayed inactivation kinetics (compared to control channels) under 

baseline conditions.  

In the presence of C. sculpturatus venom, the decay slopes of all species 

appeared less steep than baseline (Figure 5C, 5D). To determine the extent of change 

in the decay slope, I measured the proportion change in slope from baseline (Figure 

5B). At voltages ranging from -10 mV to +30 mV, there was a significant species effect 

on proportion change in decay slope (F-value = 4.289; df = 2, 37; P = 0.021). The 

proportion change of decay slope from baseline for grasshopper mice was significantly 

lower than rats and house mice across the range of voltages. These results show that a 

toxins have a significantly smaller debilitating effect on the inactivation mechanism of 

grasshopper mouse Nav1.4 channels compared to controls.  

	

34	

A

B

2

.

1

8

.

0

/

)
s
m
A
u
(
 

l

e
p
o
S
 
y
a
c
e
D

4

.

0

0

.

0

−25

Rat
Mouse
Gmouse
10 20 30

−10 0
Voltage (mV)

2

Mouse
Mouse+Venom
) Gmouse
s
Gmouse+Venom
m
A
u
(
 

1

/

.

C

8
0

.

4

.

0

l

e
p
o
S
 
y
a
c
e
D

6

.

0

e
p
o
s
 

l

n

i
 

4

.

0

2

.

0

e
g
n
a
h
c
 

n
o

i
t
r
o
p
o
r
P

0

.

0

−10

0

10

20
Voltage (mV)

30

Rat
Rat+Venom

D

2

.

1

8
0

.

4

.

0

/

)
s
m
A
u
(
 

l

e
p
o
S
 
y
a
c
e
D

0

.

0

−20

0

10

Voltage (mV)

20

30

0

.

0

−20

0

10

Voltage (mV)

20

30

 

Figure 5: Effects of C. sculpturatus venom on the decay slope of Nav1.4 current. 

A) Decay slope of Na+ current plotted across voltage steps ranging from -25mV to 

+30mV for grasshopper mice (black) and house mice (purple) before (solid symbols) in 

the absence of venom. B) Proportion change in slope of Na+ currents due to venom 

application, calculated as [(pre venom slope – post venom slope)/ pre venom slope]. C) 

Decay slope of Na+ current plotted across voltage steps comparing grasshopper mouse 

to house mouse channels before (solid symbols) and after (open symbols) the 

application of venom. D) Decay slope of Na+ current plotted across voltage steps 

comparing grasshopper mouse to rat channels before (solid symbols) and after (open 

symbols) the application of venom.  

	

35	

Steady-state inactivation and recovery: In addition to voltage dependence of 

activation and fast inactivation kinetics, I also measured the voltage dependence of 

steady-state inactivation and recovery from fast inactivation. I found no differences 

between the three species in their voltage dependence of steady-state inactivation or 

rate of recovery from fast inactivation either in the presence or absence of C. 

sculpturatus venom (data not shown).  

 

Role of unique amino acid modifications in conferring resistance to C. 

sculpturatus venom 

Models of molecular evolution found seven amino acids to be under positive 

selection in the Onychomys lineage. Of these, I333 (S5-SS1 loops of DI) and Y1182 

(S5-SS1 loops of DIII) are located in extracellular loops that are in close proximity to a 

and b toxin binding sites on the Nav channel. To test the role of DI and DIII amino acids 

substitutions in reducing the Nav1.4 channel sensitivity to C. sculpturatus toxins, I 

changed two amino acids in each of these locations (i.e., DI: I333 to E333 and E334 to 

G334; DIII: Y1182 to S1182 and K1183 to E1183) to the presumed ancestral residue 

(as seen in house mice and rats). Further, the wildtype grasshopper mouse Nav1.4 also 

carries a unique 48-amino acid insert in its C-terminus region. Because amino acids in 

the C-terminus play an important role in regulating the inactivation kinetics of Nav 

channels, this insert may be important for reducing sensitivity to scorpion toxins. 

Therefore, I removed the 48-amino acid C-terminus insert from the previously mutated 

grasshopper mouse Nav1.4 channel (referred to as the “triple mutant” channel). Other 

than the mutations I introduced into DI, DIII and C-terminus, the triple mutant channel 

	

36	

was identical to the wildtype grasshopper mouse Nav1.4. Using previously described 

voltage-clamp technology and protocols, I tested the electrophysiological properties of 

this triple mutant channel and compared it to the wildtype grasshopper mouse Nav1.4 

channel. 

 

 

Table 2: Boltzmann parameters for normalized conductance vs. voltage 

relationships of wildtype grasshopper mouse and triple mutant Nav1.4 channels. 

V50a and V50b are the voltages of half maximal activation for the proportion of channels 

affected and not affected by C. sculpturatus venom, respectively. K1 and K2 are the 

slope parameters, P is the proportion of channels unaffected by venom, n is the number 

of oocytes recorded.  

 

b toxin effects # 1 (hyperpolarizing shift in voltage dependence of 

activation) on triple mutant channels: I plotted conductance-voltage curves for triple 

mutant channels and compared them to wildtype grasshopper mouse channels (Figure 

6A). As before, I fit conductance-voltage curves to a Boltzmann equation and 

statistically compared the V50 parameters. In the absence of venom, I found a small but 

significant difference in the Boltzmann fit parameter, V50a (Table 2), between the 

wildtype grasshopper mouse and triple mutant channels. On average, the V50a of the 

triple mutant was shifted 1.6 mV in the negative direction compared to the wildtype 

	

37	

grasshopper mice channel (z = -13.68; P << 0.01). This hyperpolarizing shift in the V50a 

of the mutant channel suggests that the amino acid modifications in grasshopper mouse 

Nav1.4 channels are responsible for the baseline difference in V50a between 

grasshopper mice and control species.  

In the presence of venom, the difference between the V50b parameters of 

grasshopper mice and the triple mutant was only marginally significant (z = -1.87; P = 

0.06, Table 2). However, the conductance-voltage curve showed that the triple mutant 

had higher conductance than grasshopper mouse channels at voltages ranging from -

40 to -10 mV. These results are shown in Figure 6B which are enlarged data from 6A to 

which the post-venom tends have been added. Indeed, the conductance of the triple 

mutant channels was significantly higher than grasshopper mouse channels at -20 mV 

in the presence of venom (t = 3.97; df = 1, 168; P << 0.01). These data suggest that 

triple mutant channels are more sensitive than grasshopper mouse channels to the 

effects of b toxins on the voltage dependence of channel activation. 

b toxin effects # 2 (reduction in peak Na+ current amplitude) on triple 

mutant channels: Next, I measured the b-toxins effects of C. sculpturatus venom on 

peak Na+ current at depolarized membrane potentials (Figure 6C and 6D). I found that 

C. sculpturatus venom decreases peak Na+ currents in both grasshopper mice and 

triple mutant channels (overlapping ± 1SEs). The difference between pre- and post-

venom currents at +5 mV for grasshopper mice and triple mutants is shown in Figure 

6D (t = -0.121; df = 1, 342; P = 0.904). 

 

	

38	

A 

B 

Gmouse
Triple mutant

−70

−30

−50
−10
Voltage (mV)

5

)

A
N
g
(
 
e
c
n
a
t
c
u
d
n
o
C
 
d
e
z

i
l

a
m
r
o
N

0

.

1

8

.

0

6

.

0

4

.

0

2

.

0

0

.

0

C 

0

.

0

t

n
e
r
r
u
C
d
e
z

 

3

.

0
−

6

.

0
−

i
l

a
m
r
o
N

9

.

0
−

2

.

1
−

−80

−40

0

30
Voltage (mV)

60

90

)

A
N
g
(
 
e
c
n
a
t
c
u
d
n
o
C
 
d
e
z

i
l

a
m
r
o
N

6

.

0

5

.

0

4

.

0

3

.

0

2

.

0

1

.

0

0

.

0

D 

V
m
5
 
@

 
k
a
e
p
 
n
i
 
e
c
n
e
r
e
f
f
i

D

8
.
0

4
.
0

0
.
0

Gmouse
Gmouse + Venom
Triple
Triple + Venom

−55

−45

−35

−25
Voltage (mV)

−15

Gmouse

Triple_Mutant

 
Figure 6: Effect of C. sculpturatus venom on the activation of triple mutant Nav1.4 

channels expressed in Xenopus laevis oocytes. A) Baseline conductance-voltage 

relationship of triple mutant channels (gold triangles) overlaid over wildtype grasshopper 

mouse channels (black diamonds). Data are fit to a single Boltzmann equation. B) 

Conductance-voltage relationship of triple mutant channels (gold triangles) compared to 

wildtype grasshopper mouse channels (black diamonds) in the absence (solid symbols) 

or presence (open symbols) of venom. Connecting lines do not represent fits. C) 

	

39	

Current-voltage relationship for triple mutant (gold triangles) and grasshopper mouse 

(black diamonds) channels. D) Difference between pre and post venom peak current 

observed at +5 mV for grasshopper mouse (black) and triple mutant channels (gold). 

Data are reported as mean ± 1SE.  

 

Effect of a toxin on tau: To test the effects of a toxins on the triple mutant 

channel’s inactivation kinetics, I measured tau for triple mutant and wildtype 

grasshopper mouse Nav1.4 channels and found no difference either in the absence (F-

value = 1.634; df = 11, 583; P = 0.086) or presence of venom (df = 11; 635; F = 0.163; P 

= 1.0; Figure 7A).  

Effect of a toxin on Na+-current decay slope: Finally, I measured the Na+ 

current decay slope for grasshopper mouse and triple mutant channels (Figure 7B) and 

found no difference between the two types of channels either before or after venom 

application. Further, there was no difference in the proportion change in post-venom 

decay slope (Figure 7C, overlapping ± 1SE; df = 1, 12; F-value = 1.609; P = 0.2287). 

Figure 7: Effect of C. sculpturatus venom on the inactivation kinetics of triple 

 

mutant Nav1.4 channels to measure a-toxin effects. A) Tau plotted across voltage 

	

40	

for triple mutant (gold triangles) and grasshopper mouse (black diamonds) channels 

before (solid symbols) and after (open symbols) venom application. B) Na+ current 

decay slope plotted across voltage steps for triple mutant (gold) and wildtype 

grasshopper mouse channels (black) in the presence (open symbols) and absence 

(solid symbols) of venom. C) Proportion change in post-venom Na+ current decay slope 

plotted across voltage steps for triple mutant (gold) and wildtype grasshopper mouse 

channels (black). 

 

Relative contributions of DI, DIII and C-terminus insert towards reduced C. 

sculpturatus venom sensitivity 

           The electrophysiology recordings from the triple mutant construct suggested that 

structural modifications to grasshopper mice Nav1.4 are involved in reducing sensitivity 

to C. sculpturatus venom b-toxin effects. To understand the relative contributions of the 

different regions (DI, DIII and the C-terminal insert) in reducing sensitivity to venom, I 

made three individual mutant channels: 1) Di: two amino acid residues I333 and E334 

were changed to the presumed ancestral state (E333 and G334, respectively) in domain 

I; 2) Diii: two amino acid residues Y1182 S1182 and K1183 changed to the presumed 

ancestral type E1183 in domain II; 3) NoC: only the C-terminus insert was removed. 

This series of experiments focused on the electrophysiology property that varied 

between wildtype grasshopper mouse and triple mutant Nav1.4 channels in the 

presence of venom – the voltage dependence of activation. 

	

41	

A

C

.

6
0

.

5
0

)

A
N
g
(
 

e
c
n
a

t
c
u
d
n
o
C
d
e
z

 

i
l

a
m
r
o
N

.

4
0

.

3
0

.

2
0

.

1
0

.

0
0

6
.
0

5
.
0

4
.
0

3
.
0

2
.
0

)

A
N
g
(
 

e
c
n
a

t
c
u
d
n
o
C
d
e
z

 

i
l

a
m
r
o
N

1
.
0

0
.
0

Gmouse
Gmouse + Venom
Triple
Triple + Venom

−55

−55

−45

−35

−25
Voltage (mV)

Diii
Diii + Venom

−45

−35

−25
Voltage (mV)

−15

−15

B

.

6
0

.

5
0

)

A
N
g
(
 

e
c
n
a

t
c
u
d
n
o
C
d
e
z

 

i
l

a
m
r
o
N

.

4
0

.

3
0

.

2
0

.

1
0

.

0
0

D

6
.
0

5
.
0

4
.
0

3
.
0

2
.
0

)

A
N
g
(
 

e
c
n
a

t
c
u
d
n
o
C
d
e
z

 

i
l

a
m
r
o
N

1
.
0

0
.
0

Di
Di + Venom

−55

−55

−45

−35

−25
Voltage (mV)

NoC
NoC + Venom

−45

−35

−25
Voltage (mV)

−15

−15

 

Figure 8: Effect of C. sculpturatus toxins on the voltage dependence of activation 

of Di, Diii and NoC mutant channels. Conductance-voltage relationships before (solid) 

and after (open symbols) venom for A) Triple mutant channels (gold triangles) and 

wildtype grasshopper mouse (black diamonds) Nav1.4 channels; B) Grasshopper 

mouse, triple mutant and Di mutant channels (navy blue); C) Grasshopper mouse, triple 

mutant and Diii mutant channels (indigo); D) Grasshopper mouse, triple mutant and 

NoC mutant channels (sky blue). Electrophysiology and activation protocols are 

described previously. Data represent mean ± 1SE.  

 

	

42	

Table 3: Boltzmann parameters comparing normalized conductance vs. voltage 

relationships for wildtype grasshopper mouse, triple mutant and single mutant 

(Di, Diii, NoC) Nav1.4 channels. V50a is the voltage of half maximal activation, K1 is the 

slope parameter and n is the number of oocytes recorded. Only pre-venom data is 

 

shown. 

 

Effect of DI mutations on sensitivity to C. sculpturatus venom: To test the 

sensitivity of the Di mutant to venom b toxin effects, I compared the voltage 

dependence of activation for the Di mutant to the wildtype Nav1.4 before and after 

venom. In the absence of venom, I found baseline differences in the V50a of Di mutants 

compared to grasshopper mice, but not triple mutant channels (Table 3). On average, 

the V50 of Di channels was shifted in the hyperpolarizing direction from grasshopper 

mouse channels by 1.22 mV (z = 5.07; P << 0.01). These data suggest that Di mutant 

channels have a higher baseline excitability than grasshopper mouse channels (but not 

triple mutant channels). In the presence of venom, there were no differences between 

V50b values of the Di, triple mutant and wildtype Nav1.4 channels. However, the 

conductance of Di mutant channels was significantly higher than that of wildtype 

	

43	

grasshopper mouse channels at -20 mV (t = 2.79; df = 2, 57; P < 0.01, Figure 8A), but 

did not differ from the triple mutant (t = -1.39; df = 2, 57; P = 0.17; Figure 8A and 8B). 

This suggests that the Di mutant is more sensitive to b toxin effects (similar to the triple 

mutant) than the wildtype grasshopper mouse Nav1.4 construct.  

Effect of DIII mutations on sensitivity to C. sculpturatus venom: To test the 

sensitivity of the Diii mutant to venom b-toxin effects, I compared the voltage 

dependence of activation for the Diii mutant to the wildtype Nav1.4 before and after 

venom. In the absence of venom, I found baseline differences in the V50a of Diii mutants 

compared to both grasshopper mice and triple mutant channels (Table 3); Diii had an 

intermediate V50a value (Table 3). On average, the V50a of Diii was lower than the 

grasshopper mouse channels by 0.77 mV (z = 2.79; P = 0.005) but higher than the triple 

mutant channels by 2.41 mV (z = -8.67; P << 0.01). These data suggest that Diii mutant 

channels have a higher baseline excitability than grasshopper mouse channels, and a 

lower baseline excitability than triple mutant channels. In the presence of venom, there 

were no significant differences between the V50b values of Diii, triple mutant or 

grasshopper mouse channels. However, the Diii mutant channels had a marginally 

higher conductance than the wildtype channels at -20 mV (t = -1.81; df = 2, 56; P = 

0.07, Figure 8C), but not the triple mutant channels (t = 1.16; df = 2, 56; P = 0.25). This 

suggests that a larger proportion of the Diii mutant channels are sensitive to b toxin 

effects than the wildtype grasshopper mouse Nav1.4 channels. 

Effect of C-terminal insert on sensitivity to C. sculpturatus venom: To test 

the sensitivity of NoC mutant to venom b-toxin effects, I compared the voltage 

dependence of activation of NoC mutant and the wildtype Nav1.4 before and after 

	

44	

venom. I found baseline differences between the V50a of NoC mutant and grasshopper 

mouse channels but not the triple mutant channels. On average, the V50a of NoC 

channels was shifted in the hyperpolarizing direction from grasshopper mouse channels 

by -1.38 mV (Table 3; z= 4.75; P << 0.01). These data suggest that NoC mutant 

channels have a higher baseline excitability than grasshopper mouse channels, but not 

the triple mutant channels. In the presence of venom, there were no differences 

between the V50b values of all species. Additionally, the Na+ conductance for NoC 

mutants at -20 mV was only marginally different from grasshopper mice (t = -1.77; df = 

2, 54; P = 0.08; Figure 8D), but there was no difference between NoC and triple mutant 

channels (t= 0.10; df = 2, 54; P = 0.32). This suggests that the NoC mutant has an 

intermediate level of sensitivity to b toxin effects compared to the wildtype and triple 

mutant Nav1.4 channels.  

 

DISCUSSION 

Grasshopper mouse Nav1.4 channels are less sensitive to the a- and b-toxin 

effects of C. sculpturatus venom 

Nav1.4 is the only Nav channel paralog expressed on the surface of mammalian 

skeletal muscle cells (myocytes), where its function is crucial for muscle contraction 

(Goldin et al. 2000). Previous studies showed that Nav1.4’s activation and inactivation 

(or gating) mechanisms are disrupted by the application of a and b toxins from 

Centruroides elegans and C. vittatus venoms (Vandendriessche et al. 2010; Rowe et al. 

2011).Venom-induced modification of the Nav1.4 gating mechanism hyperexcites the 

myocytes of sensitive mammals (e.g. house mice, rats, humans) (Bosmans & Tytgat 

	

45	

2007). Hyperexcitation leads to a block of membrane depolarization and loss of muscle 

contraction. Impaired contraction of the diaphragm can cause respiratory failure and 

death.  

Grasshopper mice are resistant to C. sculpturatus venom, both natural stings and 

controlled injections (Rowe et al., 2005 and 2008). Preliminary data showed 

grasshopper mouse muscle contraction was unaffected by C. sculpturatus venom, 

whereas house mouse muscles experienced a block in contraction (A. Rowe, 

unpublished). In this study, I tested the hypothesis that grasshopper mouse muscles are 

resistant to C. sculpturatus venom because they express structural modifications in 

Nav1.4 that impart resistance to a- and b-toxin effects. 

Centruroides b toxins contribute to membrane hyperexcitability by interacting with 

specific amino acids in Nav domain II (DII) voltage sensors, causing a hyperpolarizing 

shift in the voltage dependence of channel activation (Cestèle et al. 1998; Cestèle & 

Catterall 2000; Mantegazza & Cestèle 2005). This b-toxin effect on activation makes 

channels more likely to open at resting membrane potentials. I tested the b-toxin effects 

of C. sculpturatus venom on the wildtype Nav1.4 channels of grasshopper mice and 

compared their functional properties to the Nav1.4 channels of house mice and rats 

(control species). As predicted, venom had a significantly smaller b-toxin effect on the 

voltage dependence of activation of grasshopper mouse Nav1.4 channels compared to 

rat channels (Figure 2). Moreover, b toxins had a significantly smaller inhibitory effect on 

the peak current of grasshopper mouse channels compared to rat, but not house mouse 

channels. My results show that grasshopper mouse Nav1.4 channels are less sensitive 

than rat channels to the b toxin effects of C. sculpturatus venom. However, house 

	

46	

mouse Nav1.4 were also less sensitive to b-toxin effects compared to rats (Figure 2C). 

This might suggest that house mouse Nav1.4 channels carry a different set of structural 

modifications, perhaps in less conserved regions of the channels, that impart reduced 

sensitivity to b toxins. Alternatively, house mice and grasshopper mice may share a 

subset of substitutions in other extracellular loops or less conserved regions (absent in 

rat channels) that confer reduced sensitivity to b toxins. Understanding the molecular 

mechanism underlying this reduced b toxins sensitivity in house mice compared to 

grasshopper mice may provide key insights into the evolvability of highly conserved and 

functionally important ion channel proteins. 

Centruroides a toxins contribute to membrane hyperexcitability by targeting 

amino acids in the Nav DIV voltage sensor, impairing the channel’s fast inactivation 

mechanism (Catterall 1979; Bosmans & Tytgat 2007). By preventing the outward 

movement of the DIV voltage sensors, venom immobilizes the inactivation gate, and 

causes the channel to remain conductive for longer than physiologically normal 

(Catterall 1980; Kharrat et al. 1989; Campos et al. 2008; Wang et al. 2011). This effect 

manifests as a decrease in the slope of the Na+ current decay; thus, a toxin-bound Nav 

channels produce Na+ currents that activate normally, but that do not inactivate within 

milliseconds, causing a shallow decay phase (Campos et al. 2008). I found that venom 

reduced the decay time constant (tau) similarly in grasshopper mice as well as the 

control species (Figure 4A). Only at -25 mV was the tau of grasshopper mouse Nav1.4 

channels significantly higher than house mouse channels in the presence of venom. 

However, the effect size of this difference was small (difference in effect size between 

grasshopper mouse vs. house mouse tau = 0.98 ± 0.25 ms) and is therefore unlikely to 

	

47	

represent a biologically meaningful difference. Although venom had similar effects on 

the tau values in all three species, I found that the baseline tau of grasshopper mouse 

Nav1.4 channels was significantly higher than that of control species at -20 and -25 mV 

(Figure 4B). Additionally, the proportion change in post-venom Na+ decay slope of 

grasshopper mouse channels was significantly lower than for control species (Figure 

5B). Collectively, these results suggest that, although grasshopper mouse Nav1.4 

currents decay more slowly under baseline conditions, they are less perturbed by a 

toxins in C. sculpturatus venom than the controls. However, the biological significance 

of this finding is unclear because the absolute values of post-venom decay slopes are 

similar in all three species.  

C. sculpturatus venom causes pain-related behavior, muscle spasms and death 

in house mice, but has little effect on grasshopper mice (Rowe & Rowe 2008). Ex vivo 

analyses showed that C. sculpturatus venom initially increased the force of contraction, 

and then blocked contraction in house mouse muscle, while having virtually no effect on 

grasshopper mice. While the current study shows that rat Nav1.4 channels are 

significantly more sensitive to the a- and b-toxin effects of C. sculpturatus venom than 

grasshopper mice, grasshopper mice are not completely resistant to these effects. 

Moreover, house mice are only slightly more sensitive to b toxins than grasshopper 

mice. These findings suggest two critical points. First, a reduction in the sensitivity of 

grasshopper mouse Nav1.4 to a and b toxins will not likely provide their muscle with 

complete physiological resistance to scorpion venom. Centruroides venom targets a 

range of ion channels, including voltage-gated potassium, calcium, and chloride 

channels, as well as other receptors involved in neuromuscular function (Valdivia & 

	

48	

Possani 1998; Tytgat et al. 1999; Escalona & Possani 2013). Future work should focus 

on investigating other molecular targets of C. sculpturatus venom in grasshopper mice 

muscle. Second, although house mice are identical to rat Nav1.4 channels at the highly 

conserved sites shown in Figure 1, there are other extracellular loops and less 

conserved regions in which house mice either carry unique amino acid substitutions or, 

at least, differ from rats and grasshopper mice. These house mouse Nav1.4 structural 

variants may have arisen due to random mutations that accumulated in the CD-1 

lineage during the creation and/or maintenance of pure genetic lines. Conversely, the 

variants may represent relicts of ancestral selective pressures from scorpions prevalent 

in areas where the wild ancestors of house mice evolved. Fossil records suggest that 

the earliest ancestors of the genus Mus, lived on the Indian subcontinent approximately 

5.5 million years ago (Boursot et al. 1993, Suzuki et al. 2013), during a time when 

scorpions likely prevailed in the area.  

 

Three distinct regions of the Nav1.4 channel contribute to reduced venom 

sensitivity 

My second goal was to identify the amino acids involved in reducing sensitivity to 

C. sculpturatus venom. Because grasshopper mice express several amino acid variants 

in their Nav1.4 protein, I used models of molecular evolution to guide my search for 

functionally important residues. The branch-site model of nucleotide substitutions 

(CODEML package, PAML) predicted that seven amino acid residues might be under 

positive selection in the Onychomys lineage. Of these, I333 in DI S5-S6 and Y1182 in 

DIII S5-S6 are in extracellular loops close to known Centruroides toxin binding sites 

	

49	

were associated with a >65% probability of being under positive selection. While I333 is 

positioned in the a toxin receptor site, Y1182 lies close to known b toxin binding sites. It 

is noteworthy that the amino acids adjacent to I333 (E334) and Y1182 (K1183) are also 

unique to the Onychomys lineage. However, these residues were not predicted to be 

under positive selection by the relatively conservative parameters of the branch-site 

model.  

To maximize the probability of identifying residues responsible for reducing toxin 

sensitivity, I used electrophysiology to test the role of two amino acids in DI (I333 and 

E334) two in DIII (Y1182 and K1183), and the C-terminus insert (unique to grasshopper 

mice) by creating a triple mutant construct. I expected to observe differences between 

mutant and wildtype channels in baseline properties because the amino acids 

exchanged between the constructs are located in conserved regions. Indeed, I found 

the triple mutant channels to activate at more hyperpolarized membrane potentials than 

wildtype grasshopper mouse channels under baseline conditions (Table 2, Figure 6A). 

These findings are significant because they indicate that substitutions in DI, DIII and the 

C-terminus make wildtype grasshopper mouse Nav1.4 channels (and likely their 

myocytes) less excitable. In all other measured baseline properties, triple mutant Nav1.4 

channels were similar to grasshopper mouse channels.  

In the presence of C. sculpturatus venom there was a small but significant effect 

on the voltage dependence of activation for both channels. In the triple mutant, there 

was a greater increase in conductance at negative voltage steps compared to wildtype 

grasshopper mouse channels (Figure 6B). However, the triple mutant and grasshopper 

mouse channels experienced similar levels of post venom decreases in peak Na+ 

	

50	

current amplitude at positive membrane potentials. Additionally, the triple mutant 

channels did not differ from grasshopper mouse channels in their sensitivity to a toxins 

i.e., the two channels had similar Na+ current decay slopes and decay constants (tau). 

These findings indicate that the amino acid variants seen in DI S5-S6, DIII S5-S6 and 

the C-terminus play some role in alleviating the effects of b- but not a-toxins in the 

venom of C. sculpturatus. Future work should identify additional regions of the 

grasshopper mouse Nav1.4 channel involved in reducing sensitivity to the b (effects on 

peak current) and a-toxin effects. Some of these undiscovered regions of importance 

may be shared between grasshopper mice and house mice; this would explain why 

house mouse Nav1.4 channels are less sensitive to C. sculpturatus venom than rat. 

Whether shared mechanisms of resistance are a result of convergent evolutionary 

forces or genetic drift acting on in-bred lines of CD-1 house mice are merely a matter of 

conjecture until further research is conducted. 

 

Effect of structural modifications on resistance to a and b toxins may be additive 

Next, I investigated the individual contributions of DI, DIII and C-terminus 

modifications towards reducing the wildtype grasshopper mouse Nav1.4’s sensitivity to b 

toxins. In the presence of venom, I found that the Di and Diii mutants were similar to the 

triple mutants (Figure 8B and 8C) in their voltage dependence of activation (b toxin 

effect), whereas the voltage dependence of activation for NoC channels was 

intermediate to grasshopper mice and triple mutants (Figure 8D). These results show 

that the amino acid variants in grasshopper mouse Nav1.4 DI, DIII and C-terminal are all 

involved in reducing sensitivity to the effects of b toxins on voltage dependence of 

	

51	

activation. These findings are significant because they suggest that three separate, 

structurally modified regions in the grasshopper mouse Nav1.4 channel may contribute 

to reduced sensitivity to C. sculpturatus venom. 

 

Proposed mechanism of resistance: role of amino acids in DI, DIII and C-terminus 

Amino acids in extracellular loops of Nav channels can influence the dissociation 

constant (Kd) of b toxins. Cestèle et al. (1998) found that chimeric mutagenesis 

replacements of amino acids in the pore loop of DI and DIII dramatically increased toxin 

Kd. Therefore, I propose that the DI and DIII amino acid substitutions seen in the Nav1.4 

channels of grasshopper mice reduce sensitivity to b toxins by lowering toxin Kd. Further 

investigation of dissociation constants using isolated C. sculpturatus venom peptides 

will be necessary to empirically elucidate the exact mechanisms involved in reducing 

venom sensitivity. 

Although previous studies have shown that the intracellular C-terminus of Nav 

channels plays a crucial role in determining the rate of fast inactivation, there is no prior 

evidence that this region affects activation properties. My study is the first to show that 

amino acids in the C-terminus region of Nav channels may influence the interaction of 

venom toxins with voltage sensors during activation. Computational protein models that 

can elucidate structure-function relationships would help to unravel potential 

mechanisms through which the C-terminus interacts with voltage sensors during 

activation and venom binding. 

 

	

52	

Physiological costs associated with modifications that reduce sensitivity to C. 

sculpturatus venom 

         Structural modifications to highly conserved proteins, even if beneficial, are often 

accompanied by physiological costs. One such example of an evolutionary cost is seen 

in garter snakes (Thamnophis sirtalis) that feed on toxic newts (Taricha granulosa). 

Garter snakes carry unique amino acid modifications in their Nav1.4 proteins that make 

them resistant to tetrodotoxin (TTX), a Na+ pore-blocking neurotoxin found in newts 

(Geffeney 2002). However, the amino acids substitutions that make garter snake Nav1.4 

channels resistant to TTX have adverse effects on both the conductance of Nav1.4 

channels and their kinetic properties (Yoshida 1994; Brodie & Brodie 1999). In my own 

system, I found baseline differences in the voltage dependence of activation of 

grasshopper mouse, house mouse and rat Nav1.4 channels. Grasshopper mouse 

Nav1.4 channels activated at significant more depolarized potentials than rat and house 

mouse channels. Further, I could empirically demonstrate that the same unique 

structural modifications in the grasshopper mouse Nav1.4 that make these channels 

partially resistant to C. sculpturatus venom are also responsible (to varying degrees) for 

shifting the channel’s baseline activation threshold.  

            I also found that grasshopper mouse and house mouse Nav1.4 channels have 

slower baseline inactivation kinetics compared to rat channels. While this inhibition of 

inactivation in grasshopper mouse Nav1.4 may represent a cost associated with 

reduced sensitivity to C. sculpturatus venom, my data shows that it does not arise from 

the unique structural modifications identified in this study. Therefore, I propose that 

improper association between grasshopper mouse and house mouse Nav1.4 alpha 

	

53	

subunits with rat beta subunits affects their inactivation kinetics. However, if these 

differences in inactivation kinetics do represent a trade-off, it likely arises from amino 

acid variants shared between grasshopper mice and house mice that also have 

consequences for venom resistance. 

 

Summary and Significance 

My study is the first to sequence the complete coding region of the grasshopper 

mouse (Onychomys spp.) skeletal-muscle Nav1.4 gene (Scn4a), and to report the 

effects of Arizona bark scorpion (C. sculpturatus) venom on heterologously expressed 

Nav1.4 channels from southern grasshopper mice, house mice and rats. Using 

comparative sequence analyses, molecular biology and electrophysiological recordings, 

I show that C. sculpturatus venom induces a- and b-toxin effects on rat Nav1.4, 

disrupting channel activation and inactivation. However, as predicted, grasshopper 

mouse Nav1.4 is significantly less sensitive to certain effects of these toxins. Further, I 

show that the molecular mechanism of reduced sensitivity involves amino acid variants 

at three distinct locations within the channel protein: two major and one minor region of 

contribution. These findings are significant because there are no other examples of ion 

channels that are resistant to distinct classes of peptide toxins simultaneously targeting 

different gating mechanisms (activation, inactivation), nor are there examples of 

resistance that involve structural modifications to multiple regions within a channel 

protein. Further, I empirically demonstrate that structural modifications to Nav1.4 

proteins result in significant trade-off to their functional properties that may represent 

physiological costs to muscle excitability. More broadly, the discovery of specific amino 

	

54	

acids influencing the voltage range over which ion channels activate may be important 

in the search for therapeutic targets for treating neuromuscular disorders that affect 

myocyte excitability.  

 

	

 

55	

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56 

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CHAPTER 2: Role of grasshopper mouse beta subunits in modulating the 

function of skeletal-muscle voltage-gated ion channels (Nav1.4) 

 

INTRODUCTION 

Transmembrane proteins called voltage-gated sodium ion (Nav) channels play a 

crucial role in mediating action potentials in the electrically excitable cells of nerves and 

muscles (Hodgkin & Huxley 1952). The mammalian Nav protein complex is 

heterotrimeric; it comprises a large, heavily glycosylated, pore-forming alpha subunit (~ 

260 kD) and two smaller accessory proteins called beta subunits (~36 kD) (Catterall 

2000; Yu & Catterall 2003). The four homologous domains (DI-DIV) of a single alpha 

subunit polypeptide undergo complex three dimensional folding to form a 

transmembrane channel that is highly selective for Na+ ions (Noda et al. 1984; Catterall 

2000; Goldin 2002). While the expression of alpha subunit protein alone can produce 

functional Na+ currents in heterologous cells (Goldin et al. 1986; Noda et al. 1986; 

Catterall 2000), to achieve physiologically relevant gating kinetics and current density, 

co-expression with beta subunits is essential (Winters & Isom 2016; Brackenbury et al. 

2011) 

Four beta subunit paralogs (Scn1b- Scn4b) in mammalian genomes encode the 

accessory beta 1 – beta 4 proteins (Isom et al. 1992; Isom et al. 1995; Morgan et al. 

2000; Yu et al. 2003.). These beta subunit proteins, classified as type I transmembrane 

glycoproteins, share several structural features; they comprise of a large extracellular 

Ig-like N-terminus, one transmembrane segment and a short intracellular C-terminus 

region (Isom 2001; Winters & Isom 2016). Whereas beta 1 and beta 3 share 57% 

	

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sequence homology and associate non-covalently with the alpha subunit, beta 2 and 

beta 4 are 35% homologous, and associate with the alpha subunit through covalent 

disulfide bonds on the extracellular surface (Winters & Isom 2016; Chahine & O'Leary 

2011). In heterologous cells, beta subunit co-expression increases cell surface 

expression of Nav channels, changes the voltage range over which activation and 

inactivation occurs, as well as enhances fast inactivation and recovery from inactivation 

(McEwen et al. 2004; Isom et al. 1992; Isom et al. 1995; Patino et al. 2009; Yu & 

Catterall 2003). Indeed, the importance of beta subunits in regulating normal channel 

physiology can be seen in the range of human diseases that result from mutations in 

genes encoding beta subunits (Medeiros-Domingo et al. 2007; Dulsat et al. 2017; 

Patino et al. 2009; Escayg & Goldin 2010; Ogiwara et al. 2012).  

Given their crucial role in regulating the excitability of cells, Nav channels are 

often targets of offensive and defensive neurotoxic venoms produced across the animal 

kingdom. One such venom is produced by Centruroides sculpturatus or the Arizona 

bark scorpions in the family Buthidae (Bosmans & Tytgat 2007). When threatened, C. 

sculpturatus scorpions use their sharp stingers to deliver a cocktail of painful and lethal 

toxins into potential predators. Based on their effects on Nav channels, these toxins are 

broadly classified as: 1) a toxins that impair the inactivation mechanism to prolong the 

channel’s open state (Campos et al. 2008; Bosmans & Tytgat 2007; Thomsen & 

Catterall 1989; Kharrat et al. 1989), and 2) b toxins that promote premature activation of 

the channel (Mantegazza & Cestèle 2005; Cestèle & Catterall 2000; Escalona & 

Possani 2013). While a and b toxins bind to different regions of the Nav1.4 alpha 

subunits, they work synergistically to cause prolonged depolarizations, and 

	

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spontaneous action potentials. In sensitive mammals (including human infants), the 

effects of venom on Nav1.4 (expressed on skeletal muscles) can manifest as muscle 

paralysis, asphyxiation, respiratory failure and death. 

Carnivorous rodents called grasshopper mice (Onychomys torridus) routinely 

encounter and consume bark scorpions in the wild (Rowe & Rowe 2006). In staged 

behavioral trials, grasshopper mice are not deterred by C. sculpturatus stings, having 

evolved physiological resistance to their venom (Rowe & Rowe 2006; Rowe & Rowe 

2008). Previously, I sequenced the coding region of the gene that encodes the 

grasshopper mouse Nav1.4 alpha subunit (Scn4a) and found several unique amino acid 

modifications in highly conserved regions of the channel. I demonstrated that these 

modifications made grasshopper mouse Nav1.4 channels less sensitive to the a and b 

effects of C. sculpturatus venom compared to control channels representing venom 

sensitive rodents, Rattus norvegicus (rat) and Mus musculus (house mouse). However, 

grasshopper mouse Nav1.4 channels were not completely resistant, exhibiting small, 

venom-induced shifts in their gating kinetics.  

Given that grasshopper mice are completely resistant to natural C. sculpturatus 

stings and controlled venom injections (A. Rowe unpublished), what could explain their 

Nav1.4’s partial sensitivity to toxins? One hypothesis (of many) is that grasshopper 

mouse Nav1.4 alpha subunits require association with their own beta subunits to exhibit 

complete venom resistance. Because beta subunits are important contributors to Nav 

function, association with species-specific beta subunits may be necessary to mimic 

physiologically relevant gating kinetics. Therefore, in this study, I tested the role of 

	

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grasshopper mouse beta subunits in modulating their Nav1.4 activity and response to 

venom. 

The expression of beta subunits is tissue specific and differentially regulated 

across development (Sutkowski & Catterall 1990). Two beta subunits are expressed in 

skeletal muscles: beta 1 and beta 4 (Makita et al. 1996, Yu et al. 2003). Studies show 

that beta 1 is highly expressed in skeletal muscles and its association has notable 

impacts on the voltage dependent properties of Nav1.4 (Makita et al. 1996). Work on 

beta 4 association with Nav1.4 is limited; however, the co-expression of beta 4 with 

Nav1.4 channels in heterologous cells has significant effects on voltage dependence of 

activation (Yu et al. 2003.; Chahine & O'Leary 2011).  

I detected structural modifications in the beta 1 and beta 4 by sequencing the 

genes that encode these proteins: Scn1b and Scn4b. However, my work shows that 

grasshopper mouse beta subunits affect neither the voltage dependent properties of the 

grasshopper mouse Nav1.4 channels nor their response to venom.  

 

MATERIALS AND METHODS 

Molecular biology 

I used the Onychomys torridus skeletal muscle tissue, RNA extraction and cDNA 

synthesis protocols described in Chapter 1. A gene specific reverse primer along with 

Oligo d(T)20 primed the reverse transcription reaction for cDNA synthesis. I designed 

degenerate PCR primers by identifying conserved 5’ and 3’ regions in a Scn1b/4b 

alignment of multiple closely related species, and amplified the gene in one piece. Then 

I ran the amplified fragments on a 0.8% w/v agarose gel, excised and purified the 

	

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appropriate sized bands using Wizard SV Gel and PCR Clean-Up System (Promega). 

Such purified PCR samples from three O. torridus mice were sent to Genewiz for 

Sanger Sequencing of PCR products, sequence synthesis and cloning into pcDNA3.1+ 

expression vector. House mouse Scn1b and Scn4b were downloaded from the NCBI 

database and the full-length genes were synthesized and cloned into pcDNA3.1+ vector 

by Genewiz. Rat Scn1b and Scn4b channels are same as those described Chapter 1. I 

could not obtain rat Scn4b construct due to shortage of time. However, studies show 

that Scn2b shares structural and functional similarities with Scn4b, and likely modulates 

alpha subunit activity in similar ways (Winters & Isom 2016); therefore, I used the rat 

Scn2b gene in place of rat Scn4b for all experiments in this chapter. To synthesize 

Capped mRNA (cRNA) from both plasmid constructs, I used the mMESSAGE 

mMACHINE™ T7 Transcription Kit by ThermoFisher Scientific.  Plasmids were 

linearized at the 3’ end of the gene, past the stop codon, using NotI restriction enzyme. I 

used a nanodrop spectrophotometer (NanoDrop™ 2000, ThermoFisher Scientific) to 

quantify the synthesized cRNA. 

 

Electrophysiology 

Expressing Scn1b and Scn4b in Xenopus laevis oocytes: I purchased 

Xenopus laevis frog oocytes from Xenopus1, MI. I removed follicles surrounding 

oocytes using forceps and incubated these oocytes in ND-96 culture media as 

described in Chapter 1. Then I co-injected Nav1.4 alpha subunit cRNA from 

grasshopper mice with cRNA generated from the grasshopper mouse Scn1b and Scn4b 

construct. To compare the effects of native beta subunits versus foreign beta subunits 

	

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on the grasshopper mouse Nav1.4 activity, I compared the electrophysiological 

properties of this construct combination to: 1) grasshopper mouse Nav1.4 alpha co-

injected with rat beta 1 and rat beta 2, and 2) grasshopper mouse Nav1.4 alpha co-

injected with house mouse beta 1 and beta 2. For all construct combinations, alpha 

subunit and the two beta subunits were co-injected in a 1:1:1 ratio. All injections were 

performed as described in Chapter 1. Injected oocytes were stored at 16oC for a 

maximum of 4 days from injection.  

Recording: I used the standard two-electrode voltage clamp technique at room 

temperature to record sodium currents expressed by Xenopus oocytes after 8–48 hours 

of expression. Electrodes, solutions, recording chambers, instruments, and leak 

subtraction protocol were identical to those described in Chapter 1. Similarly, lyophilized 

crude C. sculpturatus venom extraction, storage and dilution protocols were identical to 

those described in Chapter 1. 

Data extraction and analysis: I measured voltage dependence of activation, 

voltage dependence of Na+ current decay slope, Na+ current decay constant, voltage 

dependence of steady-state inactivation and recovery from inactivation. All 

electrophysiology protocols, methods of data extraction and statistical analysis are 

similar to those described in Chapter 1.  

 

 

 

 

 

	

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RESULTS 

Sequence of genes encoding grasshopper mouse beta 1 and beta 4 subunits 

from skeletal muscle 

To determine the role of accessory beta subunits in resistance to scorpion 

venom, I addressed two questions: 1) Do the grasshopper mouse beta 1 and beta 4 

subunits differ from the beta subunits of control species (rats and house mice) in their 

effects on the grasshopper mouse Nav1.4 channels? 2) Do grasshopper mouse beta 1 

and beta 4 subunits play any role in reducing the Nav1.4 channel’s sensitivity to C. 

sculpturatus venom? To answer these questions, I sequenced the complete coding 

region of genes encoding beta 1 (Scn1b) and beta 4 (Scn4b) subunits from the skeletal 

muscles of grasshopper mice. By comparing these sequences to homologs from closely 

related species, I found that both Scn1b and Scn4b genes of grasshopper mice coded 

for interesting amino acid variants. For example, in the transmembrane segment of the 

beta 1 protein, there were four amino acid differences between the rat and grasshopper 

mouse homologues (figures not shown). One amino acid change from a polar 

uncharged asparagine (N) to another polar uncharged serine (S) at location 131 was 

unique to grasshopper mice. The beta 4 protein of grasshopper mouse differed from its 

rat homolog in eight amino acids. However, the most remarkable feature of the 

grasshopper mouse beta 4 was the presence of a unique amino acid insert located in 

the distal part of the protein’s C-terminus (Figure 9, bolded amino acids). This insert 

comprised two polar uncharged amino acids (asparagine, N, and threonine, T), a 

negatively charged glutamic acid (E), and three positively charged arginines (R). 

	

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C- terminus

C- terminus

Beta 4 
Beta 4 
O. torridus
O. torridus
Deer mouse   
Deer mouse   
Chinese hamster  
Chinese hamster  
Prairie vole     
Prairie vole     
House mouse      
House mouse      
Rat  
Rat  
Human 
Human 

SSSGNDNTENGLPGSKAEEKPPTNETRRRRH       
SSSGNDNTENGLPGSKAEEKPPTNETRRRRH       
SSSGNDNTENGLPGSKAEEKPPT------KV   
SSSGNDNTENGLPGSKAEEKPPT------KV   
SSSGNDNTENGLPGSKAEEKPPT------KV      
SSSGNDNTENGLPGSKAEEKPPT------KV      
SSSGNDNTENGLPGSKAEEKPPT------KV       
SSSGNDNTENGLPGSKAEEKPPT------KV       
SSSGNDNTENGLPGSKAEEKPPT------KV   
SSSGNDNTENGLPGSKAEEKPPT------KV   
SSSGNDNTENGLPGSKAEEKPPT------KV             
SSSGNDNTENGLPGSKAEEKPPT------KV             
SSSGNDNTENGLPGSKAEEKPPS------KV
SSSGNDNTENGLPGSKAEEKPPS------KV
 
Figure 9: A partial multi-species amino acid sequence alignment of the beta 4 

protein. Bolded region shows the C-terminus insert unique to O. torridus grasshopper 

mice.  

 

Electrophysiological properties of GaGb, GaMb and GaRb channels 

To test the influence of species-specific beta subunits on the grasshopper mouse 

Nav1.4’s alpha subunit activity and response to venom, I compared the grasshopper 

mouse Nav1.4 alpha subunit co-expressed with grasshopper mouse beta 1 and beta 4 

(referred to as GaGb) to grasshopper mouse Nav1.4 alpha co-expressed with: 1) rat 

beta 1-beta 2 (GaRb) and 2) house mouse beta 1-beta 4 (GaMb) subunits. I co-

expressed all alpha-beta combinations in Xenopus laevis oocytes and used the two-

electrode voltage clamp technique to measure their electrophysiological properties 

before and after the application of whole C. sculpturatus venom.  

 

Voltage dependence of activation 

In the absence of venom, there was a significant difference in the V50 parameters 

of GaGb and GaMb channels (2.39 mV difference; z = 5.11; P << 0.01, Table 4) but not 

GaGb and GaRb channels (0.96 mV difference; z = 1.58; P = 0.11, Table 4). These 

	

71	

data suggest that GaMb channels have a lower baseline excitability than GaGb and 

GaRb channels. However, due to small sample sizes and overlapping 1*SEs in Figure 

10A, these results must be interpreted with caution. 

Effect of C. sculpturatus venom on voltage dependence of activation: b 

toxins in C. sculpturatus venom induce a hyperpolarized shift in the voltage dependence 

of Nav1.4 activation (and V50). Because the post-venom shift in the normalized 

conductance for all channels was negligible (Figure 10B-D), I fit the post-venom data to 

a single Boltzmann equation. As expected, venom did not significantly shift the V50 

values of any of the channels (Table 4). This shows that b toxins in C. sculpturatus 

venom had a negligible effect on GaGb, GaRb and GaMb channels. 

 

	

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Figure 10: Influence of grasshopper mouse beta subunits on the voltage 

dependence of activation of grasshopper mouse Nav1.4 channels. A) Normalized 

conductance-voltage relationship for GaGb (black) and GaMb (purple) and GaRb 

(green) before venom application. Effect of 0.15 µg/µl whole C. sculpturatus venom 

(open circles) on the normalized conductance voltage relationship of: B) GaRb (green) 

C) GaMb (purple) D) GaGb (black) channels. For all data collected with grasshopper 

mouse beta subunits, n = 10, for house mouse beta subunits n = 14 and for rat beta 

subunits n = 7.  

	

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Table 4: Boltzmann parameters for normalized conductance vs. voltage 

relationships of GaGb, GaMb and GaRb channels. V50 is the voltage of half maximal 

activation, K is the slope of the relationship, P is the proportion of channels unaffected 

 

by venom and n is the number of oocytes sampled.  

 

Effect of C. sculpturatus venom on peak current: b toxins in C. sculpturatus 

venom bind with Nav channels and reduce peak Na+ currents at depolarized voltage 

steps (see Chapter 1). This effect is demonstrated in the relationship between 

normalized peak current and voltage (Figure 11A and 11B). I found that GaGb, GaMb 

and GaRb channels experienced similar levels of peak current reduction in the 

presence of venom, especially at +5 mV (Figure 11C); a two-way repeated measures 

ANOVA found no significant differences between the peak Na+ currents of GaGb and 

GaMb channels (P = 0.41; t-value = 0.41; df = 29) or GaGb and GaRb channels (P = 

0.65; t-value = -0.74; df = 29) at voltages ranging from -5 mV to +45 mV in the presence 

of venom. These data show that GaGb, GaRb and GaMb channels do not differ in their 

sensitivities to b-C. sculpturatus toxins that reduce peak Na+ current. 

	

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Figure 11: Effects of species- specific beta subunits on modulating Nav1.4 

activation and peak current in the presence of C. sculpturatus venom. All peak 

currents were normalized by pre-venom peak currents at different voltage steps. 

Voltage-current curves for: A) GaRb (green) and GaGb (black) B) GaMb (purple solid) 

and GaGb (black solid) before and after (open symbols) the application of venom. C) 

Difference between pre and post venom peak current observed at + 5 mV. All data 

represent mean ± 1SE.  

 

Fast inactivation kinetics   

a toxins in C. sculpturatus venom bind to the S3-S4 extracellular loop of DIV and 

disrupt the inactivation mechanism of Nav channels. a-toxin bound channels experience 

prolonged ion flow well beyond the normal timeframe of inactivation (typically 4-5 ms 

after activation). To determine the a-toxin effect of C. sculpturatus venom on Nav1.4, I 

applied a single depolarizing potential of 0 mV for 50 ms to oocytes expressing either 

GaGb, GaMb or GaRb channels, and then overlaid the current traces produced before 

and after the application of venom. In the absence of venom (solid lines), representative 

	

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current traces from all channels demonstrate rapid inactivation of Na+ currents (Figure 

12A). In contrast, the application of 0.15 µg/µl C. sculpturatus venom (dashed lines) 

slowed the decay of Na+ current in GaGb, GaMb and GaRb channels, suggesting a-

toxin inhibition of the fast inactivation mechanism in all three channels.  

To quantify the effects of beta subunit association on fast inactivation kinetics, I 

measured Na+ current decay constants (tau) and the slope of decay over a range of 

voltage steps (Figure 12B). A three-way repeated measured ANOVA suggested no 

baseline differences in the tau between GaGb and GaRb channels or GaGb and GaMb 

(F value = 0.293; df = 2, 29; P = 0.748). Similarly, there were no baseline differences in 

the decay slopes of GaGb and GaRb or GaMb channels (P > 0.1; df = 28, Figure 12C). 

These results suggest that species-specific beta subunits do not differentially affect 

Nav1.4 alpha subunit’s inactivation kinetics in the absence of venom. 

Figure 12: Effects of species-specific beta subunits on modulating Nav1.4 alpha 

subunit baseline inactivation kinetics: A) Representative Na+ current traces for 

GaGb (black), GaMb (purple) and GaRb (green) channels in the absence (solid line) 

 

and presence (dashed line) of venom. B) Tau plotted as a function of voltage for GaGb, 

	

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GaMb and GaRb channels. C) Na+ current decay slope for GaGb, GaMb and GaRb 

channels. 

 

Next, I applied C. sculpturatus venom to the oocytes expressing GaGb, GaMb 

and GaRb channels, and re-measured Na+ current tau and decay slope. There were no 

differences in the tau value of GaGb, GaRb and GaMb channels in the presence of 

venom (Figure 13A-B; F-value = 1.5223; df = 2, 348; P = 0.2197). Venom reduced the 

slope of Na+ current decay in all three channels (figure not shown), and the proportion 

change in slope before and after the application of venom was similar in the three 

channels (Figure 13C). A three-way repeated measures ANOVA showed that there 

were no significant differences in the proportion change in slope post venom for GaGb 

vs. GaRb (P = 0.29; df = 28; t-value = -1.078) or GaGb vs. GaMb (P = 0.38; df = 28; t-

value = 0.89). Together, these results show that grasshopper mouse beta subunits do 

not influence the interaction of Nav1.4 channels with C. sculpturatus venom in the 

Xenopus oocytes expression system. 

 

 

	

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Figure 13: Effects of species-specific beta subunits on modulating Nav1.4 alpha 

subunit’s inactivation kinetics in the presence of C. sculpturatus venom. Na+ 

current decay constant for A) GaGb (black) and GaRb channels (green). B) GaGb 

(black) and GaMb (purple) channels. C) Proportion change in post-venom Na+ current 

decay slope calculated as described in Chapter 1. All points represent mean ± 1SE. 

 

Voltage dependence of steady-state inactivation and recovery from fast 

inactivation  

There were no differences between the steady-state inactivation or recovery from 

fast inactivation of GaGb, GaRb and GaMb either in the presence or absence of venom 

(overlapping ±1SE, figure not shown).   

 

DISCUSSION  

In Chapter 1, I showed that the grasshopper mouse Nav1.4 alpha subunit has 

evolved amino acid modifications that reduced its sensitivity to C. sculpturatus venom. 

During all experiments in Chapter 1, I co-expressed Nav1.4 alpha subunits with rat beta 

	

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subunits in order to make specific comparisons between alpha subunits of grasshopper 

mice and control species. Although it is well known that beta subunits can modulate the 

gating kinetics of alpha subunits, few studies have examined the consequences of co-

expressing Nav channels with their species-specific beta subunits in heterologous cells. 

In this chapter I examined the role of grasshopper mouse beta subunits in modulating 

the grasshopper mouse Nav1.4 channel’s baseline functional properties and response 

to C. sculpturatus venom.  

In mammals, each Nav alpha subunit protein associates with up to two beta 

subunits either through non-covalent interactions (with beta 1 and beta 3) or covalent di-

sulfide bonds (with beta 2 or beta 4) (Chahine & O'Leary 2011; Winters & Isom 2016). 

These alpha-beta subunit associations are specific to different tissues and 

developmental stages. Skeletal muscles express only one Nav alpha subunit protein 

(Nav1.4) and at least two beta subunits: beta 1 and beta 4 (Goldin et al. 2000; Yu et al. 

2003; Sutkowski & Catterall 1990; Chahine & O'Leary 2011). Although their in vivo 

associations with the Nav1.4 alpha subunits are poorly understood, beta 1 and beta 4 

proteins have proven effects on the gating properties and excitability of Nav1.4 channels 

in heterologous cell lines (Makita et al. 1996; Yu et al. 2003.; Aman et al. 2009; Wallner 

et al. 1993). Using comparative sequence analyses, I found several amino acid 

differences between the beta 1 subunits of grasshopper mice versus rat and house 

mice. One amino acid change in the transmembrane segment of beta 1, N131 to S131, 

was unique to grasshopper mice. Both serine (S) and asparagine (N) are polar 

uncharged amino acids, but differ slightly in their side chains.  

 

	

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Unlike the beta 1 protein, the beta 4 protein of grasshopper mice did not carry 

any unique amino acid substitutions in its extracellular or transmembrane segments. 

However, I found a unique amino acid insert in the C-terminus region of the beta 4 

protein. Of the six amino acids in this insert, there were three positively charged 

arginines (R), one negatively charged glutamic acid (E) and two polar uncharged 

residues (asparagine, N, and threonine, T). Presence of charged amino acids in the 

insert suggests that this region could interact electrostatically with the intracellular 

components of the Nav1.4 alpha subunits, and/or modify the function of channel 

components that work from the intracellular end (e.g. the intracellular inactivation gate 

of Nav channels). Therefore, I predicted that the structural modifications in both beta 1 

and beta 4 proteins of grasshopper mice would influence their association with the 

alpha subunit and alter baseline functional properties of the channel. Additionally, there 

is evidence to suggest that beta 1 associates with Nav channels on the extracellular 

surface, and in a region that likely overlaps with scorpion a-toxin binding sites on the 

alpha subunit (Makita 1996). Therefore, my second prediction was that structural 

modifications in grasshopper mice beta subunits would result in conformational changes 

that affect the interaction of Nav channels with C. sculpturatus toxins.  

Contrary to my predictions, I found that grasshopper mouse beta 1 and beta 4 

subunits play no specific role in shaping the baseline or post-venom electrophysiological 

properties of grasshopper mouse Nav1.4 channels in the Xenopus oocyte expression 

system. These findings suggest that minor changes to the side chain of certain amino 

acids (e.g. asparagine, N to serine, S) in transmembrane segments of beta 1 do not 

affect the alpha-beta associations in significant ways.  

	

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Similarly, the presence of a unique C-terminus insert in the grasshopper mouse 

beta 4 protein does not affect the baseline properties of Nav1.4 channels or their 

response to venom. These findings were surprising because the insert is absent from 

the beta 4 sequence of closely related rodents (Figure 9), implying its association to a 

specific role in grasshopper mice. Additionally, the presence of charged amino acids in 

this region strongly indicates some physiological role. Future studies should test the role 

of the beta 4 C-terminus insert in regulating other cellular processes unrelated to gating, 

e.g., channel trafficking or subcellular localization. Alternatively, the C-terminus insert’s 

ability to modulate channel gating properties may be contingent on the presence of 

mammalian regulatory proteins or transcription factors that are absent in frog oocytes. 

To test this hypothesis, future work should express Nav channels and their beta 

subunits in a mammalian expression system (e.g. human embryonic kidney cells or 

Chinese hamster ovary cells).  

Lastly, the C-terminus of beta 4 subunits is known to regulate neuronal 

excitability though its role in open channel block or OCB (Barbosa et al. 2015; Lewis & 

Raman 2013). Although OCB has never been described in muscle tissue, it is likely that 

the beta 4 C-terminus insert of grasshopper mice has been co-opted to serve as an 

OCB. As described in Chapter 1, Nav channels inactivate within a few milliseconds of 

activation by employing an intracellular loop that acts as a hinged lid (Stühmer et al. 

1989; West et al. 1992; Armstrong 2006). Once inactivated, Nav channels require 

prolonged hyperpolarization in order to reset to “resting state” and activate again. While 

the inactivation mechanism helps prevent excessive action potential generation in most 

excitable cells, certain neurons (e.g. cerebral Purkinje cells) are required to produce 

	

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rapid trains of action potentials for normal functioning. Therefore, rapidly firing neurons 

by-pass the inactivation gate by employing the beta 4 subunit to form an OCB (Raman 

& Bean 1999; Barbosa et al. 2015). The OCB competitively binds to the docking site of 

the inactivation gate in a voltage-dependent manner and prevents further influx of Na+ 

ions. However, unlike the inactivation gate, the OCB detaches from its docking site at 

more depolarized potentials (i.e. at potentials when the channel should be refractory 

under classical inactivation conditions). The release of the OCB is accompanied by a 

resurgence of transient Na+ currents that can cause surrounding Nav channels to open 

and elicit the next action potential (Barbosa et al. 2015; Schiavon et al. 2006; Patel et al. 

2015). Therefore, my hypothesis is that the C-terminus insert in beta 4 of grasshopper 

mice enables this subunit to act as an OCB and regulate myocyte excitability when the 

channels are under attack by toxins that impair the inactivation mechanism. If this 

hypothesis is supported, it will uncover a potentially novel mechanism of a toxin 

resistance. However resurgent currents cannot be detected in the Xenopus oocytes; 

resurgent currents are fast and transient and do not last long enough to surpass the 

large capacitive currents generated in the Xenopus oocyte expression system. Future 

experiments aimed at understanding the role of the beta 4 C-terminus insert in C. 

sculpturatus toxins resistance should use a different heterologous expression system 

(e.g. Human Embryonic Kidney cells) which can detect resurgent currents.  

	

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CONCLUDING REMARKS  

The aim of my dissertation research was to understand the molecular and 

physiological mechanisms through which the muscles of grasshopper mice (Onychomys 

torridus) are resistant to lethal neurotoxins in Centruroides sculpturatus venom. My 

study is the first to sequence the complete coding region of the genes encoding the 

Nav1.4 alpha, beta 1 and beta 4 subunits from skeletal muscles of grasshopper mice. 

Using electrophysiology recordings, I show that grasshopper mouse Nav1.4 channels 

are significantly less perturbed than the sensitive Nav1.4 channels of house mice and 

rats to C. sculpturatus a and b toxin effects. Further, I could employ an interdisciplinary 

approach; by integrating techniques from molecular evolution, bioinformatics and 

molecular genetics, I identified specific amino acid modifications in three separate 

regions of the grasshopper mouse Nav1.4 alpha subunit that confer reduced sensitivity 

to C. sculpturatus venom. My work is the first to reveal that amino acids in the Nav1.4 

channel’s C-terminus can play a role in setting the channel’s activation threshold.  

Interestingly, although the amino acid modifications that confer reduced 

sensitivity are located in conserved regions of the channel, they have minimal effects on 

the channel’s baseline functional properties. My findings suggest that highly conserved 

and functionally important Nav channel proteins can be modified to encode alternative 

phenotypes without compromising overall function. Such proteins could serve as 

therapeutic targets for the design of precision medications to treat neuromuscular 

disorders.  

	

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