. a sum... n fi‘i‘zcat .. V . . , “.155... .. .:1 .v 95.3% 2: fl ‘5fi.?.§&m..§t , may». .r . I. Ag»... » .... t V 11.4.} (flu - u.\a. l 11? . THEBlS «9 4m «,1 7-. 1'? gég?oiés This is to certify that the thesis entitled REGULATION OF Cl'lHCOa' TRANSPORT AND EXTRACELLULAR pH BY CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR presented by MARIJA KRHA has been accepted towards fulfillment of the requirements for the MS. degree in Physiology /- Dyed, 3 AMA; / Major Professor’s Signature 57/2 7 /0 7/ / / / Date MSU is an Affirmative Action/Equal Opportunity Institution LlBRARY l Michigan State University PLACE IN RETURN Box to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 c I' > F' (Anderson et al., 1991b). It also has been shown that HCOa' ions pass through CFTR, even though the importance of such a function is not yet well characterized (Poulsen et al., 1994). The AF508 mutation of CFTR and consequent loss of Cl' conductance can explain the pathophysiology of many organ systems altered in CF, where reduced Cl' permeability leads to dehydrated and clogged ducts (WIne, 1991; Bear et al., 1992; Quinton, 1983; Welsh, 1990). All of the consequences leading to fatality in CF, however, have been difficult to rationalize solely as a defect in Cl' secretion (Kunzelmann, 1999). The majority of CF patients (about 90%) will die of lung disease (Welsh and Smith, 1995). This appears to be a consequence of a high bacterial infection rate as a result of the thick mucus buildup and low clearing by cilia (Welsh and Smith, 1995). This buildup of mucus and preferential infection by certain pathogens (like Staphylococcus aureus and Pseduomonas aeruginosa) has been hard to explain solely by abnormal CI' transport and is not completly understood. 2. Pathophysiology of CF The exact origin of CF is unknown, although, CF may have first occurred around 5,000 years ago in Eastern Europe and was further expanded in the population through human migration, gene mutations, and new diet conditions (Busch, 1990). Historical evidence of the disease was first recorded in folk stories and diaries in Europe as early as the 16005’ (Busch, 1990). Children with the disease did not survive early childhood and as result were often thought to be “bewitched” if they had a salty forehead. These historical documents mention both the salty forehead in children, as well as some early autopsy results which note damage observed in the pancreas (Busch, 1990). In fact, the disease is sill commonly diagnosed by excessive salt in the sweat. Clinically, however, CF affects more organs than just the skin. It also affects the lungs, liver, pancreas, small intestine and reproductive tract. Overall, the disease is Characterized by defective electrolyte transport and Clogging of ducts with thick, sticky mucus. In all organs affected in CF, mutations in the CFTR gene directly lead to abnormal CI' permeability as well as indirectly to abnormalities in Na” and HCOa' balance (Quinton, 1983; Smith and Welsh, 1992; Seidler et al., 1997; Quinton, 1999; Shumaker et al., 1999; Ballard et al., 1999; Lee et al., 1999a; Ahn et al., 2001). 2.1. Observations on the Skin The skin in CF is affected by a malfunction of sweat glands and electrolyte imbalance. This is also the only organ involved where thick mucus does not play a role (Quinton, 1999). Sweat glands in the skin normally secrete NaCl in the secretory coil of the sweat gland as an isotonic fluid (Quinton, 1999). Water, Na‘ and CI’ are transported out of the body and into the gland across the membranes of the epithelial cells lining the secretory coil. This isotonic fluid (sweat) accumulates in the lumen of the coil, and then, as the sweat travels up through the absorptive duct on a way to the skin surface, Na" and CI‘ are reabsorbed back into the body, making sweat hypotonic when it arrives at the skin surface. The reabsorption of Na+ and Cl' occurs through channels in the epithelial cells lining the absorptive duct (specifically epithelial Na+ channels, ENaC, and CFTR respectively) (Reddy and Quinton, 2003). In a healthy individual the reabsorption of CI’ is mediated through CFTR, while in CF this doesn‘t occur; the Cl’ stays in sweat. Interestingly, the conductance of Na+ through ENaC in sweat glands appears to be associated with the function of CFTR in some way because in CF the reabsorption of Na+ is also reduced (Reddy and Quinton, 2003). As a result, in diseased individuals, the lack of CFTR is manifest as an abnormally salty sweat (still isotonic) due to the defect in reabsorption in the sweat duct (Quinton, 1983). In the skin, the malfunctioning or absent CFTR Cl" channel can reasonably serve as the sole and sufficient explanation of its pathophysiology in CF. 2. 2. CF Effect in the Airway The airway epithelium consists of surface epithelium and secretory submucosal glands. It is believed that in healthy individuals Na+ and CI' are secreted out of epithelial cells in the submucosal gland and absorbed back into the cells on the surface epithelium of lung, thus maintaining proper electrolyte balance (Na+ and Cl“) and “healthy” ainrvay surface fluid (Boucher et al., 1983; Jiang et al., 1993; Smith and Welsh, 1993; Smith et al., 1994; Smith et al., 1996). Na” and CI’ are secreted by the submucosal gland as a mechanism to get water moving into the gland, which in turn will secrete salt, water and mucus. Na+ is absorbed back at the surface epithelium with Cl' and excess water passively follows. Airway surface fluid (ASF) is defined as a thin layer of liquid that covers the lung surface epithelium (Smith et al., 1996). Some research groups believe that in the disease, as a result of the lack of CFTR on the lung surface epithelium and its regulation of the Na+ channel (ENaC), Na+ absorption is increased, while Cl' absorption is decreased (Welsh and Smith 1995, Mall et al., 2004). It has been Shown that, as a result of the abnormal electrolyte balance and transport, the ASF in CF patients has increased concentrations of salt and in particular Cl‘ (Joris et al., 1993; Gilljam et al., 1989; Smith et al., 1996). In addition, in healthy individuals, ASF appears to contain the bactericidal factors known as defensin molecules (Zhang et al., 2001; Smith et al., 1996). In CF, however, it has been shown that these factors are lacking or less effective, leading to chronic airway infections (Smith et al., 1996). How these factors are inhibited in CF is somewhat controversial and currently there are two competing hypotheses. The “compositional hypothesis” focuses on the role of CFTR as a Cl' channel and its role in maintaining the normal electrolyte balance of ASF (by regulating electrolyte transport across the membrane) (Smith et al., 1996; Zabner et al., 1998). Antibacterial factors, normally contained within the ASF, are activated with low salt concentrations (Smith et al., 1996; Zabner et al., 1998). The malfunctioning CFTR and hence defective electrolyte transport will result in high salt concentration of ASF thus inactivating its bactericidal activity and leading to chronic infections (Smith et al., 1996; Zabner et al., 1998). As a side- effect, the buildup of neutrophils and macrophages in the airways in response to infection also occurs, which leads to an inflammatory environment eventually causing hypersecretion of mucus and its build up (Smith et al., 1996). On the other hand, the “low-volume hypothesis" focuses on CFTR as both a Cl' channel and a regulator of ENaC and therefore CFTR affects both Na+ and water transport (Mall et al., 2004). This hypothesis emphasizes the importance of proper ASF volume or depth to promote lung clearance by cilia and thus aid in clearance of mucus and infection. In CF patients, a decrease of Cl’ absorption at the surface epithelium leads to an increase in absorption of Na+ and consequently water (Matsui et al., 1998; Knowles and Boucher, 2002; Mall et al., 2004). As a result the ASF volume is reduced, and according to the “low-volume hypotheses,” this causes the build up of mucus. In healthy individuals, the ASF volume depth is slightly greater than the height of outstretched cilia, which aids in the free movement of cilia and therefore the mucus clearance out of the lungs (Mall, 2004). Vlfith the reduction of ASF volume and depth, cilia can not move freely and there is consequently a decreased mucus transport, and buildup of bacteria (Mall, 2004). The buildup of bacteria leads to chronic infections and recruitment of neutrophils, which can trigger goblet cell metaplasia and mucin hypersecretion (Mall, 2004). Airway pathophysiology in CF is characterized by thick, sticky mucus on the surface epithelium. Due to thick mucus, clogging of bronchial passages also occurs, leading to difficulties in breathing. Thick mucus coupled with the lack of apparent ASF antibacterial function leads to reduced clearing of normal airway mucus and build up of bacteria. CF patients often sustain infections that progressively destroy the normal functioning of lungs. About 90% of CF patients will eventually die due to lung disease infections (Welsh and Smith, 1995). The bacteria most often found to cause severe infections and/or are colonized at the time of death in the ainrvays of CF patients are Staphylococcus aureus and Pseduomonas aeruginosa (Welsh and Smith, 1995). AS opposed to the skin pathophysiology, lung disease is more difficult to explain solely due to a defect in Cl' secretion. 2. 3. CF Effect in the GI Tract The liver and pancreas of CF patients also are characterized by plugging and obstruction of ducts, which results in impairments in digestion. While the liver is affected in only a rather small portion of patients (about 5%), the pancreas is one of the more severely affected organs (Welsh and Smith, 1995). CF frequently leads to a condition termed “pancreatic insufficiency” (Welsh and Smith, 1995). Only about 10% of CF patients will escape pancreatic insufficiency, this being due to different genotypes which result in milder forms of CF (Quinton, 1999). In healthy individuals, the acini in the pancreas secrete a fluid rich in enzymes and other proteins (Berne et al., 2004). The pancreatic duct cells are mainly responsible for secretion of a HCOi-rich fluid in response to hormones such as vasoactive intestinal peptide (VIP) and secretin (Ahn et al., 2001). Proteins secreted by acinar cells are kept inactive and in solution while flowing through the ducts by this copious secretion of alkaline fluid (Quinton, 1999; Lee et al., 1999a). In CF patients there is a decrease in CI' and HC03'- dependent fluid secretion and an increase in macromolecule concentration (i.e. protein) in pancreatic secretions (Quinton, 1999; Ahn et al., 2001). When there is a reduction of anion secretion, and thus water volume, the proteins become insoluble, precipitate and clog the smaller pancreatic ducts (Quinton, 1999; Ahn et al., 2001). In most patients (about 85%), the obstruction of the pancreatic ducts and resulting lack of digestive enzymes delivered to the duodenum results in pancreatic insufficiency and necessitates consumption of supplements providing additional digestive enzymes (Welsh and Smith, 1995). Loss of HCOg' homeostasis in the pancreas of CF patients requires a more complex explanation than solely a loss of CFTR and its function as a Cl' channel. The small intestines are only affected in a small percentage of newborns (about 10%) where they are obstructed by thick stool, a condition termed meconium ileus (Welsh and Smith, 1995). Prior to the 20th century, this condition was lethal. Now, however, it can often be relieved quickly by simple procedures, but sometimes does require surgery in newborns. Many CF patients also are at risk of developing deficiencies of fat-soluble vitamins (A, D, E and K) due to malabsorption, especially those individuals with pancreatic insufficiency and/or hepatobiliary disease (Rashid et al., 1999). 2.4. CF Effect in the Reproductive Tract The reproductive tract also is often negatively affected in CF. In males, this is expressed through a condition called congenital bilateral absence of vas deferens (CBAVD) resulting in incomplete development of fine ducts (vas deferens) (Timmreck et al., 2003). This condition, thought to be the result of blockage of ductules in utero, leads to sterility, and was only recently linked to mutations in CFTR (Quinton, 1999; Timmreck et al., 2003). In females the disease is exhibited through a prevalent thick mucus secretion obstructing the cervical canal and resulting, in many cases, in infertility. 3. Molecular Biology of CF When expressed, the CFTR protein is localized in the plasma membrane of epithelial cells. CFTR belongs to the family of membrane proteins known as ATP-binding cassette (ABC) transporters (Riordan et al., 1989). ABC transporters are integral membrane proteins composed of two transmembrane domains, that in bacteria, utilize the energy of ATP to pump nutrients across the membrane (Jones and George, 2004). The amino acid sequences of the two transmembrane domains vary considerably (Jones and George, 2004). In addition, they also can be involved in the pumping of chemotherapeutic drugs from cancer cells, leading to development of drug-resistance (Jones and George, 2004). All ABC transporters contain a conserved ATP-binding cassette region, also known as the nucleotide binding domain (NBD), which is involved in ATP binding and hydrolysis (Walker et al., 1982). The NBDS of ABC transporters contain conserved Walker A (GXS/TGXGKS/T) and B (Rxwhhth) motifs as well as a C motif or signature sequence (LSXGXR/K), where the symbols “X” stands for any amino acid residue, and “h” for any hydrophobic residue (Zou and Hwang, 2001). Walker A and B motifs have been shown to be involved in ATP IO binding (Saraste et al., 1990). The C motif is thought to participate in transfer of the energy of ATP hydrolysis to the conformational change leading to transport function (Ames and Lecar, 1992). The human CFTR contains 1480 amino acid residues and has a molecular mass of ~168 kDa when fully glycosylated at two residues (Riordan et al., 1989). CFTRcontainS several structural parts typical for the family of ABC transporter; two sets of six helical membrane-spanning domains (MSD1 and MSDZ), a cytoplasmic regulatory (R) domain, and two cytoplasmic nucleotide (ATP) binding domains (NBD1 and N802) (Figure 1) (Sheppard and Welsh, 1999; Zou and Hwang, 2001). Despite conserved structural domains and three conserved sequences in NBDs, CFTR has very little similarities in function with other members of this superfamily. The opening of the channel and its function as primarily a Cl' channel, appears to be allowed only upon phosphorylation of the R domain and cleavage of ATP by the nucleotide binding domains (Sheppard and Welsh, 1999). The two MSDS are believed to form the actual channel pore (Sheppard and Welsh, 1999). The R domain and its phosphorylation by PKA allow for the activated channel, while ATP hydrolysis by NBD domains appears to control Channel gating and, hence its open cycle (Sheppard and Welsh, 1999). The unphosphorylated R domain seems to have an inhibitory effect on the channel (Rich et al., 1991). When removed experimentally, it results in a constitutively active Channel (Rich et al., 1991). The channel will open only with rising levels of intracellular CAMP and subsequent phosphorylation of R domain 11 MSD1 MSD2 PDZ domain Figure 1. CFTR Structure. CFTR belongs to the family of membrane proteins referred to as ATP-binding cassette (ABC) transporters (integral membrane proteins composed of two transmembrane domains). The human CFTR contains 1480 amino acid residues. It contains several structural parts typical for ABC transporters, two sets of six helical membrane-spanning domains (MSD1 and MSD2), a cytoplasmic regulatory (R) domain, and two cytoplasmic nucleotide (ATP) binding domains (N801 and NBDZ). The two MSD domains are believed to form the actual Channel pore. The opening of the channel is allowed only upon a rise in CAMP levels and subsequent phosphorylation of the R domain by PKA at serine and threonine residues, and cleavage of ATP by the nucleotide binding domains. The R domain and its phosphorylation by PKA allows for the activated channel, while ATP hydrolysis by NBD domains appears to control channel gating and, hence its open cycle. Once the phosphorylation of the R domain occurs, the channel open cycle is controlled by hydrolysis of ATP by NBD domains and is closed by phosphatases’ dephosphorylation of the R domain. The CFTR is said to have two open (01 and 02) and two closed states (C1 and CZ), defined by ATPs and/or ADPS bound to the NBD domains. At the C-terminal tail of CFTR (the very last three amino acids, Thr-Arg-Leu) are so called PDZ domain(s) (protein interaction domains). Proteins with PDZ domains can bind to other proteins with corresponding PDZ domains and physically influence each other’s cellular functions, hence playing an important role in cellular signaling. by PKA at serine and threonine residues (Zou and Hwang, 2001). Once the phosphorylation of the the R domain occurs, the channel open cycle is controlled by hydrolysis of ATP by NBD domains and is finally closed by phosphatase- mediated dephosphorylation of the R domain (Sheppard and Welsh, 1999). The CFTR is said to have two open (01 and 02) and two closed states (Cl and CZ), defined by ATPs and/or ADPS bound to the NBDs (Sheppard and Welsh, 1999; Zou and Hwang, 2001). The simplest model of the opening and closing of the CFTR is that upon the phosphorylation of the R domain, and hydrolysis of ATP on one of the NBD domains, the channel is in its 01 state (Sheppard and Welsh, 1999). Upon ATP hydrolysis on the second NBD domain, the Channel is in the 02 state and will soon be closed. Two closed states are defined at the release of two ADPS and binding of the first ATP molecule to the NBDI domain (C1) followed by consequent binding of a second ATP molecule to the N302 domain and phosphorylation of the R domain (CZ) (Sheppard and Welsh, 1999). Various models have been proposed to explain timing and order of these states such as phosphorylation of the R domain, release of ADP, binding of ATP and its hydrolysis, and crosstalk between the domains. The exact details of gating and conformational states, however, are still unknown. Another important structural feature of CFTR is the so called PDZ domain located at the C terminal tail of CFTR (Figure 1) (Raghuram et al., 2003; Haggie et al., 2004). The very last three amino acids of the C-terminus of CFTR (T hr- Arg-Leu) match the conserved PDZ motif found in many PDZ domain-containing proteins (Haggie et al., 2004). PDZ domains are protein interaction domains 13 located on many eukaryotic proteins (Sheng and Sala, 2001). Protein-protein interaction through PDZ domains is based on sequence specificity. They are used for intracellular trafficking of proteins and subcellular localization of protein complexes and hence, play an important role in cellular signaling (Sheng and Sala, 2001). Proteins with PDZ domains can bind to other proteins with corresponding PDZ domains and physically influence each other’s cellular functions. CFTR has been Shown to influence (both directly and indirectly) the function of other membrane transport proteins such as epithelial Na+ channels (ENaC), outwardly rectifying Cl' channels, Cl'lHCOa' exchangers, renal K+ channels (ROMK), aquaporin type III water channels (AQP3), Na*lH+ exchanger isoform 3 (NHE3), and Ca” activated Cl- channels, (Kunzelmann and Schreiber, 1999; Schwiebert et al., 1999). This influence has been suggested to be mediated through PDZ domain protein-protein interactions (Kunzelmann and Schreiber, 1999). It also has been shown that CFTR is confined to the apical membrane of epithelial cells and that its activity can be modulated through the PDZ domain interactions with the above mentioned complexes (Raghuram et al., 2003). The C-terminal tail of CFTR has been shown to interact with, and be regulated by, PDZ domains of Na‘lH+ exchanger regulatory factors (NHERF) and CAP70 (Wang et al., 2000; Raghuram et al., 2001). Malfunction of CFTR can be caused by numerous mutations in any of the above-mentioned domains, leading to various degrees of disease severity. So far, more than 1,000 mutations have been identified in CF that could lead to various defects in CFTR biosynthesis, its function on the membrane, and its 14 stability (Amaral, 2004). The most common mutation, deletion of phenylalanine at position 508 (AF508), accounts for about 70% of all cases of CF (Cheng et al., 1990). This mutation is located within the first nucleotide binding domain (Cheng et al., 1990). Cheng and colleagues were one of the first groups to report what occurs as a result of this particular mutation. They Showed that the AF508 mutation leads to a large decrease of fully glycosylated CFTR in cells (Cheng et al., 1990). They concluded that this mutation results in the protein being retained within the endoplasmic reticulum (ER) due to incomplete processing that eventually leads to protein degradation. The effect of this mutation is “incomplete processing” by the endoplasmic reticulum early in the translation process (prior to translation of the ATP binding site) and a consequent lack of trafficking to the membrane. Sato et al. (1996) later showed that this defect does not result in a dysfunctional protein if it is successfully processed to the membrane. They demonstrated the release of AF 508 CFTR protein from the ER and its correct processing to the plasma membrane when cells were treated with 10% glycerol. The protein was shown to be partially functional with the expected increase of CAMP-activated chloride current. In fact, recent findings indicate that more than 50% of the wild type (WT) CFTR translated becomes a misfolded protein and is retained within the ER and subsequently degraded (Sharma et al., 2001). The remainder of the WT protein (25—50%) will be released from ER and will undergo further glycosylation and processing in the Golgi apparatus and finally be exported to the cell membrane (Sharma et al., 2001). Glycerol treatment of cells was shown to stabilize the mature protein, and not only corrects trafficking of 15 AF508 CFTR, but also facilitates the processing of the WT (Sato et al., 1996). The stabilization defect of AF508 mutation was also shown to be a temperature- sensitive defect (Sharma et al., 2001 ). The introduction of other chemical chaperones, reduced temperature, and down-regulation of Hsp70 (a molecular chaperone) were found to partially correct the effects of the AF508 mutation (Sharma et al., 2001). Specifically, misfolded AF508 CFTR does not process properly, most likely due to association with molecular chaperones such as Hsp70/de-1 and calnexin, and as a result enters the ubiquitin-proteosome degradation pathway (Pind et al., 1994; Amaral, 2004). 4. Contemporary Views on CFTR The first direct demonstration of CF as a ‘Cl' transport defect’ was reported in 1983 (Quinton, 1983). This study reported that in isolated sweat ducts low Cl‘ permeability in ducts resulted in poor reabsorption of NaCl and high NaCl concentration in the sweat of CF patients. Most of the research done in the field of cystic fibrosis so far has been focused on this interrupted chloride transport of CFTR. It has been Shown that many CF mutations do cause this defective Cl‘ transport (Ahn et al., 2001). It also has been shown, however, that high or normal Cl' transport is maintained in several CFTR mutations which still cause mild and in some cases severe forms of CF (Ahn et al., 2001). This suggests that CFTR most likely has more roles than previously proposed by contemporary evidence of a Cl‘ transport defect. Since the first reports indicating a CI‘ transport defect as the underlying problem of CF, CFTR has been found to be a global regulator of I6 epithelial fluid and electrolyte transport influencing and/or regulating various functions including 1) Cl' secretion through Cl' channels, 2) Na” absorption through ENaC, 3) K+ secretion through Iuminal K+ channels, and 4) HCOa' secretion through CI'IHCOa' exchangers, and 5) HCOa' salvage through Na*/H' exchangers (Lee et al., 1999b; Ahn et al., 2001). The influence and role of CFTR on the many facets of epithelial fluid and electrolyte transport has been a focus of much recent research. 5. Shmuel Muallem’s Work Recently, groups from the University of Texas Southwestern and Yonsei University in Seoul, Korea, headed by Shmuel Muallem published new findings that have made dramatic impact in the field of cystic fibrosis (Choi et al., 2001a; Choi et al., 2001b). Muallem's group was interested in impaired HCOa' secretion in all CFTR expressing tissues and in CF in general, which was, and still is, poorly understood. HCOa' rich fluid secretion in these tissues is normally high (containing 100 — 140 mM of HCOg') and is important for maintenance of protein stability and/or their inactive state (for example, digestive enzymes in pancreas and mucins in other tissues) (Lee et al., 1999a). Previously it was hypothesized that HCOg’ secretion in CFTR-expressing tissues is mediated through electrogenic as well as electroneutral pathways (Lee et al., 1999a). An electrogenic pathway could involve either an unknown HCOa‘ channel or CFTR itself (lllek et al., 1997; Seidler et al., 1997). Electroneutral secretion most likely would involve a Cl'lHCOa' anion exchanger (AE) (Lee et al., 1999a). 17 In their 1999 studies, Lee and colleagues examined the regulation of H003: secretion by CFTR. In their first study, they used HEK 293 and NIH 3T3 cell lines expressing CFTR. From patch clamp and internal pH studies, they concluded that the major pathway for HCOa' secretion in these cells was through an AE that was directly regulated by CFTR (Lee et al., 1999a). They found that secretion was dependent on membrane expression and CAMP-activated confomnatlon of CFTR, independent of CFTR function as a Cl‘ conductor, and inhibited by mutations in NBDZ of CFTR. Later that year, they published another set of studies in which they evaluated the physiological importance of their previous findings(Lee et al., 1999b). CFTR regulation of CI'IHCO3' exchange was examined in cells naturally expressing high levels of CFTR in the luminal membrane (the human colonic T84 cell line, mouse submandibular glands [SMG] and pancreatic ducts) (Lee et al., 1999b). Similar findings as previously were recorded where: 1) stimulation with forskolin (a CFTR stimulator) exerted positive effects on AE in T84 cells independent of Cl‘ conductance through CFTR, and 2) in studies using SMG and pancreatic ducts isolated from transgenic mice, CFTR was found to regulate luminal AE activity but not basolateral AE’s. The luminal AE in question was not identified, but they speculated that it might be AE3 or a yet unidentified isoform. HCOa‘ homeostasis (impaired in many CFTR expressing tissues) is sustained through both HCOa' secretion and salvage mechanisms (Ahn et al., 2001). Concurrently with their studies examining CFTR control of H005 secretion, this group also was interested in HCOg' salvage mechanisms. In their 18 2001 study, Ahn and colleagues studied mouse pancreatic ducts and heterologous expression systems and hypothesized that CFTR also was involved in the control of these salvage mechanisms, specifically by influencing the activity of the Na“/H+ exchanger (NHE3) (Ahn et al., 2001). They found that CFTR control of NHE3 activity was mediated through two mechanisms: 1) by increasing CAMP dependent inhibition of NHE3, and 2) by increasing expression of NHE3 in luminal membranes of pancreatic duct cells (Ahn et al., 2001). The molecular mechanism of this control, they hypothesized, was signaled through PDZ interaction and binding of CFTR and NHE3 to one of the PDZ scaffolding proteins. This also was further supported by CFTR and NHE3 co- immunoprecipitation in PS120 cells and observation that the interaction was dependent on the C-terminal PDZ motif of CFTR (Ahn et al., 2001) In more recent work (Choi et al., 2001a) this group studied HEK293 cells transiently transfected to express various pancreatic sufficient and insufficient mutations (mutations that result in a functional pancreas and those resulting in complete pancreatic dysfunction respectively) of CF. Pancreatic sufficient mutations used were as follows: E193K, 65518, DG48V, A8006, H949Y and R1070Q; pancreatic insufficient mutations used were: I148T, G178R, R297Q, G551 D, H620Q, GQ70R, A1067T, 61244E, $1255P, and 613490 (Choi et al., 2001a). Surprisingly, they Showed that some pancreatic insufficient mutations had normal CI' transport while HCOa’ transport and pH regulation were aberrant (Choi et al., 2001a). These results showed that HCOa' transport and pH control might have an equal if not greater role than chloride transport in the pancreatic l9 pathophysiology of CF. In a follow up paper (Choi et al., 2001b), the authors hypothesized that CFTR may also be a CI'I HCOa' exchanger. These findings renewed interest in alternative defects linked to CFTR malfunction, especially dysregulation of H005 conductance and extracellular pH (pHo). 6. Working Hypothesis in This Study The primary research aims of our laboratory are to test the human cystic fibrosis transmembrane conductance regulator (CFTR) for the capacity to regulate extracellular pH. As a result of previous reports in the literature and findings in our laboratory, our working hypothesis is that the extracellular pH of the milieu surrounding CF cells (CFTR- “minus”) is more acidic than that of normal cells (CFTR+ “plus”) and that under optimum conditions a very sensitive pH probe can differentiate CF from normal cells. This study, in particular, aimed to characterize the bicarbonate transport function of CFTR. We designed an experimental approach that enabled us to closely examine the capacity of CFTR to carry bicarbonate and chloride, and to test CFTR as a possible Cl‘l HCOg' exchanger. In our data collection, we utilized microphysiometer technology to determine the importance of pH and H005 in CF. We utilized a pH biosensor to detect extracellular pH changes around CF cells in culture. This project was made feasible by a technology in the form of a silicon chip-biosensor, the microphysiometer (Molecular Devices Corporation, Sunnyvale CA), that can detect subtle changes in extracellular pH (McConnell et al., 1992). The 20 microphysiometer, a semiconducter-based instrument, detects the rate at which a living cell acidifies its extracellular environment. The device can detect changes in extracellular pH as a result of transient acid/base fluxes such as those caused by short-lived transporter activity, alterations in metabolic rate, and changes in intracellular pH (McConnell et al., 1992). We also used an ORION iodide electrode system to measure and directly record anion flux through the CFTR channel expressed in mammalian cell lines. In early 2001, our laboratory published findings using CFTR-expressing (2WT2 and 3T3WT) and non-expressing cell lines (C127, NIH/3T3) that indicated that CFTR-expressing cells alkalinize extracellular media in response to forskolin while CFTR-deficient cells (as is the case in the disease) acidify it. This could be the result of either increased base efflux or decreased acid efflux in CFTR- expressing cells (Luckie and Wm, 1998; Luckie et al., 2001). Results from our studies and from the literature suggest that the CFTR may normally be involved in significant HCOa' conductance, and that in the disease state, pH regulation is disrupted resulting in the acidification of the environment of the CF lung (Smith and Welsh, 1992; Poulsen et al., 1994; Luckie et al., 2001; Coakley et al., 2001; Tate et al., 2002; Coakley et al., 2003). Interestingly, changes in extracellular pH (pHo) in pancreatic secretions lead to premature activation of zymogens while changes of pHo of the surface fluid in the CF lung may contribute to the inactivation of natural immune defenses that occurs in CF (Smith et al., 1996). Hence, an understanding of CFTR HCOa' conductance may prove critical to 21 understanding CF lung and pancreatic disease, the lethal components of cystic fibrosis. 7. Specific Aims of the Research The purpose of this study was to characterize bicarbonate conductance through CFTR and to examine the “Muallem hypothesis” that CFTR is an anion exchanger (AE). The specific aims of my research were; 1) to characterize the Cl' channel conductance properties of CFTR in selected cell lines, 2) to examine the conditions necessary for CFTR-dependent HCOa' conductance, and 3) to test the Muallem hypothesis. My study focused on two cell lines, 2WT2 and AF508-8. Both cell lines are derived from C127 mouse mammary epithelial cells. They are stable cell lines transfected with the BPV vector carrying wild type (2WT2) and mutant CFTR CDNA (AF508-8) (Marshall et al., 1994). Direct characterization of the Cl' channel conductance properties of CFTR in our cell lines was achieved through iodide efflux experiments. We used an ORION iodide electrode to measure and record iodide (anion) flux through CFTR in transfected cells. Iodide efflux-characterized Cl' channel conductance properties and activity was assessed by monitoring the responses to inhibitors (glibenclamide, DPC and DIDS) and activators of this channel (forskolin). We assumed that the majority of iodide efflux observed was mediated by CI' channels (mainly CFTR). AE mechanisms prefer CI' over l', theoretically by a factor of ~260, thus the transport of I' through such mechanisms would be insignificant (Venglarik et al., 1990). Experimentally researchers have shown that 22 the I' does not compete with CT with AE mechanisms, further supporting this assumption (Humphreys et al., 1994). Iodide efflux was expected to be observed in both cell lines but it was also expected to be much higher in wild type cells in response to forskolin (CFTR stimulator). Examination of the conditions necessary for CFTR dependent HCO3‘ conductance were achieved through the use of microphysiometry. The microphysiometer, a semiconducter-based instrument, detects the rate at which a cell acidifies its extracellular environment. HCO3' is the cells’ most important physiological pH buffer, and thus, by detecting extracellular pH changes, we were able to interpret HCOa' movement through CFTR. Cells were perfused with physiological buffers containing various CFTR and AE activators and inhibitors (as listed above) and resulting changes in pH, were dynamically monitored. Finally, examination of the Muallem hypothesis (the third aim of my research), was achieved through the above mentioned cytosensor and iodide studies as well as by extensive literature research and review. In evaluating Muallem’s hypothesis we assumed that CFTR channel and exchanger properties, as proposed by this group, are two separate entities. We focused on three predictions proposed by Muallem’s group; 1) bicarbonate transport should be CI' dependent and stimulated by forskolin, 2) glibenclamide (a known CFTR inhibitor) should stop efflux of chloride through the channel but should not prevent the action of CFTR as an exchanger, and 3) DIDS (an anion exchanger blocker) should affect and block bicarbonate movement thorough CFTR’S anion exchanger in cells expressing CFTR. 23 MATERIALS AND METHODS 1. Cell Lines In the current studies of iodide efflux and extracellular acidification, we used three different cell lines of C127 (also referred to as C127i) mouse mammary epithelial origin. The lines were developed by Dr. Seng Cheng, supplied by Genzyme Corporation, and have been well Characterized (Denning et al., 1992). These cell lines are C127 parental cells stably transfected by the calcium phosphate precipitation method. The cells were transfected with bovine papilloma-based vector (BPV) alone, or containing the CDNA of wild type or AF508 mutated CFTR (pBPV-CFTR and pBPV-AF508 respectively) under the control of the mouse metallothionein MT1 promoter (Figure 2) (Marshall et al., 1994). Expression vectors were made by inserting a fragment from the high copy number plasmid pMT-CFTR4 containing the entire human CFTR CDNA into eukaryotic expression vector CLH3AXBPVXT-NEO (Reddy et al., 1987; Cheng et al., 1990; Marshall et al., 1994). AF508 mutant CFTR was previously constructed by oligonucleotide—directed mutagenesis into the pMT vector and was then transferred to CLH3AXBPVXT-NEO using the same procedures as in the case of wild type CFTR (Cheng et al., 1990; Marshall et al., 1994). The pBPV vectors also contained the BPV genome, neomycin resistance gene under the control of another copy of the MT1 promoter, pML (derivative of pBR322), and a 1.9-kb intron of the or- subunit of human fertility hormones (Figure 2) (Marshall et al., 24 SV40 late polyA BPV sequences ha [vs pBPV-CFTR ECO” 24,400 bp Kpnl MT promoter Kpnl MT promoter EcoRl EcoRl Pvul PML SV40 early Inn-on and polyA Figure 2. Mammalian expression vector pBPV-CFTR. The pBPV vectors were used to transfect C127 mouse mammary epithelial cells to express wild type or mutated AF508 CFTR (Marshall, et al., 1994). The vector was composed of human CFTR CDNA under the control of mouse metallothionein MT1 promoter obtained from high copy number plasmid pMT-CFTR4 (Cheng, et al., 1990). It also contained BPV genome to promote self-replication and maintenance of plasmid in C127 cells, neomycin resistance gene under the control of another copy of MT1 promoter for selection purposes in mammalian cells, pML (derivative of pBR322) for propagation and selection of plasmid in Escherichia coli, and 1.9-kb intron of the a-subunit of human fertility hormones shown that when placed at 5’ untranslated region it increases efficiency of RNA processing and cytoplasmic accumulation (Marshall et al., 1994). The C127 cells were transfected by the calcium phosphate precipitation method and cells were selected for by G418 treatment. AF508 mutant CFTR was previously constructed by oligonucleotide-directed mutagenesis into pMT vector and was then transferred to CLH3AXBPVXT-NEO using the same procedures as in the case of wild type CFTR thus obtaining pBPV-AF508 (Cheng et al., 1990; Marshall et al., 1994). 25 1994). Transfected C127 cells used in this study are denoted as BPV cells (transfected with BPV vector alone), 2WT2 cells (transfected with BPV vector containing wild type CFTR CDNA) or 508-8 cells (transfected with BPV vector containing CDNA of CFTR containing deletion of three bases resulting in absence of phenylalanine at position 508). In the current work, they will are referred to as wild type (2WT2 cells), mutant (508-8 cells) and control (BPV cells). The pBPV-CFTR and pBPV-AF508 transfected C127 cells were shown to produce high levels of recombinant human CFTR (in case of wild type cells pretreated with butyrate, the limit of production was up to 1-5 x 105 mature CFTR molecules/cell) (Marshall et al., 1994). The processing of mature, glycosylated CFTR, however, has been shown to be ineffective. Only about 40% of produced wild type CFTR was converted to the mature form and expressed as functional Cl‘ conducting Channel at the plasma membrane (Marshall et al., 1994). The rest of the produced protein was rapidly degraded in a pre-Golgi compartment. Similarly, AF508 mutated CFTR in 508-8 cells was found to be arrested in the endoplasmic reticulum and not detected at the plasma membrane (Marshall et al., 1994). 2. Cell Culture Cell lines were stored in liquid nitrogen until needed. They were thawed over the course of 5 min in the standard Dulbecco’s Modified Eagle Medium (DMEM). When cultured, they were only used up to passage number 43 (for wild type cells), 37 (for mutant cells) and 21 (for control cells). Thawed cells were 26 maintained in an incubator at 37°C in a humidified atmosphere of 95% air and 5% C02 in DMEM supplemented with 10% fetal bovine serum (F BS), 5% penicillin-streptomycin cocktail (PIS), prepared with 10,000 units/ml penicillin G sodium and 10,000 ug/ml streptomycin sulfate in 0.85% saline, and 0.2 mg/ml G418. The medium was changed every 2-3 days. Cell growth was monitored and when 95-100% confluency was reached, cells were passaged after detachment with a Ca2+- and Mg2*-free phosphate-buffered saline wash for 5 min at 37°C and by 10 min of 0.25% trypsin, 1 mM EDTA treatment at 37°C. Detached cells were passaged to either a desired flask (for further culturing), 35 mm cell culture dishes (for iodide efflux experiments) or 12 well plate inserts (for microphysiometer studies). 3. Iodide Efflux The presence of functional CFTR was assayed by CAMP-dependent activation of Cl' channels in iodide efflux experiments. We used an ORION® iodide electrode to measure and record iodide (anion) flux through CFT R in transfected cells. The C127 cells (wild type, mutant and control) were maintained in T25 flasks in a 37°C incubator in standard DMEM supplemented with 10% F BS and 5% PIS. When confluency of 100% was reached, they were trypsinized and passaged into five, 35 mm sterile polystyrene cell culture dishes and grown in the same conditions until 95-100% confluency was reached. At this point cells were treated with 0.2 mg/ml G418 and left in the incubator over night. On the day of the experiment, the cells were first incubated in 1.0 ml of 37°C efflux buffer 27 (5.4 mM KCI, 0.8 mM MgSO4, 10.0 mM HEPES, 1.0 mM NaHzPO4, 1.8 mM CaCIz, 150.0 mM NaCl and 1.0 mglml glucose) for 20 min. Efflux buffer was then removed and dishes were filled with 1.0 ml of loading buffer (5.4 mM KCI, 0.8 mM MgSO4, 10.0 mM HEPES, 1.0 mM NaHzPO4, 1.8 mM CaClz, 150.0 mM Nal and 1.0 mglml glucose). The cells were then placed in a 37°C incubator (in a humidified atmosphere of 95% air and 5% C02) for an hour to allow equilibration of sodium iodide inside and out of cells. Following the loading period, iodide efflux through CFTR was measured using the complete sample replacement technique of Venglarik et al. (1990). Drug solutions prepared in efflux buffer (EB) were: 1) 10 DM forskolin, 2) 400 (TM glibenclamide + 100 (M Diphenylamine-2—dicarboxylate (DPC) + 10 uM forskolin, 3) 500 (M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) + 10 DM forskolin, and as vehicle control for glibenclamide treatments 4) dimethyl sulfoxide (DMSO) + 10 DM forskolin (Table 1). Stock glibenclamide was diluted in DMSO and when added to EB the final DMSO concentration equaled 0.4%. For each 35 mm culture dish of cells, there were 12 measuring points including the collection of the remaining loading buffer through cell lysis at the end of the experiment. The first collection vial (wash) included the loading buffer and four washes with 1 ml EB. Following the wash, 1 ml of EB was placed on each cell dish for 30 seconds and the efflux fluid was collected (vials 2-6). VIaIS 7-11 were collected in the same way, however, they included 30 sec with 1 ml of EB with appropriate drug treatment (for experimental vials) and control. For the last treatment (at the five minute point), lysis buffer (2N NaOH), was used in order to 28 Table 1. Drug treatments andlor buffers used in iodide efflux and microphysiometer studies. The following drug treatments and/or solutions were used in studies as designated in order to characterize CFTR as channel or exchanger. Not all treatments were used in both set of studies. At times treatments were combined and used simultaneously as denoted in method section and figures representing the results. Drug treatments . . . . an d Ior buffers Function Iodide Efflux MicrophySIometry 10 uM forskolin CFTR agonist Applied Applied 400 (M glybenclamide . . . + 100 “M DPC CFTR antagonist Applied Applied Anion exchanger . . 500 uM DIDS antagonist Applied Applied Vehicle control for o 400 mM . 0.4 /o DMSO glybenclamide Applied Not used treatment To examine CFTR CI‘ free buffer as a CI‘IHCO3' Not used Applied exchanger To load cells with Loading buffer iodide the efflux . (with iodide) rate of which will App'wd N°t used then be observed To create Efflux buffer concentration . (no iodide) gradient for iodide Applied N°t used to exit cells 29 determine the amount of sodium iodide that remained in the cells. All of the previous time points could then be evaluated relative to the iodide lysis count. Iodide measurements for each vial were recorded in the next 24-48 hr using the ORION® iodide electrode (Model 9653 ionplusTM Series) following the instructions provided by the manufacturer. Before each “reading session,’ the iodide electrode was calibrated with Nal standards (10 (M, 100 uM, 1 mM, 10 mM, and 100 mM), and an ionic strength adjuster (5.0 M NaN03) was added to all samples per manufacturer’s instructions to adjust to a constant background ionic strength. Data are presented as rate constant of efflux flow at each data collection point graphed as iodide efflux rate vs. time. Rate is defined as change in iodide concentration over time. We used the following equation to calculate the rate constant: r = [ln(R1) - ln(R2)]l(t1-t2) where R1 and R2 represent the percent of iodide concentration remaining in the cell layer at times 1 (t1) and 2 (t2) respectively, as compared to iodide concentration in the lysis vial (Venglarik et al., 1990). When discussing the iodide efflux results, data are described as change in rate (iodide concentration) over time for the three different cell lines (wild type, mutant and control). 4. Measurement of Extracellular Acidification (Cytosensor Microphysiometry) 4.1. General Operating Principles The microphysiometer, a semiconducter-based instrument, detects the rate at which a cell acidifies its extracellular environment. The device can detect 3O changes in extracellular pH (pHo) as a result of transient acid/base fluxes (McConnell, et al., 1992). Cells are in diffusive contact with a semiconductor- based pH sensor, the light-addressable potentiometric sensor (LAPS) (Hafeman, et al., 1998; Parce et al., 1989) (Figure 3). The LAPS determines the pH in a Nernstian fashion (61 mV per pH unit change at 37°C) from changes in voltage and the voltage at the surface of the Chip relative to acidification (McConnell et al,1992) Two fluid paths are associated with each cell chamber (four total), and both data recording and fluid path selection are computer controlled (Figure 4). In this study, cell chambers were used in which adherent epithelial C127 cells were grown on porous polycarbonate filters and during the assay retained between this and a second microporous polycarbonate membrane (Figure 3). Extracellular acidification rates were determined as the rate of change of sensor output during periodic interruptions of fluid flow that causes transient acidifications of < 0.1 pH unit. Acidification rates were calculated using a least-squares regression of data obtained during periodic interruptions of fluid flow (40 sec) and were reported in microvolts per second (LIV/sec). One microvolt per second (uV/sec) corresponds Closely to an acidification rate of 0.001 pH unit/min (at pH 7.4). Since acidification rate varies with absolute pH, we always started the experiment at pH 7.4 and the total pH never varied by more the 0.1 pH unit for the duration of the experiment. When rates of acidification were compared across different chambers in which the absolute number of cells (and thus basal acidification rate) varied, the data were normalized to account for variation in 31 CellChamDer ; Capsule Cup Capsulelnsert O-Hn / g Spacer SensorChip CeHs LED Figure 3. Assembled microflow (sensor) cell chamber. The cells were assembled in a microflow chamber (a 1.4 ul cylindrical space of 50 um height and 6 mm diameter) in which they are in diffusive contact with a semiconductor- based pH sensor, the light-addressable potentiometric sensor (LAPS) (Hafeman, et al., 1988; Parce et al., 1989). The LAPS determines and records the extracellular acidification rates as the rate of change of sensor output during periodic interruptions of fluid flow. A two mm diameter circular region in the center of the cell capsule is used for pH recordings by a light emitting diode (LED) located underneath the sensor chamber. Cell chambers were used to grow adherent epithelial C127 cells on porous cell capsules. During the assay cells were retained between two microporous polycarbonate membranes (capsule cup and capsule insert). The cells and sensor chambers were first incubated with the running media. The spacer was then placed on the capsule cell surface and the capsule insert was placed on top of the spacer. Once the cell assemble was complete, the cell capsules were transferred to equilibrated sensor chambers with a silicon chip (pH detector) on the bottom and placed on to the recording instrument. The cell capsule was held to the bottom of the sensor Chamber by a plunger and a gantry. The plunger was used to deliver continuous fluid flow to the cell layer using microflow cylinders that perfuse cells, bringing the media from a specified source and removing it to the waste container. 32 Cell Chamber - q Pump Valve 2 Fluid Paths To f I a I Waste Figure 4. Microphysiometer fluid path. Since HCOg‘ is one of the most important physiological pH buffers, with microphysiometer technology we were able to detect HCOa‘ activity through extracellular pH changes. Cells were perfused with physiological buffers containing various stimulators and inhibitors and resulting pHo was monitored. On the day of the experiment the cells were prepared as per manufacturer’s recommendations and transferred into sensor chambers. To enhance the detection of subtle pH changes, sensor chambers were perfused with nominally bicarbonate-free medium with a low buffering capacity. Media was also degassed/debubbled and warmed to 37°C before perfusing the cells. Adherent cells were not manipulated in any way during an experiment, however pHo was continually monitored. Two fluid paths are associated with each cell sensor chamber (four total), and both data recordings and fluid path selection is computer controlled. Reservoirs of media were connected to the flow chamber by tubing in which the flow was set to 100 ul/min. Directing flow from a given reservoir was done by software driven valves, one path perfusing the cells, and the other one directly being removed to the waste. Each recording cycle lasted two min and consisted of a perfusion phase and an interruption phase. During the perfusion phase, the media was continuously passed through the chamber for an interval of 80 sec. During the next phase (interruption phase), the pumps were inactivated for 40 sec, during which the rate of acidification within the chamber was calculated, recorded and plotted by the software (rate data). The flow was then resumed and the next cycle begun, with rates of acidification returning again to baseline. Once the rate of pHo was constant drug treatments followed. 33 acidification levels between chambers. Normalization was performed by Molecular Devices’ cytosensor software “Cytosoft 2.0.1 .” For normalization,steady state acidification rates of about 10 min prior to drug application were established as baseline and rate values (uVIs) were converted to percentage (%) change. Since HCOa‘ is one of the most important physiological pH buffers, with microphysiometer technology we were able to study HCOa'flux through extracellular pH changes. Cells were perfused with physiological buffers containing various stimulators and inhibitors and resulting pHo was monitored. Buffers used were nominally bicarbonate free with a low buffering capacity (2.0 mM HEPES and ~100 uM HCO3' from atmospheric C02), with the assumption that intracellular cell production of HCOa' should create a large driving force for HCOa' efflux (in our HCOg‘ free extracellular buffer) (Luckie, et al., 2001). 4.2. Specific Experimental Procedures The C127 cells (control, wild type or mutant) were cultured in T25 flasks at 37°C incubator in standard DMEM supplemented with 10% FBS and 5% PIS (as described above). When confluency of 100% was reached, they were trypsinized with 0.6 ml of 0.25% trypsin, 1 mM EDTA solution, followed by a 1.0 ml DMEM wash and finally 0.2 ml of the resulting cell mixture was passaged into six 12-mm diameter disposable 3.0 mm porous polycarbonate cell capsules (Corning Incorporated). The cells were grown in the same conditions until 95-100% confluency was reached (about 2-3 days). Once confluency of 95-100% was 34 reached, they were treated with 0.2 mglml G418 and left in the incubator overnight. On the day of the experiment, the cells were prepared as per manufacturer’s recommendations before transfer into sensor chambers (Figure 3). The cells and sensor chambers were first equilibrated with the running medium (148 mM NaCl, 5.0 mM KCI, 1.0 mM M9012, 1.0 mM CaCI2, and 2.0 mM HEPES, 1.8 mglml glucose; pH 7.4) for about 10 min. A spacer, which defines the internal size of the cell area used for recording, was then placed on the capsule cell surface (Figure 3). A capsule insert, which traps the cells between a pair of miroporous membranes (capsule and insert) and a Spacer, was then placed on top (Figure 3). Once the cell assembly was completed, the cell capsules were transferred to equilibrated sensor chambers with a silicon chip (pH detector) on the bottom and placed on to the recording instrument. The cell capsule was held to the bottom of sensor chamber by a plunger and gantry. The plunger also was used to deliver continuous fluid flow to the cell layer (Figure 3). A two mm diameter circular region in the center of cell capsule was used for pH recordings by a light emitting diode (LED) located underneath the sensor chamber (Figure 3). To enhance the detection of subtle pH changes, sensor chambers were perfused with a nominally bicarbonate-free running media with a low buffering capacity. Although bicarbonate is not added, atmospheric CO2 will contribute to the solution. Concentration of bicarbonate can be estimated given a solubility of 0.592 ml CO2/ml water and assuming room air is 740mm Hg with 0.0314% CO2 (Brookes and Turner, 1994). The media also was degassed/debubbled and 35 warmed to 37°C before perfusing the cells. Adherent cells were not physically manipulated in any way during the experiment, but pHo was continually monitored. The perfusion media also contained drugs where indicated. Reservoirs of cell media were connected to the flow chamber by tubing in which the flow rate was set to 100 ul/min (Figure 4). Directing flow from a given reservoir was done by a software driven valve system. Each cycle lasted two min and consisted of a perfusion phase and an interruption phase. During the perfusion phase the medium was continuously passed through the chamber for an interval of 80 sec. During the interruption phase, the pumps were halted for 40 sec, during which the rate of acidification within the chamber was calculated, recorded and plotted by the software (termed rate data). The flow was then resumed and the next cycle begun, with rates [of acidification returning again to baseline. The standard protocol during an experiment is to allow all cells to stabilize prior testing. Once the rate of acidification stabilized and was constant for 10-20 min, drug treatments followed. Drug treatments used in the microphysiometer experiments were as follows: 1) 10 DM forskolin (10 min), 2) 400 uM glibenclamide + 100 (M DPC (10 min), 3) 400 (M glibenclamide + 100 uM DPC + 10 uM forskolin (10 min), 4) 500 (M DIDS (10 min), 5) 500 uM DIDS+ 10 DM forskolin (10 min), 6) Cl' free buffer (148 mM Na gluconate, 5.0 mM K gluconate, 1.0 mM Mg gluconate, 1.0 mM Ca gluconate, and 2.0 mM HEPES, 1.8 mglml glucose; pH 7.4) (28 min followed by 10 min Cl' free buffer + 10 DM forskolin and finished by another 12 min of Cl' free) (Table 1). All drugs were dissolved in 36 running media unless otherwise specified. Each time perfusing media was changed, the cells were allowed to equilibrate until rate measurements become constant. Following completion of an experiment, rate data was normalized (10 min prior to drug treatment of interest) and data was exported to spreadsheet software Microsoft Excel, 2001 (Microsoft Corporation). The data was plotted as normalized acidification rate vs. time, and any differences in the normalized acidification rates in various cells (control, mutant and wild type) were determined. 5. Statistical Analysis Except where noted, all data are reported as mean :I: SEM. Statistical significance was assessed by performing two-tailed Student’s t-tests as applied in Microsoft Excel. When dealing with microphysiometer data, the notation “n=x(y)” was used, where “x” stands for traditional trial number and denotes the number of cell capsules used in a single experiment. The “y” stands for number of multiple replications performed on a single cell capsule for the duration of a Single experiment. For example n=2(4) would indicate two different cell capsules, each tested twice for a same condition. Multiple curves of normalized acidification rate obtained through the multiple replications would then be averaged to represent a response from a cell capsule under a certain condition. For all microphysiometer data, each capsule was represented as the mean of all trials, however, only the number of capsules tested was used as a trial number in statistical tests. In iodide efflux studies, data was obtained from 35 mm cell 37 culture dishes and each dish of cells was used as a single trial number. The data were again represented as average curves of all trials for a Single experimental condition. 38 RESULTS 1. Iodide Efflux In testing the effect of inhibitors (glibencamide and DPC) and an activator (forskolin) of CFTR as well as an inhibitor of anion exchangers (DIDS), studies using iodide (anion) efflux were performed (Table 1). As described above, cells were grown in 35 mm cell culture dishes and left in an iodide-containing buffer to allow for equilibration of extracellular and intracellular iodide concentrations. Following the loading period, the cells were removed from the incubator and iodide efflux through CFTR was measured using the complete sample replacement technique of Venglarik et al. (1990). 1.1. CF TR Activation Increased Iodide Efflux Rates The cAMP elevating agent, 10 uM forskolin, elicited a significant increase in iodide efflux in wild type cells (transfected with wild type CFTR), when compared to mutant (transfected with AF508 CFTR) and control (vector transfected) cells (Figure 5). Neither mutant or control cells exhibited any Significant increase in iodide efflux (Figure 5). Forskolin activates CFTR by causing an increase in [CAMP];, thereby activating the PKA pathway and (presumably) phosphorylating the R domain of the CFTR protein (Rommens et al., 1991). Looking at iodide efflux rate (change in concentration over time) of wild type cells before stimulation (at 2.5 minutes) and at the second time point during forskolin stimulation (at 3.5 minutes), 10 DM forskolin caused about 3.5 39 # -——0-- BPV IOuM forskolin(n=6) .7 .I 0 s —-O— 2WT2 10quorskoIin(n=20) I + 5(B-8 IOuM forskolin (n=20) 0.6-l 05... .° :3 9 U l Iodide Efflux Rate 0.2 . T 0.1-I lOuM forskolin T I T T I I l 1.5 2 2.5 3 3.5 4 4.5 Time (min) vi-I 5.5 Figure 5. Effect of 10 (M forskolin treatment on iodide efflux rate of control cells transfected with vector alone (BPV), and cells stably expressing wild type CFTR (2WT2) or AF508 mutant CFTR (508-8). When compared to mutant and control cells, 10 DM forskolin (cAMP elevating agent) exposure elicited a rapid and Significant increase in iodide efflux in wild type cells. Forskolin caused a total change in rate (change in concentration over time) of 0.42 (wild type), 0.07 (mutant) and -0.01 (control) in between 2.5 minutes (before stimulation) and 3.5 minutes (second time point of forskolin treatment). Three and half fold rapid increase in rate of iodide efflux indicated an increase in CFTR activation in wild type cells, and lack thereof, in both mutant and control cells. Each point represents an average of 20 experiments for wild type and mutant cell lines and six experiments for the control cell line (* P < 0.01 compared to 508-8; # P < 0.01 compared to BPV; error bars as SEM). fold increase in efflux rate and yielded a change in rate of 0.42 (Figure 6). Wild type cells not receiving forskolin stimulation had a Slight increase between those same points, causing a non-significant change in rate of 0.08 (Figure 6). This minor drift in iodide efflux (observed in non-stimulated cells) was, however, significantly different from the much higher forskolin-elicited response. By contrast, forskolin caused only a slight increase in iodide efflux from mutant cells (equaling to a total of 0.07 change in rate between 2.5 and 3.5 minute points) and no change in control cells (Figure 7; Figure 8). The iodide efflux rate of mutant cells stimulated with 10 DM forskolin was comparable to the background noise level of non-stimulated wild type cells (Figure 6; Figure 7). Similarly, mutant and control cells that did not receive forskolin stimulation did not have any significant increase in efflux (Figure 7; Figure 8). 1.2. Anion Exchanger (AE) Inhibition Does Not Decrease Iodide Efflux Rates Simultaneous treatment of wild type cells with 500 uM DIDS (AE inhibitor) and 10 pM forskolin resulted in a similar mean iodide efflux response to that seen with forskolin alone (Figure 9A). This time the efflux increased about 6.5 fold when compared to the pretreatment state (in between 2.5 and 3.5 minutes) and yielded a change in rate of 0.46 (Figure 9A). The mean increase in efflux rate of wild type cells treated with both DIDS and forskolin was higher (though not Significantly different) than the mean increase in wild type cells treated only with forskolin (3.5 fold increase) (Figure 9A). Due to an observed unequal pretreatment iodide efflux rate, the figure was redrawn representing only those 41 —D— 2WT2 no stimulation (n=6) 0.7 -I —O— 2WT210quorskolin (n=20) -—-I=III= 0.6- 0.5.. 3 0.4. 5 E .. 0 1. § 0.3- . .. I 0.2 - T 1 ' 1 l 4 T 2 1 J- .L o l-l lOuM forskolin 0'0 T l r I r I T fi I I5 2 2.5 3 3.5 4 4.5 5 5.5 Time (min) Figure 6. Effect of 10 uM forskolin treatment on iodide efflux rate of cells stably expressing wild type CFTR (2WT2). Treatment with 10uM forskolin elicited a rapid and significant increase in iodide efflux in wild type cells thus indicating functional and active CFTR. Comparing the time points (t) prior to stimulation (t=2.5) with the second time point during forskolin stimulation (t=3.5) a total change in rate of 0.42 (3.5 fold increase) was observed. Cells not receiving any drug stimulation had a slight increase between those same points, causing a total change in rate of efflux of 0.08 that was deemed insignificant and part of background level. Each point represents an average of 20 experiments for 10 (M forskolin treatment and six experiments for control studies with efflux buffer alone (* P < 0.05; # P < 0.01; error bars as SEM). 42 0 7 —D— 538 no stimulation (n=6) —0— SIB-8 IOuM forskolin (11:20) 0.6.. 0.5-1 0.4- 0.3 -I Iodide Efflux Rate 0.2-I 0.1.l lOuM forskolin UI-I I 1 I 2.5 3 3.5 4 4.5 Time (min) in N— 5.5 Figure 7. Effect of 10 (M forskolin treatment on iodide efflux rate of cells stably expressing AF508 mutant CFTR (508-8). When compared to cells not receiving treatment, application of 10 (1M forskolin did not cause any significant change in rate of iodide efflux. The rate of efflux varied over time but did not indicate functional membrane expressed CFTR. Background drift was actually comparable to un-stimulated wild type cells. Each point represents an average of 20 experiments for 10 DM forskolin treatment and six experiments for control studies with efflux buffer alone (error bars as SEM). 43 0.7 .. —O— BPV no stimulation (n=6) —0— BPV IOuM forskolin (n=6) 0.6- 0.5- .° 5 1 .° u 1 Iodide Efflux Rate 0.2 -l IOuM forskolin “- 315 1 4.5 Time (min) 5.5 I; N-I N UI t» Figure 8. Effect of 10 jiM forskolin treatment on iodide efflux rate of cells transfected with vector alone (BPV). Treatment of control cells with 10 (M forskolin did not cause any significant Change in rate of iodide efflux. The rate of efflux remained steady for the duration of the experiment (almost identical with un-stimulated control cells) indicating no functional membrane expressed CFTR. Each point represents an average of six experiments (error bars as SEM). fl —n-— 2WT2 IOuM forskolin (0:20) 0.7. 0'7 ‘ B —o—— 2WT2 SOOuM DIDS + IOuM forskolin (n=6) 0.6- 0.5- 0.6- 0.4- T 0.3 .. 0.2 3 l T .I. 0-5" 0.1 ~ 0 i r T I T T T T 'l 3 I l 5 2 2.5 3 3.5 4 4 5 5 5 5 :1 0.4- - g -\ . f I g 0.3 ~ ' 0.2 3 T T l I. l T l L .. 'r 0.i - _ A SOOuM DIDS + IOuM forskolin IOuM forskolin 0 I I T I I I I I I I5 2 2.5 3 3.5 4 4.5 5 5.5 Time (min) Figure 9. Effect of 500 (M DIDS and 10 DM forskolin treatment on iodide efflux rate of cells stably expressing wild type CFTR (2WT2). (A.) 500 (TM DIDS (an AE inhibitor) and 10 uM forskolin treatment of wild type cells resulted in an almost identical iodide efflux rate as that elicited by treatment with 10 (M forskolin alone. The increase in rate was about 6.5 fold as compared to the pretreatment state (in between t=2.5 and t=3.5 minutes) and yielded a change in rate of 0.46. The increase in efflux rate of wild type cells treated with both DIDS and forskolin was higher (though not statistically significantly different) than in cells treated only with forskolin (mean 3.5 fold increase). Each point represents an average of 20 experiments for 10 (TM forskolin treatment and Six experiments for studies with 500 (TM DIDS and 10 0M forskolin (error bars as SEM). (8.) Due to differences in iodide efflux rates prior to drug treatment, a graphical comparison was created representing only those experiments performed Simultaneously. The iodide efflux rates of the treatments were more similar, indicating no difference between the two treatments. The pretreatment state paralleled each other as well. Each point represents an average of six experiments (error bars as SEM). 45 experiments performed Simultaneously (Figure 98). The iodide efflux rates of the treatments were even more similar when presented in this fashion, indicating no difference between the two treatments. The pretreatment states paralleled each other as well. Mutant cells, on the other hand, did not Show any significant change in iodide efflux as a result of treatment with 500 uM DIDS and 10 DM forskolin (Figure 10). Both sets of mutant cells (receiving DIDS and forskolin or only forskolin) did exhibit a small, steady increase in iodide efflux rate over time, which was found to be significantly different from each other (Figure 10). The efflux rate of these cells increased by approximately two fold for the duration of the entire treatment (2.5 to five minutes) (Figure 10). 1.3. CF TR Inhibition Reduced Iodide Efflux Rates Application of 400 uM glibenclamide and 100 uM DPC (CFTR inhibitors) along with 10 DM forskolin, decreased iodide efflux in wild type cells indicating, at least partial CFTR inhibition (Figure 11). Glibenclamide and DPC reduced the increases in iodide efflux elicited by forskolin to only about 1.5 fold (50%) in wild type cells, causing a total change in rate of 0.12 (Figure 11). Due to data variations, however, when compared to a 3.5 fold (350%) increase in rate of efflux caused by forskolin treatment alone, reduction of increase in efflux rate of cells treated with CFTR inhibitors was found to be insignificant (p=0.06). The same treatment in mutant cells did not cause any significant change or difference in the overall rate of efflux in either cells treated with forskolin alone, or treated with both forskolin and glibenclamide and DPC (Figure 12). Both treatments did, —D-— 5(8—8 IOuM forskolin (n=20) 0-7 - -—o—- SIB-8 SIDUM DIDS + IOuM forskolin (n=6) 0.6 - 0.5- # 3 g 0.4-1 g S T . T 0.2-4 l 'L . 0.1-I SOOUM DIDS + IOuM forskolin IOuM forskolin 0 T i T T F T i I .5 2 2.5 3 3.5 4 4.5 5 5.5 Time (min) Figure 10. Effect of 500 (M DIDS and 10 DM forskolin treaunent on iodide efflux rate of cells stably expressing AF508 mutant CFTR (508-8). Mutant cells did not Show a sudden increase in iodide efflux as a result of treatment with 500 TM DIDS and 10 DM forskolin as did wild type cells (Figure 9). Both sets of mutant cells (receiving DIDS and forskolin or only forskolin) did exhibit a parallel, steady increase. Even though the differences in mean efflux rates were statistically significant, the efflux rates increased equally, by only two fold for the duration of the entire experiment (2.5 to five minutes) in both drug treatments. Each point represents an average of 20 experiments for 10 (TM forskolin treatment and six experiments for studies with 500 TM DIDS and 10 uM forskolin (* P < 0.05; # P < 0.01; error bars as SEM). 47 —D— 2WT2 IOuM forskolin(n=20) 0'7 '1 —o— 2WT2400uM glib lOOuM DPC IOuM for (n28) 0.6 '1 0.5 .. Iodide Efflux Rate .__ 4000M glib 1000M DPC IOuM forskolin IOuM forskolin i I I l I l I I5 2 2.5 3 3.5 4 4.5 Time (min) Mu: 5.5 Figure 11. Effect of 400 M glibenclamide, 100 pM DPC and 10 pM forskolin treatment on iodide efflux rate of cells stably expressing wild type CFTR (2WT2). Application of 400 TM glibenclamide, 100 pM DPC (CFTR inhibitors), and 10 TM forskolin decreased iodide efflux in wild type cells, indicating partial CFTR inhibition. Treatment with CFTR inhibitors and forskolin caused a maximum 1.5 fold (50%) increase in efflux rate of wild type cells causing a total change in rate of efflux of 0.12 (between t=2.5 and t=3.5). Due to large data variations, when compared to a 3.5 fold (350%) increase in rate of efflux caused by forskolin treatment alone, the mean increase in efflux rate was not found to be significant (p=0.06). Each point represents an average of 20 experiments for 10 pM forskolin treatment and six experiments for studies with 400 ttM glibenclamide, 100 pM DPC and 10 pM forskolin (error bars as SEM). 48 —D— 5(B-8 IOuM forskolin (n=20) 0.7-4 -—0— 5(8-8400uM glib lOOuM DPC IOuM for (n28) 0.6- 0.5.. 0.4- lodide Efflux Rate 0.3. 0.2-4 0.1.4 400uM glib lOOuM DPC IOuM forskolin IOuM forskolin r I i I I T 2.5 3 3.5 4 4.5 5 5.5 Time (min) Figure 12. Effect of 400 (TM glibenclamide, 100 “M DPC and 10 pM forskolin treatment on iodide efflux rate of cells stably expressing AF508 mutant CFTR (508-8). Treatment of mutant cells with 400 MM glibenclamide, 100 pM DPC (CFTR inhibitor cocktail) and 10 TM forskolin did not cause any significant change in the overall rate of efflux in either cells treated with forskolin alone, or treated with inhibitor cocktail and forskolin. Both drug treatments did exhibit a parallel, steady increase for the duration of the experiment (in between t=2.5 and t=5). Glibenclamide, DPC and forskolin treatment caused only about a 1.7 fold (70%) increase in the rate of efflux, while forskolin treatment alone caused about a two fold increase (100%). Their differences in rates were not overall statistically significant (p=0.24 at t=3; p=0.02 at t=3.5; p=0.13 at t=4; p=0.06 for at t=4.5). Each point represents an average of 20 experiments for 10 TM forskolin treatment and Six experiments for studies with 400 pM glibenclamide, 100 TM DPC and 10 (M forskolin (error bars as SEM). 49 however, cause a steady increase in efflux rate for the duration of the experiment (1.7 fold increase with forskolin alone and two fold increase with inhibitors and forskolin) (Figure 12). No significant difference between the two treatments was observed. If CFTR inhibition occurred, wild type cells treated with CFTR inhibitors (400 TM glibenclamide and 100 (M DPC) and the stimulator (10 pM forskolin) were expected to behave similarly to mutant cells treated with a stimulator. A significant difference was, however, observed between the two treatments. Wild type cells exhibited a rate of efflux increased by about 50%, causing a change in rate of efflux by 0.12, while mutant cells showed a change in rate of only 0.07, thus again indicating only partial inhibition of CFTR (Figure 13). 1. 3.1 Vehicle Controls Studies for CF TR Inhibition A last set of iodide efflux studies were carried out as a vehicle control for 0.4% DMSO. Studies were performed where cells were treated with 0.4% DMSO and forskolin and compared to DMSO treatment alone. In wild type cells, 0.4% DMSO and 10 1.1M forskolin treatment caused a significant increase in iodide efflux rate, increasing 5.5 fold (causing a 0.87 increase in change of rate) (Figure 14). The same DMSO and forskolin treatment in mutant cells did not cause any significant change and remained rather steady for the duration of the experiment (Figure 15). When compared to the wild type treatment it was significantly lower (Figure 16). The 0.4% DMSO treatment alone did not cause any significant change in iodide efflux rate in either wild type or mutant cell lines (Figure 14; SO —O— 2WT2 400uM inb ImuM DPC IOuM for (n28) 0.71 ——D— 508-8 IOuM forskolin (n=20) 0.6-4 0.5-1 0.4- lodide Efflux Rate 0.3 .. 0.2- 4OOUM glib lOOuM DPC IOuM forskolin IOuM forskolin I I I IS 2I 2.5 3 3:5 4 4.5 Time (min) "'1 5.5 Figure 13. Comparison of CFTR inhibition in cells stably expressing wild type CFTR (2WT2) with CFTR activation in cells stably expressing AF508 mutant CFTR (508-8). Vlfild type cells treated with CFTR inhibitors (400 pM glibenclamide and 100 TM DPC) and stimulator (10 (TM forskolin) exhibited an increase when compared to mutant cells treated with forskolin. Wild type cells exhibited a rate of efflux increased by about 50% and causing a change in rate of efflux by 0.12, while mutant cells Showed a change in rate of only 0.07 (between t=2.5 and t=3.5). If complete CFTR inhibition had occurred, it would be expected that these two treatments would elicit the same results. Each point represents an average of 20 experiments for mutant cells and eight experiments for wild type cells (* P < 0.05; error bars as SEM). 51 "1 i T —o— 2WT2 + DMSO (n=6) -—O- 2WT2 + DMSO + IOuM forskolin (n=6) 0.9a 0.8.. 0.7.. 0.6.. 0.53 Iodide Efflux Rate 0.4., 0.3-4 0.2-I 0.1- 5.5 Figure 14. Effect of 0.4% DMSO and 10 uM forskolin treatment on iodide efflux rate of cells stably expressing wild type CFTR (2WT2). DMSO studies were performed as a vehicle control for CFTR inhibitor experiments. Wild type cell treatment with 0.4% DMSO and 10 pM forskolin caused a significant increase in iodide efflux rate of 5.5 fold (0.87 increase in rate of efflux) in between t=2.5 and t=3.5. This forskolin-elicited response was higher than the 3.5 fold increase observed in wild type cells that did not receive DMSO (Figure 6.) The 0.4% DMSO treatment alone did not cause any significant change in iodide efflux rate. Each point represents an average of six experiments. (# P < 0.01; error bars as SEM) 52 —D— 5(B-8 DMSO (n=6) —0— 508-8 DMSO + IOuM forskolin (n=6) 0.9.. 0.8. 0.7-I 0.6.. 0.5. Iodide Efflux Rate 0.4... 0.3- 0.2 - 0H DMSO + IOuM forskolin DMSO I I5 2l 2.5 3 315 4 4.5 Time (min) “'1 5.5 Figure 15. Effect of 0.4% DMSO and 10 [1M forskolin treatment on iodide efflux rate of cells stably expressing AF508 mutant CFTR (508-8). Mutated cells treated with 0.4% DMSO and 10 TM forskolin did not exhibit any significant change in rate of iodide efflux for the duration of the trial. No significant change in efflux was noted with mutant cells treated with 0.4% DMSO alone. Each point represents an average of Six experiments (error bars as SEM). 53 T —o— 2WT2 DMSO + IOuM forskolin (n=6) —0— 508-8 DMSO + IOuM forskolin (n=6) 0.9.1 0.8- 0.7—I 0.6.. 0.5.. Iodide Efflux Rate 0.4.. 0.3 .I 0.2- I uh 0.1-i DMSO + IOuM forskolin U I I I l I I 1.5 2 2.5 3 3.5 4 4.5 s 5.5 Time (min) Figure 16. Comparison of CFTR stimulation with 0.4% DMSO and 10 P." forskolin treatment in cells stably expressing wild type CFTR (2WT2) and AF508 mutant CFTR (508-8). V\fild type cells treated with 0.4% DMSO and 10 11M forskolin exhibited a Significant increase in iodide efflux rate of 5.5 fold. The same treatment in mutant cells did not cause any significant change in efflux rate. Each point represents an average of six experiments (# P < 0.01; error bars as SEM). 54 Figure 15). 2. Extracellular Acidification Rates The microphysiometer was used to detect changes in extracellular pH (pHo) as a result of alterations in metabolic rate, changes in intracellular pH and transient acid/base fluxes such as those caused by Short-lived transporter activity. In our studies, cells were perfused with physiological buffers containing various stimulators and inhibitors of CFTR and anion exchangers (glibenclamide, forskolin, Cl’ free medium and DIDS). The resulting pH, was monitored via microphysiometer technology to characterize bicarbonate conductance through CFTR and to examine the possibility of CFTR serving as a CI'IHCOg' exchanger (Table 1). 2.1. CF TR Activation Decreases Extracellular Acidification Rates It has been shown that forskolin causes increased extracellular alkalinization of CFTR-expressing cells and increased extracellular acidification of CFTR-deficient control cells (Luckie et al., 2001). In the current studies, we compared acidification rates of cells transfected with vector alone (control), and of cells stably expressing wild type CFTR or mutated CFTR (wild type and mutant, respectively). A ten-minute exposure to 10 TM forskolin caused decreased acidification rate in wild type cells when compared to mutant and control cells (Figure 17). An initial increase in acidification rate for mutant (90%), wild type (35%) and control cells (35%) was observed. Four minutes following 55 210 # + 2WT2 IOuM forskolin n=26(39) 200- I —o— BPV IOuM forskolin n=13(20) -—D— 5(B-8 IOuM forskolin n=26(38) I90 - '80-I § 170. 3 a 160.. E O 2: § 150. 5.: E T T < 140% l T E. I - r a 130-: T T T l T E ° T it Z 120- . i104 Toot IOuM forskolin 90 T I I I r I T 0 5 IO I5 20 25 30 35 Time (min) Figure 17. Effect of 10 TM forskolin treatment on normalized acidification rates of control cells (BPV) and cells stably expressing wild type CFTR (2WT2) or AF508 mutant CFTR (508-8). A ten-minute exposure to 10 TM forskolin caused decreased acidification rate in wild type cells when compared to mutant and control cells. An initial increase in acidification rate for mutant (90%), wild type (35%) and control cells (35%) was observed. Four minutes following the exposure to forskolin, acidification rate for mutant cells was reduced to 50% compared to the pretreatment state. Acidification rate remained high for the duration of the treatment and further increased to about 65%. Acidification rate of control cells continued to rise to final level of 45%. Acidification rate of wild type cells was reduced to approximately 25% and was significantly lower than the other two cell lines for the duration of the forskolin exposure. Even after the forskolin washout, acidification rate of all three cell lines remained separated from each other. Each point represents an average of n=26(39) for wild type cells, n=26(38) for mutant cells, and n=13(20) for control cells (* P < 0.01 compared to 508-8 and BPV; # P < 0.01 compared to 2WT2 and BPV; “ P < 0.01 compared to 508-8 and 2WT2; - P < 0.01 compared to 508-8; error bars as SEM). 56 the exposure to forskolin, acidification rate for mutant cells was reduced and was now increased by only 50% compared to the pretreatment state. Acidification rate in these cells remained higher for the duration of the treatment and was even further increased to a final value of about 65%. Acidification rate of control cells increased and continued to rise to a final 45% increase, while the rate in wild type cells decreased and was significantly lower than the other two cell lines for the duration of forskolin exposure. Wild type cells' final acidification rate increase, as compared to the pretreatment state, was only 25%. They exhibited a distinctly different acidification rate from both control and mutant cell lines. Even after the forskolin washout, acidification rate of all three cell lines remained different from each other. 2. 2. AE Inhibition Decreases Extracellular Acidification Rates A general anion exchange (AE) inhibitor DIDS was tested for its ability to inhibit CFTR. Treatment with 500 (TM DIDS alone in wild type cells resulted in decreased acidification rate by about 25% (Figure 18). When this treatment was combined with 10 (M forskolin, the effect was amplified yielding a 55% decrease in acidification rate, suggesting a still functional HCOa' efflux (Figure 19). DIDS treatment alone in control cell lines also elicited a decrease in acidification rates, by as much as 150% (Figure 18). The same treatment in mutant cell lines caused a less consistent reponse, however, it did exhibit an overall pattern of decreased acidification rate (Figure 18). When DIDS was combined with 10 (1M forskolin treatment, mutant and control cell lines showed decreased acidification rates in 57 300 + 2WT2500uMDlDSn=4 —-O—- BPV SWuM DIDS n=5(9) 250- —o— mmM DIDS n=4 200.. I ISO-1 - I A; -:.:‘.—.-.—— ‘ ./ I '—'—:‘._° § l00 . . . 1k * # # - . ° ‘1' ' E _ \i T I T .l I l a 5,- ‘ 1 .. I . o ,l 2: " ‘1 i o- - 2 50., . o g IOO-i 1 ‘. l O . Z .L -150. -200.l -250 « SOOuM moi '300 I 1 I I I r T I 0 5 I o l 5 20 25 30 35 40 Time (min) Figure 18. Effect of 500 [M DIDS treatment on normalized acidification rates of control cells (BPV) and cells stably expressing wild type CFTR (2WT2) or AF508 mutant CFTR (508-8). Treatment with the general AE inhibitor (500 (M DIDS) in wild type cells resulted in decreased acidification rate by about 25%. In control cells, the same treatment yielded a decrease in acidification rate of 150%. The same treatment in mutant cell lines caused a less consistent reponse, however, it did exhibit an overall pattern of decreased acidification rate. Each point represents an average of n=4 for wild type and mutant cells, and n=5(9) for control cells (# P < 0.05 compared to BPV; * P < 0.05 compared to 508-8; error bars as SEM). 58 34 + 2m 500uM DIDS + new for n=8 ---0— BPV WM DIDS + IOuM for n=4(7) 300 + 5(8-8 SCXMM DIDS + IOuM for n=7 280- 32m 260- 240. 220. 200- I 80- I60- I40- Normalized Acidification Rate (%) SOOuM DIDS + lOuM forskolin I I I I I I I r 10 15 20 25 3o 35 4o 45 50 Time (min) 0 'JI-i Figure 19. Effect of 500 pM DIDS and 10 pM forskolin treatment on normalized acidification rates of control cells (BPV) and cells stably expressing wild type CFTR (2WT2) or AF508 mutant CFTR (508-8). Treatment with the AE inhibitor, 500 (M DIDS, in wild type cells resulted in a decreased acidification rate of about 25% (Figure 18). However, when this treatment was combined with 10 uM forskolin the effect was amplified, yielding about a 50% decrease in acidification rate, thus suggesting still functional forskolin stimulated HCOa' efflux. Simultaneous DIDS and forskolin treatment in mutant and control cell lines also resulted in decreased acidification rate in the range of about 50-70%. Each point represents an average of n=8 for wild type cells, n=7 for mutant cells, and n=4(7) for control cells (# P < 0.05 compared to 2WT2; * P < 0.01 compared to 508-8; error bars as SEM). S9 the range of about 50-70% (Figure 19). Overall, there was not much significant difference observed between acidification rates in different cell lines (wild type, mutant and control) in any of the DIDS studies (Figure 18; Figure 19). 2. 3. CF TR Inhibition Increases Extracellular Acidification Rates We used 400 (M glibenclamide and 100 (M DPC (commonly used CFTR inhibitors) along with 10 (M forskolin to test their ability to inhibit CFTR and therefore bicarbonate efflux, thus affecting the pHo. If CFTR is inhibited, we would expect to see an increase in acidification rates. The simultaneous inhibitor and stimulator treatment yielded about a 60-120% increase in acidification rate in all three cell lines (60% for wild type, about 90% for control and 120% for mutant cell lines) (Figure 20). When compared to only 20-25% increase in acidification rates of wild type cells with only forskolin treatment, these results (60% increase) indicate inhibition of CFTR in wild type cells. Treatment with 400 (M glibenclamide and 100 uM DPC alone in all three cell lines elicited similar results as a combined treatment with forskolin (45-100% increase in acidification rates), supporting previous indications of CFTR inhibition (Figure 21). There was no significant difference in any of the three cell lines in both studies involving 400 uM glibenclamide and 100 (M DPC (Figure 20; Figure 21). 2. 4. Cf Free Buffer and CF TR Activation Significantly Reduce Extracellular Acidification Rates Our final studies monitored acidification rates of cells in CI' free media with and 60 375 + 2WT2 4(DuM glib quM DPC IOuM for n=lO —-0-—- BPV W glib imuM DPC iOuM for n=4(8) —D— SIB-84WM glib lmuM DPC IOuM for n=9 350‘ 325- 300- 275- 250 .. 225.. 200- I75. lSO. 125- IOO > 75. 50‘ Normalized Acidification Rate (%) 25- ~25-l -50- -75~ 400m glib lOOuM DPC + lOuM forskolin 1 -IOO I I j I I 0 5 l0 IS 20 25 30 35 4O 45 Time (min) Figure 20. Effect of 400 ullll glibenclamide, 100 uM DPC and 10 (1M forskolin treatment on normalized acidification rates of control cells (BPV) and cells stably expressing wild type CFTR (2WT2) or AF508 mutant CFTR (508-8). Treatment with 400 (M glibenclamide and 100 (M DPC (CFTR inhibitors) along with 10 (M forskolin was found to cause about a 60-120% increase in acidification rate in all three cell lines (60% for wild type, and about a 90—120% for control and mutant cell lines respectively). When compared to only a 25-35% increase in acidification rates with just forskolin treatment, this increased response indicates inhibition of CFTR in wild type cells. There were no sustained differences among any of the three cell lines. Each point represents an average of n=10 for wild type cells, n=9 for mutant cells, and n=4(8) for control cells (# P < 0.01 compared to BPV; * P < 0.05 compared to 508-8; error bars as SEM). 61 400 + 2WT2400uMglib100uMDPCn=4 350. —-0— BPV 400uM gliblOOuM DPC n=2(4) —-O— Sin-SngibIOOuMDPCM 300- 250. 53200. 8 I a g oo __‘=:' I . . . ,7 ,- 5% 7 - g < 50. 1 ul- 1- z '50- “D l l " " ' 'IOO- II 1 .1505 ' / l .L -200. 400uM glib lOOuM DPC .. '250 I T r 1 1* I I I I 0 5 ID I s 20 25 30 35 4o 45 Time (min) Figure 21. Effect of 400 uM glibenclamide and 100 uM DPC treatment on normalized acidification rates of control cells (BPV) and cells stably expressing wild type CFTR (2WT2) or AF508 mutant CFTR (508-8). Treatment with 400 (M glibenclamide and 100 (M DPC (CFTR inhibitors) elicited similar results to a combined treatment with forskolin in all three cell lines (45- 100% increase in acidification rates) thus supponing the previous indication of CFTR inhibition. There were no statistically significant differences among any of the three cell lines. Each point represents an average of n=4 for wild type and mutant cells, and n=2(4) for control cells (error bars as SEM). 62 without 10 (1M forskolin. By introducing a Cl' free environment on the extracellular side, thus causing a concentration gradient for CI' to exit the cells and HC03' to enter, we can examine the possibility that CFTR is a CI'IHC03' exchanger. If CFTR has anion exchanger qualities, when comparing wild type cells to mutant and control cells, we would not expect to see decrease in acidification rate. HCOg‘ would only be able to enter the cell while Cl' would be exiting the cell, thus maybe even causing a higher increase in acidification rate as compared to mutant and control cells. Following the switch to Cl‘ free medium containing 10 uM forskolin, the acidification rate of wild type cells decreased by about 140% while there was no change in either mutant or control cells (Figure 22). This decrease in acidification rates of wild type cells was short lived (Figure 22). When compared to 10 (M forskolin treatment in running medium, the 10 uM forskolin in CI' free medium was shown to have a much higher impact on acidification rate (Figure 23). An exposure to 10 (M forskolin in running media elicited an increase in acidification rate in wild type cells of about 35% (Figure 23). Four minutes following the drug exposure acidification rate in wild type cells was reduced to a final increase, as compared to pretreatment state, of approximately 25% (Figure 23). This difference in acidification response in these two media was found to be significantly different. The data suggests continued, and even increased, alkalinization of extracellular environment in wild type cells despite the Cl‘ free media. 63 200 + 2WT2 Cl-frcc buffer 4» lOuM for n=8 I80~ —0— SW CHrcc bufch lOuM for n=4(8) —0— 508-8 Ci-frcc buffer + lOuM for n=lO 160-1 I 120: T . mo .53": _: 7 . ~ . . 0 ° ' , . . ...-. s ‘- T . l E 80.. * - - - . ‘1. ° * E 40.. * . .l. L :3 20- * .L < 0. g -20- E 40... -60.. -30. lOuM forskolin 100.1 .120“ J” F5 2'0 25 3'0 35 Time (min) o ”'1 c Figure 22. Effect of Cl' free media and 10 uM forskolin treatment on normalized acidification rates of control cells (BPV) and cells stably expressing wild type CFTR (2WT2) or AF508 mutant CFTR (508-8). By introducing a Cl' free extracellular environment, we examined CFTR as a possible Cl'lHCOa' exchanger. During CI‘ free conditions throughout the application of 10 (M forskolin, the acidification rate of wild type cells rapidly decreased by about 140%. No such decrease was observed in either mutant or control cells. Evidence points to a continued alkalinization of the extracellular environment despite the Cl' free media. Despite the observed differences between wild type and mutant cells, however, they were not found to be statistically significant. Each point represents an average of n=8 for wild type cells, n=10 for mutant cells, and n=4(8) for control cells (* P < 0.01 compared to BPV cells by both 2WT2 and 508-8; error bars as SEM). 200 + 2WT2 lOuM for n=26(39) ‘80 - I —D— 2WT2 Cl-free buffer + lOuM for n=8 Normalized Acidification Rate (%) 4204 lOuM forskolin I I I r I I IS 20 25 30 35 o Md 9 Time (min) Figure 23. Comparison of normalized acidification rates of cells stably expressing wild type CFTR (2WT2) in normal and Cl‘ free media. An exposure to 10 (M forskolin in running media elicited an increase in acidification rate in wild type cells of about 35%. Four minutes following the exposure to forskolin acidification rate in wild type cells was reduced to approximately 25%. In CI‘ free media containing 10 (M forskolin, the acidification rate of wild type cells was rapidly decreased by about 140%. This decrease in acidification rates of wild type cells was short lived but significantly lower than of those in running media. The data suggests continued, and even increased, alkalinization of the extracellular environment in wild type cells despite the Cl' free media. Each point represents an average of n=8 for CI' free buffer, n=26(39) for running media (* P < 0.05; error bars as SEM). 65 DISCUSSION Cystic fibrosis (CF) is the most common fatal recessive genetic disease effecting caucasians in the United States (Quinton, 1999). About 5% of the caucasian population are asymptomic carriers and about 1 in every 2,500 children born are affected by the disease. CF is caused by the mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The gene is found on the long arm of chromosome seven, and when expressed, CFTR codes for a small, cAMP-stimulated Cl' channel found on the apical surfaces of epithelial cells (Wine, 1991). This channel facilitates the free movement of salts and water in and out of cells (Quinton, 1999). The mutation of the CFTR gene causes CF and affects many organs in the body such as the skin, lungs, liver, pancreas, small intestine and reproductive tract. Overall, CF is characterized as a disease with defective electrolyte transport and clogging of the epithelial lined ducts and surfaces with thick, sticky mucus. The majority of CF patients, as much as 90%, eventually die of lung disease due to high bacterial infection rates (Welsh and Smith, 1995). Our previous results indicated that the expression of functional CFTR may significantly alter extracellular pH via control of HCOa' efflux (Luckie et al., 2001). Recent reports even suggest that CFTR in fact may be a Cl'chog' exchanger (Choi et al., 2001a). In our current research we looked at four possible models of CFTR in the transport of H003]: 1) CFTR as a Cl' channel; 2) CFTR as a Cl“ /HCO3' anion exchanger (AE); 3) CFTR as a Cl' channel and an AE; and 4) 66 CFTR as a Cl' channel that also transports HCOa‘ and regulates CI'IHCO;; exchanger activity. In these studies, I looked at the effect of stimulators and inhibitors of CFTR and AEs' via parallel studies of iodide (anion) efflux and changes in extracellular acidification (pHo). The rationale for my study was that recently a group headed by Shmuel Muallem published new findings that have shaken up the field of cystic fibrosis research (Choi et al., 2001a; Choi et al., 2001 b). Muallem’s group was interested in the secretion of HC03' in CFTR expressing tissues. They studied pancreatic sufficient and insufficient CF mutations by comparing the Cl' transport and HC03' transport in cells expressing these mutations (Choi et al., 2001a). Their results indicated that some pancreatic insufficient mutations (as found in patients with severe disease symptoms) exhibited normal Cl' transport, while their HCOg' transport and in turn pH regulation were aberrant. This suggested that defective H005 transport and pH regulation might have an equal if not greater role than chloride transport in the pathophysiology of CF (Choi et al., 2001a). In a later publication, they hypothesized that CFTR may, in fact, also be a Cl'lHCOa' exchanger (Choi et al., 2001b). 1. Specific Aims of the Research The purpose of the current study was to characterize bicarbonate and chloride transport through CFTR and examine specific tenets of the “Muallem hypothesis," both experimentally and through a literature review, that may imply that CFTR acts as a CI‘/ HCOa' anion exchanger (AE). The specific aims of my 67 research were: I. to characterize the Cl' channel conductance properties of CFTR in selected mammary epithelial cell lines; II. to examine the conditions necessary for CFTR dependent HCOa‘ conductance; III. to test the Muallem hypothesis through both my own experiments and through a literature review of studies in other laboratories. Our study focused on two C127 mouse mammary epithelial cell lines: 2WT2 (expressing wild type CFTR) and AF508 (expressing the most common CFTR mutation, deletion of phenylalanine at position 508). They are stable cell lines transfected with the BPV vector carrying wild type or mutant CFTR cDNA. We also used control cells transfected with the BPV vector alone. In our data collection, we utilized microphysiometer technology to determine the importance of pH and H003‘ in CF and to test CFTR as a Cl' /HCO:.' exchanger. The microphysiometer can detect subtle changes in extracellular pH (pH) and the rate at which a cell acidifies its extracellular environment (McConnell et al., 1992). Cells were perfused with physiological buffers containing various stimulators and inhibitors and the resulting pHo was monitored (Table 1). Bicarbonate is one of the most important physiological pH buffers, and thus we were able to monitor its transport through transient changes in pH of the extracellular environment. We also used an ORION® iodide electrode system to measure and directly record anion (iodide) flux through the CFTR channel expressed in these mammalian cell lines. These assays were 68 used to record CFTR channel activity and its response to various inhibitors and stimulators (Table 1). 2. Proposed Models of CFTR to Examine Results published in the field of cystic fibrosis so far most often point to CFTR being solely a Cl' channel and the explanation of the pathology of the disease (dehydrated and clogged ducts) as a defect in the transport of Cl'. As discussed previously, however, there are some results that are not easily explained by a defect in CI' transport alone. The lung disease (leading to death in 90% of CF patients), characterized by a buildup of mucus and preferential infection by certain pathogens, is not well understood and has been the hardest consequence of the CFTR mutation to explain solely by abnormal Cl' transport (Welsh and Smith, 1995; Smith et al., 1996). Therefore, it may be necessary to examine CFTR as a more global regulator of electrolyte transport influencing other anions, such as H005, and possibly extracellular pH. In the current research, we attempted to characterize bicarbonate and CI' conductance through CFTR. In this study, four specific models of CFTR were tested: 2.1. CFTR as a Cl Channel This model assumes CFTR is simply a Cl' channel that does not directly influence or regulate any other transporters or transport any other ions (such as bicarbonate). This model is simplistic, but may be all that is relevant to the disease. It has been previously shown that CFTR does conduct H005 ions 69 (Poulsen et al., 1994), although the importance and amount of this transport may be insignificant. If this model is a true representation, we would expect to make the following observations: Little or no changes in pHo when CFTR inhibitors (glibenclamide and DPC) or a stimulator (forskolin) are used (Cl‘ doesn't directly affect pH); however Cl' could indirectly affect AE function and thus result in changes in pHo (use of an AE inhibitor could resolve this question); A possible change in a pHo when AE inhibitor (DIDS) is used (as a result of inhibition of other AE(s) present, for example a Cl'lHCOa' exchanger present on the apical side of epithelial cells); however if change in pHo is observed it should not be forskolin-dependent; No change in pH, of Cl' free media following forskolin exposure; Decrease in iodide efflux rate with the use of CFTR inhibitors; Increase in iodide efflux rate with the use of a CFTR stimulator; Iodide efflux rate would remain the same when compared to the control with the use of AE inhibitor (Table 1). 2. 2. CFTR as a Ci/HCOg' Exchanger (AE) In 2001 a group headed by Shmuel Muallem published new results claiming that CFTR is a CI‘IH003' exchanger (Choi et al., 2001b). Thus, one of the possible models to examine (based on Muallem's findings) could be that CFTR is simply an AE (without the CI' channel component). Multiple groups have shown that CFTR allows for transport of both Cl' (Quinton, 1983) and HCOa' ions 70 (Poulsen et al, 1994;) but no evidence thus far was reported supporting this model. If this model is correct we would expect to see: Bicarbonate transport through CFTR should be Cl' dependent and stimulated with forskolin; A decrease in acidification rate when glibenclamide and DPC are used (no evidence of inhibition of AE's); A decrease in acidification rate when a known CFTR stimulator (forskolin) is used; An increase in acidification rate when an AE inhibitor (DIDS) is used; Increase in acidification rate in CI‘ free media following forskolin exposure (due to inhibition of H005 transport); There would be no (or very little) iodide efflux rate recorded since iodide transport occurs through a channel (Table 1). 2. 3. CF TR as a Cl Channel and a CfIHCO5 Exchanger Still keeping in mind Muallem's findings, we also can assume that CFTR has an ability to be both a CI' channel as well as a Cl'/H005 exchanger. For our purposes we are presuming, though, that either of these CFTR conformations would be “expressed” only one at a time. The focus of the research was to test the ability of CFTR to conduct both Cl’ and H005 in exchanger fashion using the microphysiometer studies. In this model we predicted that in the case of CFTR being an exchanger and a channel the following should be observed: Bicarbonate transport through CFTR should be CI' dependent and 71 stimulated with forskolin; Increase in acidification rate in CI' free media following forskolin exposure (due to inhibition of H005 transport); Glibenclamide and DPC should stop efflux of chloride through the CFTR channel, but most likely would not prevent action of CFTR as an exchanger (no evidence of such action); thus acidification rate should remain decreased (not affected) as bicarbonate should still leave the cell through an AE; DIDS (an AE inhibitor) should not affect bicarbonate movement through CFTR as a channel, it should, however, inhibit bicarbonate movement through its AE; acidification rate in wild type cells should therefore increase since bicarbonate movement through CFTR's AE has been inhibited; Iodide efflux rates should be comparable to the first model presented (decrease with CFTR inhibitors; increase with CFTR stimulator; and remain the same as compared to the control with the use of AE inhibitor) (Table 1). 2.4. CF TR as a Cl Channel Wrth Regulation of a Cl/HCO5 Exchanger Activity The final model of CFTR activity tested assumes that CFTR is primarily a Cl“ channel that through its function and/or structure, affects other exchangers among those that are also 0|‘IH005 exchangers. In this model it is also assumed that CFTR, even though primarily a Cl’ channel, does allow for passage of other 72 anions as well (such as bicarbonate). In order to support this model the following would be expected: - A decrease in acidification rate when CFTR stimulator (forskolin) is used; - An increase in acidification rate when CFTR inhibitor is used; - A possible change in pHo when an AE inhibitor (DIDS) is used (as a result of inhibition of other AE(s) present); - Decrease in acidification rate of Cl' free media following forskolin exposure (due to passage of bicarbonate through CFTR); - Iodide efflux rates should be comparable to the first model presented (decrease with CFTR inhibitors; increase with CFTR stimulator; and remain the same as compared to the control with the use of AE inhibitor) (Table 1). 3. Iodide Efflux Studies 3.1. CF TR Activator Studies In order to characterize 0|" conductance properties of CFTR, we first performed iodide efflux studies where cells were treated with a CFTR activator, 10 (M forskolin. Forskolin treatment was used to validate iodide efflux studies as an appropriate method to monitor CFTR activity. As expected, 10 (M forskolin treatment increased iodide efflux in cells expressing wild type CFTR by 3.5 fold (Figure 5). The increase in forskolin-elicited iodide efflux in wild type cells was significantly different than of both mutant and control cell lines (p<0.01). Forskolin activates CFTR through an increase in [cAMP]i, hence activating the PKA 73 pathway and consequent phosphorylation of the R domain of CFTR (Rommens et al., 1991). There was no significant increase in efflux in cells transfected with mutant CFTR, or in control cells. This indicates there is a lack (or severe reduction) of CFTR cell surface expression in these cell lines, since even a small number of CFTR molecules carry a significant amount of iodide and would be detected (Figure 5). Both mutant and control cell lines had a certain level of background noise presumed to be either iodide bound to the cell membrane or iodide leaving cells through other channels. It was assumed that the majority of iodide efflux observed was recorded through Cl' channels (mainly CFTR) and as AE mechanisms prefer Cl' over I', that the transport through such mechanisms would be insignificant (Venglarik et al., 1990). It has been shown that I' does not compete with Cl“ with AE mechanisms, further supporting this assumption (Humphreys et al., 1994). These results (the forskolin-elicited increase in iodide efflux) supported all models but model II that assumes CFTR is only an AE, and thus would not allow for transport of iodide. Mutant and control cell lines had a slow steady increase in iodide efflux throughout the experiment. This was later observed in all iodide efflux experiments for all cell lines. It was found to be comparable between the different cell lines with an average increase in rate (change in concentration over time) of 0.07-0.08. This occurrence could be explained by a potential increase in temperature of media, by lysing cells that are releasing their iodide content, or by existence of other efflux mechanisms. 74 This set of experiments established that iodide efflux was a reliable method of differentiating between cells expressing wild type CFTR and mutant CFTR, or control cells not expressing CFTR. Iodide efflux was hence used to characterize CI' conductance through CFTR. We treated cells with different inhibitors and stimulators of CFTR and AE's and observed their consequent iodide efflux response as an indication of CFTR activity. 3. 2. AE Inhibitor Studies Previously it was shown that CFTR is not affected by DIDS (a general AE inhibitor) (Sheppard and Welsh, 1999). If, however, CFTR contains AE activity, DIDS, an AE inhibitor, should be able to inhibit its function. Therefore we sought to characterize 0l‘ conductance through CFTR in cells treated with 500 (M DIDS (AE inhibitor) and 10 11M forskolin. CFTR (a Cl' channel) was expected to be unaffected. If inhibition was recorded it would indicate an AE component and/or qualities of CFTR. In wild type cells, simultaneous DIDS and forskolin treatment resulted in similar mean iodide efflux as treatment with forskolin alone. DIDS and forskolin treatment in wild type cells yielded a 6.5 fold increase as compared to the pretreatment state (Figure 9A). The mean increase in efflux rate of wild type cells treated with both DIDS and forskolin was slightly higher (though not significantly different) than the mean increase in efflux of wild type cells treated only with forskolin (Figure 9A). This suggests that forskolin-elicited iodide efflux through 75 CFTR was not blocked by addition of an AE inhibitor, thus confirming the hypothesis that DIDS in not a CFTR inhibitor. When comparing the increase from pretreatment to post-treatment state for each drug combination (DIDS and forskolin vs. forskolin alone), they both yielded similar changes in rate of efflux (0.46 and 0.42 respectively). The difference of base response during the pretreatment state (higher base efflux in cells treated with forskolin alone) could be suggested to also indicate the difference between the two treatments during the drug stimulus. Thus the data were re-plotted to compare only those cells studied simultaneously (on the same day) (Figure 9B). These iodide efflux rates tightly paralleled each other, thus differences in the pretreatment state were reduced. The data again supported the idea that DIDS is not a CFTR inhibitor. The difference in base efflux (shown in Figure 9A) during steady state thus appears to be artifactual. The base efflux rates seemed to vary slightly among different cell samples. Both sets of mutant cells (receiving DIDS and forskolin or only forskolin) exhibited a small steady increase in iodide efflux rate over time, which was found to be statistically different from each other (p<0.01 and p<0.05 for different time points) (Figure 10). Responses from both treatments, however, paralleled each other and were found to have increased by only two fold in both treatments for the duration of the entire experiment (2.5-5 minutes). This increase in iodide efflux rate was presumed to be either due to media temperature increase during the experiment progression, due to lysing of cells, or to other efflux mechanisms (Figure 10). When compared to wild type cells, mutant cells did not exhibit a 76 significant increase in efflux (two fold for mutant cells vs. 6.5 fold for wild type cells). Absence of increased iodide efflux following simultaneous DIDS and forskolin treatment suggested the lack of CFTR activation in mutant cells. This was not a surprise, since mutant cells do not express functional, membrane associated CFTR. These data again confirmed the validity of the iodide efflux assay. We used 500 (M DIDS treatment as per Lee et al. (1999a), which may have resulted in broad range inhibition in these cell lines. DIDS is an AE inhibitor, and it has been shown that it does not affect CFTR when added extracellularly (Sheppard and Welsh, 1999; Reddy and Quinton, 2002). DIDS is also known as a non-specific inhibitor (Wang et al., 2004). My observations of wild type cells treated with DIDS support the hypothesis that it is not an inhibitor of CFTR (no difference was observed when compared to control). Results obtained from mutant cells however, were inconclusive (due to steady efflux increase not found statistically different from wild type cells). The mutant cells should have not had any increase in iodide efflux (due to lack of CFTR expression), so it is possible that such a high dose of DIDS resulted in increased cell lysing and hence increased extracellular iodide concentration. DIDS studies did, however, suggest that CFTR did not have an AE component thus further supporting results obtained from forskolin studies. Since iodide efflux in these cells, however, occurs mostly through a channel (i.e. CFTR), the studies did not rule out the existence of an AE that is a part of the CFTR or is regulated by it. Thus the observations from these experiments 77 support all models but model ll. Model II (CFTR as an AE) is the only one that does not account for a channel portion of CFTR. 3. 3. CF TR Inhibitor Studies CFTR inhibition studies were performed to further characterize CI' conductance through CFTR. Preliminary work showed that the best inhibition was observed with treatment of cells with 400 (M glibenclamide and 100 (M DPC (inhibitor cocktail). This drug combination was then used for further studies. Application of 400 pM glibenclamide, 100 uM DPC and 10 (1M forskolin decreased iodide efflux in wild type cells compared to wild type cells receiving forskolin alone (Figure 11). The wild type cells receiving the inhibitor cocktail and forskolin did have a steady increase in efflux starting in the pretreatment state and continuing until half way through treatment state. Comparing increase before and after forskolin treatment showed that the drug caused a maximum 3.5 fold (350%) increase in rate of efflux, while inclusion of glibenclamide and DPC resulted in only a 1.5 fold (50%) increase in rate of efflux. Mean maximum increase for these two different treatments was found to be not significantly different when performing a two-tailed student t-test (p=0.06). Different trial number (n=20 for forskolin treatment and n=8 for inhibitor treatment) required more rigorous statistical testing and could be a plausible explanation for the lack of statistical significance. None-the-less, the difference in the amount of increase rate (350% vs 50%) indicates substantial inhibition of CFTR with glibenclamide and DPC treatment. 78 The same treatment in mutant cells did not cause any significant change or difference in the overall rate of efflux in either cells treated with forskolin alone or treated with both forskolin and the inhibitor cocktail (Figure 12). There was no significant difference in the effects of the two treatments (p=0.24 at t=3; p=0.02 at t=3.5; p=0.13 at t=4; p=0.06 for at t=4.5). They both did, however, elicit a steady increase in efflux rate for the duration of the experiment (Figure 12). The efflux rate increase was comparable in both treatments (1.7 fold with forskolin treatment and 2 fold with simultaneous inhibitor and forskolin treatment). This was also comparable to the previously mentioned background drift observed in mutant cells (Figure 7; Figure 10; Figure 12) If complete CFTR inhibition was accomplished, then inhibitor and stimulator treatment of wild type cells and stimulator treatment of mutant cells should exhibit similar results. However, wild type cells treated with CFTR inhibitors and stimulators did show a significant difference (an increase) when compared to control treatment of mutant cells with forskolin, indicating again partial inhibition of CFTR (p<0.05) (Figure 13). This finding was not unexpected since according to current research findings in the literature there are no complete and specific CFTR inhibitors known (Reddy and Quinton, 2002). Since stock glibenclamide (CFTR inhibitor) was dissolved in DMSO, I also carried out vehicle control studies. Cells were treated with 0.4% DMSO and forskolin and the responses were compared to DMSO treatment alone. In wild type cells, 0.4% DMSO and 10 (M forskolin treatment caused a 5.5 fold increase in iodide efflux rate (Figure 14). The same treatment in mutant cells did not cause 79 any significant change and efflux remained steady for the duration of the study. It was however significantly different (lower) as compared to wild type cells (p<0.01) (Figure 15; Figure 16). DMSO treatment alone did not cause any significant change in iodide efflux rate in either wild type or mutant cell lines (Figure 14; Figure 16). Looking at forskolin treatment alone, it only caused a 3.5 fold increase in efflux rate of wild type cells, while DMSO and forskolin treatment caused a 5.5 fold increase. This would indicate that DMSO treatment might actually have a stimulatory effect on the efflux rate (possibly due to increased dispersal of forskolin into the cell). Forskolin increases cAMP by directly stimulating the cytoplasmic region of adenylate cyclase (Yoo et al., 2004). Therefore, it needs to be dispersed into the cell for its actions to take affect. If this is the case, the glibenclamide and DPC results (lack of complete CFTR inhibition) might be easier to explain. DMSO could have had an opposite effect on inhibition, thus yielding lower inhibition with glibenclamide and DPC. These data support the hypothesis that the glibenclamide and DPC cocktail is useful as a substantial CFTR inhibitor that could be applied in further studies. Since full inhibition was not recorded and anion flux was still observed, the results do not support model M (CFTR as an AE), while providing support for all others previously presented. 3. 4. Conclusions From Ion Flux Studies Iodide efflux studies showed that cells stably expressing wild type CFTR are stimulated by forskolin (as indicated by an increase in iodide efflux rate). The 80 AE inhibitor DIDS was not found to have an inhibitory effect on these cells, while glibenclamide and DPC treatment (CFTR inhibitors) were found to only partially inhibit forskolin-elicited iodide efflux. Considering the four models of CFTR function previously presented, only CFTR as an AE was expected to behave distinctly differently in efflux studies. If this was true, we did not expect to see any iodide efflux (as iodide is transported through channels). The other three models (CFTR as a Cl' channel; CFTR as both a CI' channel and an AE; and CFTR as a CI' channel with the regulation of an AE) were expected to behave similarly and demonstrate (i) an increase in efflux with forskolin stimulation, (ii) a decrease in efflux with CFTR inhibition and (iii) a comparable efflux between the control and AE inhibitor. Therefore, since all of these expectations were observed, our iodide efflux results do not support the CFTR as an AE model. However, they do not differentiate among the other three models. 4. Microphysiometer Studies 4.1. CF TR Activator Studies Examination of the conditions necessary for CFTR dependent H005 conductance were achieved through usage of Microphysiometry, which detects the rate at which a cell acidifies its extracellular environment. When comparing the acidification rates of cells transfected with vector alone (control), and cells stably expressing wild type or mutated CFTR, 10 (1M forskolin elicited a smaller increase in acidification rate in wild type cells than in mutant and control cells (Figure 17). An initial approximate increase in acidification rate of 90% (for 81 mutant cells) and 35% (for wild type and control cells) was observed (Figure 17). Four minutes following the forskolin treatment, acidification rate for mutant cells was still increased by 50% and it remained increased (up to 65%) for the duration of the treatment (Figure 17). Acidification rate of control cells remained increased and continued to rise to 45%, while wild type acidification rate decreased (to a 25% increase compared to pretreatment) and was significantly lower than the other two cell lines for the remainder of the forskolin exposure (p<0.01) (Figure 17). Even after the forskolin washout, acidification rate of all three cell lines remained different, although not significantly so. Forskolin treatment (presumably yielding an increase in [cAMP]i) causes an increase in metabolic activity (proton efflux), thus it would be expected to also elicit an increase in acidification rate of the extracellular environment (Luckie et al., 2001). My results indicate that the increase in acidification rate of the extracellular environment of wild type cells evoked by forskolin is significantly lower than that of mutant and control cell lines. This suggests a molecular mechanism specific to CFTR expressing cells that is able to partially blunt the forskolin-induced acid signal. We also would expect to see no difference in acidification of control cells (not expressing CFTR) and mutant cells (expressing mutant CFTR). Interestingly, our results indicate an increased acidification rate in mutant cells over the control cells. For future experimentation, it would be interesting to investigate the mechanisms of this difference. The mechanism blunting the acid signal could either be due to decreased proton efflux or increased base efflux (Luckie et al., 2001). Our laboratory has 82 previously found evidence that this difference is due to increased base (H005) efflux through CFTR (Luckie et al., 2001). CFTR’s ability to allow for bicarbonate passage, along with Cl', and its regulation of 0I'IH005 exchangers is also supported by reports in the current literature (Kopelman et al., 1988; Smith and Welsh, 1992; Poulsen et al., 1994; Chan et al., 1996; Poulsen and Machen, 1996; Clarke and Harline, 1998; Lee et al., 1998; Quinton 1999; Illek et al., 1998; Mastrocola et al., 1998; Lee et al., 19993; Lee et al., 1999b; Ko et al., 2004). The observations indicating forskolin—dependent (i.e. CFTR mediated) bicarbonate flux, do not support expectations for model I (CFTR as only a Cl' channel). However, they do not differentiate between the other three models proposed. Current research in several laboratories is aimed at trying to determine if this bicarbonate efflux utilizes a Cl'/H005 exchanger as its mode of transport. 4.2. AE Inhibitor Studies A general AE inhibitor DIDS also was used to investigate its ability to inhibit CFTR and therefore to affect pHo. Treatment with 500 pM DIDS alone in wild type cells resulted in decreased acidification rate by about 25% (Figure 18). When this treatment was combined with 10 pM forskolin, the effect was amplified to a 55% decrease in acidification rate, suggesting still functional H005 efflux that is consistent with the time course of forskolin treatment (CFTR activation) (Figure 19). These data reinforced the hypothesis that DIDS is not a CFTR inhibitor. 83 In control cells, DIDS treatment alone elicited a decrease in acidification rate by as much as 150% (Figure 18). On the other hand, when the treatment was combined with forskolin, the acidification rate was decreased only by about 50%, thus allowing for more acid efflux than before (Figure 19). This would indicate that control cells lacked a mechanism that would overcome a rapid proton efflux elicited by forskolin treatment. It would be safe to assume that the difference in the response between wild type and control cells is the result of cell surface expression of CFTR. These results, compared to the wild type response, would indicate a still functional and uninhibited CFTR in wild type cells. DIDS treatment alone in mutant cell lines caused a variable response, however, overall a pattern of decrease in acidification rate was observed (Figure 18). Since following 10 pM forskolin treatment all three cell lines showed decreased acidification rate in the range of about 50—70%, it is evident that the treatment with 500 (M DIDS did have an effect on an AE common to all three cell lines (Figure 19). It is possible that by inhibiting other anion exchangers present in these cells, DIDS treatment either caused a decrease in proton efflux or increase in base efflux. Wrth forskolin stimulation (and therefore an increase in cell metabolism), however, only CFTR expressing cells were able to even further decrease the acidification rate. The data suggest that base efflux regulated by CFTR was not mediated through an AE, which likely would have been inhibited by DIDS. Rather, the data indicate that base efflux in these cells is most likely mediated through a forskolin-stimulated non-AE pathway, such as CFTR. The data therefore do not support models I (CFTR as a CI' channel), Il (CFTR as an 84 AE) or III (CFTR as a CI' channel and an AE). Model one was excluded due to observed forskolin-elicited response (indicative of CFTR involvement), while models II and II were excluded due to lack of AE inhibition. It is, however, plausible to assume that model IV (CFTR as a Cl' channel that allows for transport of bicarbonate and that regulates an AE) is still supported by these observations. 4.3. CF TR Inhibitor Studies Treatment with 400 pM glibenclamide and 100 (M DPC along with 10 uM forskolin was found to cause a 60-120% increase in acidification rate in all three cell lines (60% for wild type, about 90% for control and 120% for mutant cell lines) (Figure 20). Treatment with 400 (M glibenclamide and 100 (M DPC alone in all three cell lines elicited results similar to a combined treatment with forskolin (45-100% increase in acidification rates) supporting previous iodide efflux results indicating CFTR inhibition with glibenclamide and DPC (Figure 21). There was no statistical difference in the response of any of the three cell lines, thus indicating that inhibition of CFTR in wild type cells increased their resemblance to the control and mutant cells. Considering that forskolin alone also elicited only a 20- 25% increase in acidification rates of wild type cells, a 60% (2.4 - three fold) increase as observed with simultaneous inhibitor cocktail treatment is significant and indicative of CFTR inhibition in wild type cells. Glibenclamide and DPC have been used frequently as CFTR inhibitors by other laboratories and have not been found to have an effect on anion exchangers (Reddy and Quinton, 2002). Hence, 85 the inhibition of CFTR shown here is not likely the inhibition of an AE component. These results further agree with AE inhibitor studies and do not support models II (CFTR as an AE) and Ill (CFTR as a CI' channel and an AE). The results, however, support both models I (CFTR as a CI' channel), and IV (CFTR as a Cl' channel that allows for transport of bicarbonate and that regulates an AE). Even though model I argues that CFTR is only a CI' channel, it would be plausible to assume that inhibition of such channel would affect Cl' concentration, and therefore affect functioning of other cell AEs. This in turn, would affect the pHo recorded by microphysiometer. 4.4. CF TR Activation in Cr Free Media Our final studies examined the acidification rate of cells in CI' free medium with and without 10 uM forskolin. Assuming that CFTR has an AE component, a Cl' free extracellular environment would create a large concentration gradient for CI' to exit the cells and H005 to therefore enter via an exchanger. Because of this, we were able to examine CFTR as a possible Cl'lH005 exchanger. In our studies, following the switch from Cl' free media alone, the media containing 10 (M forskolin elicited a decrease by about 140% in the acidification rate of wild type cells, while there was no change in either mutant or control cells (Figure 22). This evidence suggests there is a CFTR-dependent alkalinization of the extracellular environment despite the Cl' free media, and that the observed decrease in acidification rate and bicarbonate transport through CFTR is not CI' dependent. Thus, the data obtained from wild type cells do not support the 86 involvement of a CI‘IH005 exchanger in bicarbonate transport. Although the initial large decrease in acidification rates of wild type cells was short lived, wild type cells sustained a lower acidification rate than mutant and control cells for another pump cycle. Due to variation in data collected, though, the difference between wild type, mutant and control cell lines was not found to be statistically different. When compared to 10 pM forskolin treatment in running media with Cl, 10 pM forskolin in CI‘ free medium was shown to induce even a larger decrease in acidification rate in wild type cells (Figure 23). An exposure to forskolin in media with Cl' elicited up to a 35% increase in acidification rate, which was found to be significantly different from the 140% decrease observed in Cl‘ free media (p<0.05) (Figure 23). The increase in forskolin-dependent bicarbonate flux in CI' free media (as compared to CI' containing media) was unexpected. In order to equilibrate cells to a new medium and stabilize pH, cells were perfused with Cl‘ free medium for 28 minutes before introducing forskolin. It is possible that by creating a Cl' free environment that is maintained extracellularly for long periods of time (hence also creating a huge concentration gradient), the intracellular Cl' concentration was also greatly reduced. If the concentration of Cl' was reduced, the competition with H005 for passage through CFTR also would be reduced. This could be a plausible explanation for a higher H005 flux and shorter-lived decrease in acidification rate observed for wild type cells in CI' free medium. The data obtained from Cl‘ free studies supported only model IV (CFTR as a CI' channel with regulation of an AE). Due to a lack of Cl’ dependent 87 bicarbonate transport, models ll (CFTR as an AE) and Ill (CFTR as a Cl' channel and an AE) were refuted. Model I (CFTR as only a CI' channel) was also not supported. The observed forskolin dependent decrease in acidification indicated that the bicarbonate flux observed was mediated only following CFTR activation. At the same time, CI' free media excluded a possible involvement from any other cell-expressed AEs in mediating the bicarbonate efflux observed. 4. 5. Conclusions From pHo Studies Microphysiometry studies demonstrated that cells stably expressing wild type CFTR have lower acidification rates when stimulated by forskolin than cells stably expressing AF508 mutant CFTR and than control cell lines. The AE inhibitor DIDS was found not to have an inhibitory effect on these cells, allowing for further decreased acidification rates in wild type cells when DIDS and forskolin were simultaneously added. Control cell lines also had decreased acidification rates (increased alkalinization) with DIDS treatment, but this affect was reversed by addition of forskolin. The difference in the response between wild type and control cells can be assumed to be related to functional cell surface expression of CFTR in wild type cells. Glibenclamide and DPC treatment (CFTR inhibitors) were found to inhibit CFTR, as evidenced by increased acidification rates when compared to a forskolin treatment. In inhibitor studies, acidification responses of wild type cells were comparable to those of control and mutant cells. 88 Considering the models of CFTR function previously presented, only the model presenting CFTR as a CI' channel that allows for transport of H005 and regulates an AE was fully supported. This model assumed that in wild type cells we would observe: (i) a decrease in acidification rate when stimulated with forskolin; (ii) an increase in acidification rate upon inhibition with glibenclamide and DPC; (iii) a decrease in acidification rate in CI' free media; and (iv) a decrease in acidification rate upon stimulation with an AE inhibitor (DIDS). As explained above, all of these predictions were supported by microphysiometry studies. It should be noted that DIDS treatment however, was hard to differentiate in any model due to its potential influence on other cell AE's. The other three models (CFTR as a CI' channel; CFTR as both a Cl' channel and an AE; and CFTR as an AE) were not fully supported. CFTR models as an AE and as both Cl' channel and an AE assumed that bicarbonate transport would not only be forskolin elicited, but also CI’ dependent. As shown by the continued decreased acidification rate in CI' free media, that was not the case. Also, it was expected that DIDS would inhibit the decreased acidification rate and that glibenclamide and DPC would not, but neither was observed. Lastly, the model presenting CFTR as only a Cl' channel assumed that there would be little or no change in pHo with most treatments. It was expected that there would be no much change in pHo upon stimulation with forskolin alone. Forskolin is a CFTR stimulator and response elicited by such treatment would indicate a CFTR dependent bicarbonate flux. Even though Cl’ does not directly affect pH, it was predicted that the treatment potentially inhibiting other cell AE's 89 (DIDS) would elicit a response. We would not, however, expect that the response observed would be further enhanced by forskolin stimulation, as it was evident from our studies. Glibenclamide and DPC treatment resulting in inhibition of CFTR was the only microphysiometer study where the results obtained could support model I. In these experiments the increased acidification rate was recorded. It is presumed that inhibition of CFTR (as a CI' channel) would affect Cl' concentration and thus functioning of other AEs expressed by cell lines used. This in turn, could affect pHo. Overall, our microphysiometer data support only a model where CFTR is a CI' channel that allows for H005 transport and has a regulatog influence on an AE. 5. Literature Research: A Channel or An Exchanger? Our studies attempted to clarify the role of CFTR in the transport of H005 and its influence on extracellular pH. Most of the research published to date has focused on CFTR as a Cl‘ channel. Recently, there has been increased interest in CFTR as a global regulator of other channels and transporters, and even as an ion exchanger. Here I present a literature review in the areas so far most examined by the field looking at: 1) CFTR as a Cl' channel, and 2) its relationship with Cl'lHCO5 exchange. 5.1. Channel Story Though at the time not referred to as cystic fibrosis, written records and indications of CF date to as early as the 1600’s in diaries and folk stories in Europe (Busch, 1990). These records talk about “bewitched” children with salty foreheads, children presumably affected by CF (Busch, 1990). It was not until later that this characteristic was attributed to cystic fibrosis. The cause of higher salt content in sweat was first described in 1983 (Quinton, 1983). The first indication that CF was caused by a defect in CI' movement across the membrane came in the 1980’s. Prior to the 1983 research published by Paul Quinton, the physiological explanation of the disorder was unknown. Quinton carried out a study on isolated sweat ducts from control subjects and from CF patients. These ducts were microperfused and transepithelial potential differences during the microperfusions were monitored (Quinton, 1983). Quinton observed a significantly increased negative potential in the cystic fibrosis sweat duct as compared to the controls. He assumed that the differences recorded were due to differential anion permeability in the CF and control sweat ducts. He concluded that low CI’ permeability in CF ducts resulted in poor reabsorption of NaCl and hence lead to high NaCl concentration in the sweat of CF patients (Quinton, 1983). It was not until six years later in 1989, that the underlying molecular basis for CF was discovered. Collaborative efforts of two groups yielded the successful cloning of the CFTR gene (Riordan et al., 1989). The first cDNA clone was isolated from the cultured epithelial cells of the sweat glands of an individual not affected with CF (Riordan et al., 1989). Following cloning of the wild type CFTR, these groups compared the CDNA sequences from the CF and unaffected individuals and isolated two mutant clones from sweat glands of CF affected individuals. Both of 91 these clones were discovered to have a three nucleotide sequence deletion (loss of phenylalanine at position 508), a mutation that was later attributed to about 70% of the cases of CF. Analysis of the cloned sequences revealed that they coded for a polypeptide of 1480 amino acids with a molecular mass of 168,138 daltons (Riordan et al., 1989). They also concluded that this protein had two membrane spanning domains and two sequences resembling nucleotide (ATP) binding domains (Riordan et al., 1989). Based on these structural features and resemblance to other membrane proteins, defects in the protein (termed CFTR) were concluded to be directly responsible for CF (Riordan et al., 1989). Due to the previous indications of a CI' permeability defect in CF, it also was concluded that CFTR was involved in regulation of ion conductance across the apical membrane of epithelial cells. Even though the researchers speculated that CFTR could be a Cl' channel itself, there was no direct support for that prediction (Riordan et al., 1989). The isolation of CFTR cDNA pushed the CF field ahead and enabled further characterization of CFTR as a channel. The first step towards characterizing the function of the now isolated CFTR was the creation of cell expression systems. In fact, these expression systems are still being used in CF research today. In the early 1990’s, CFTR cDNA was expressed in multiple cell lines. Anderson et al. (1991a) published one of the first reports of CFTR expression and characterization. In an effort to characterize CFTR function, they expressed CFTR cDNA in HeLa, Chinese hamster ovary (CH0), and NIH 3T3 fibroblast cells with the vaccinia-T7 hybrid expression system (Anderson et al., 1991a). They then assessed its anion 92 permeability with a fluorescence microscopic assay and whole-cell patch-clamp. Following cAMP treatment, an increase in anion permeability and chloride current was recorded in CFTR expressing cells (Anderson et al., 1991a). Based on these results, they concluded that CFTR is most likely a chloride channel itself, thus further supporting the previous prediction of CFTR function. Later that year, the same research group further supported their prediction of CFTR being a channel. Trying to distinguish whether CFTR was either chloride channel or a chloride channel regulator, they used T84 epithelial cells expressing wild type and mutant CFTR and compared the properties of cAMP regulated currents (Anderson et al., 1991b). To accomplish this, they mutated several important amino acids in areas thought to form the pore of the channel from charged residues to negative ones, thereby changing the ion selectivity of the pore (Anderson et al., 1991 b.). The CFTR ion selectivity prior to these mutations was Br' 2 Cl' > I' > F', and following the mutations was changed to I' > Br' > CI' > F' (Anderson et al., 1991 b). They concluded from these data that CFTR was a Cl' channel itself and not a channel regulator. Even though the previous results of multiple groups had shown that low-conductance 0l' currents were generated with the expression of CFTR, this was one of the first studies to actually demonstrate that CFTR was a Cl‘ channel rather than a regulator (T abcharani et al, 1992). The hypothesis that the CFTR functions as a Cl' channel was even further supported with purification of CFTR and its insertion in artificial membranes (Bear et al., 1992). This group purified a recombinant CFTR from a Sf9 insect cell line 93 expressing the CFTR and reconstituted it into proteoliposomes (Bear et al., 1992). Following the formation of these vesicles, fusion with a lipid bilayer was accomplished and Cl' conductance was monitored. The activity observed (chloride currents with properties identical to those found in CFTR expressing tissues) was strong evidence that CFTR is indeed a low-conductance Cl' channel (Bear et al., 1992). Even though the evidence that CFTR was a Cl' channel was strong at this point, soon after the channel function was again being questioned. There were indications that in CF patients other transport functions may have been affected. It was noted that in CF, outwardly rectifying 0l' channels were defective, and these, typically show higher CI' conductance (Egan et al., 1992). These results did not correspond with already supported claims of CFTR being a low- conductance CI' channel. Egan et al. (1992) expressed recombinant CF genes in CF bronchial epithelial cells and noted rectification of defective 0I’ secretion that resembled that of CFTR’s low-conductance chloride current (Egan et al., 1992). They also demonstrated that the activation of outwardly rectifying Cl' channels was restored (Egan et al., 1992). The evidence again pointed to CFTR as a Cl‘ channel but one that may also regulate other cellular functions such as outwardly rectifying Cl’ channels. 5. 2. Exchanger Story Following identification of the CFTR gene, it became clear that CFTR and its function might be more complex than previously assumed. Since then, the 94 function of CFTR as a chloride channel has been confirmed by many different research groups, but it also was shown that CFTR might have a much more complex role in electrolyte transport and balance. The control and regulation of CFTR over the transport of other molecules has since been suggested, among others, that of bicarbonate. Recently there has been some indication that impaired transport of bicarbonate through CFTR might be even more important to development of CF than that of defective chloride transport alone, and that CFTR might even be a Cl'lHCO5 exchanger (Choi et al., 2001a; Choi et al., 2001b). The evidence for this claim was, however, somewhat weak. Simultaneously with the beginning of our study in 2001, Shmuel Muallem and colleagues published results of a study dealing with various pancreatic sufficient and insufficient mutations of CF. The focus of the majority of their previous research was on impaired H005 secretion in CFTR expressing tissues. Previously, they worked on both, H005 secretion as well as salvage mechanisms, and these studies indicated that CFTR was involved in both (Lee et al., 1999a; Lee et al., 1999b; Ahn et al., 2001). They used HEK293 cells transiently transfected to express wild type and various mutations of CFTR. These cells were then used for studies monitoring their intracellular pH (pH;) with BCECF dye, as well as Cl‘ channel flux with MQAE dye. Measurements of [CI‘]; with MQAE dye were shown to correspond to the measurement of CI‘ current. The cells were stimulated with 5 pM forskolin to stimulate CFTR activation and CI' free media was used in pH studies. Their results indicated that some pancreatic insufficient mutations had normal CI‘ transport while their pH 95 regulation, and in turn H005 transport, were defective (Choi et al., 2001a). They indicated that defective H005 transport and pH regulation might have an equal if not greater role compared to impaired chloride transport in the pathophysiology of CF. In a follow-up paper, the authors hypothesized that CFTR may, in fact, be a Cl'/ H005 exchanger (Choi et al., 2001b). The conductance of H005 through CFTR and pH regulation disruption in CF (resulting in the acidification of the environment of the CF lung) has long been known (Smith and Welsh, 1992; Poulsen et al., 1994; Coakley et al., 2001; Luckie et al., 2001; Tate et al., 2002; Coakley et al., 2003). This was the first time a group in a field of CF research has made such a dramatic exclamation about the importance and mode of bicarbonate transport. Following publications of these results, much 0F research has been focused on the importance of bicarbonate transport and pH regulation. The data and claims published since, even by this group, have been more indicative of regulation and communication of several members of the SL026 family of chloride-bicarbonate exchangers with CFTR (Ko et al., 2002). In 2002, Muallem and colleagues published data showing that the molecular mechanism for aberrant and yet CFTR-dependent bicarbonate transport associated with CF, is due to regulation of members of the SL026 family by CFTR (Ko et al., 2002). Their results indicated that CFTR increases activation of Cl' and H07 H005 transport by three members of this family: DRA, SL026A6 and pendrin (Ko et al., 2002). DRA was also indicated in the interaction with CFT R in mouse pancreas 96 and was found to have altered function when certain CFTR mutations were present (R117H and G551 D) (Ko et al., 2002). Interaction and regulation of a H005 salvage mechanism via Na*-HCO5 cotransporter 3 (NBC3) with CFTR also was shown by this group, again supporting the regulation of H005 transport by CFTR, but through interactive mechanisms rather than direct involvement (Park et al., 2002). In these studies, HEK cells were stably and transiently transfected with NBC3. The authors looked at bicarbonate transport and characterized it using various inhibitors and activators. The results indicated that CFTR and NBC3 collaborate in the transport of H005 and maintain a protein-protein interaction through their respective PDZ domains (Park et al., 2002). This interaction was obligatory for regulation of NBC3 and the H005 salvage mechanism by CFTR (Park et al., 2002). Early in 2004, this group published two new papers further addressing the question of bicarbonate transport through CFTR and its role in regulation of Cl‘ IHCO5 exchange. Ko et al. (2004) showed direct molecular interactions between CFTR and members of the SL026 family of CI’IHCO5 exchangers. They focused their research on two members of this family, A3 (DRA) and A6. They found a reciprocal regulation between these two 0l'/H005exchangers and CFTR. SL026 transporter activity was increased when CFTR was activated by phosphorylation (Ko et al., 2004). Interestingly, they also found that CFTR channel open probability was increased when SL026 transporters were present (Ko et al., 2004). The interaction of these proteins was maintained through their attachment to PDZ binding proteins. Both CFTR and SL026 transporters contain PDZ 97 binding domains and are found to bind to similar PDZ scaffold proteins such as EBP50 and EKARP (Ko et al., 2004; Gray, 2004). When they attach to P02 binding proteins and are in proximity, the phosphorylation of the R domain of CFTR causes its binding to a conserved STAS domain of SL026 transporters (Ko et al., 2004). K0 and colleagues found that this molecular interaction is responsible for reciprocal upregulation of CFTR and SL026 transporters and has a significant impact on regulation of chloride and bicarbonate transport in epithelial cells. They also have further proposed a mechanism for differential bicarbonate secretion in the pancreatic duct system which depends on the expression of SL026 transporters. Shcheynikov et al. (2004) examined control of Cl‘/ H005 transport by measuring membrane potential and pH; and/or current in Xenopus oocytes expressing CFTR. Their results indicated a dynamic control of the Cl'/ H005 permeability ratio of CFTR that was dependent on external CI' concentration. When CI' concentration was low or media was Cl' free, their results pointed to OH' and H005 influx but no HCO5efflux. They concluded that permeability of CFTR to H005 at Cl' free times is low. They hypothesized that CFTR exists in two conformations (a Cl' only and a CI’ and OH] H005 permeable state) and that the switch between these two states is the external CI' concentration. However, according to them, most of H005 secretion is mediated by the SL026 transporters, as discussed above, and the switch between the two conformation of CFTR channel serves as a “valve to clamp the HCO5gradient across the luminal membrane according to the CI‘ gradient” (Shcheynikov et al., 2004). They 98 suggested that this effect may be important in tissues that secret fluid with high [H005], such as the pancreas and salivary glands (Shcheynikov et al., 2004). The final conclusion of this group seems to be that CFTR is a channel with two conformational states (a Cl' only and a CI' and OH'IHCOa'permeable state), that also controls CI‘IHCO5 exchange through a PDZ domain mediated interaction with members of the SL026 transporter family. 5. 3. Conclusions From Literature Review Historically, work on CF mechanisms has mostly focused on the first model of CFTR function (CFTR as a Cl' channel). Since 1983, when the Cl' defect was first characterized, there has been much work carried out supporting CFTR's role as a channel. Recently, more interest has been directed toward looking at CFTR's relationships with other cell transporters. As presented in the literature review above, CFTR was found to be in close regulatory interaction with other cell transporters and AE's. These data clearly support the fourth model which suggests that CFTR is a CI' channel that allows for H005 transport and regulates one or more AEs. There have been no results published in the literature which indicate that CFTR is only an AE, and only one study (Choi et al., 2001 b) stating that CFTR has an AE component. This study (Choi et al., 2001b) seems to have since been refuted (Shcheynikov et al., 2004; K0 et al., 2004). Thus, based on the current literature review, the field remains divided on the issue of CFTR and its role in regulation of H005 transport and pH. In my opinion, data reported thus far could support both models I (CFTR as a Cl' channel only) and IV (CFTR as a Cl‘ channel with regulation of an AE). However, if model one is favored, the discrepancies in experimental data still need to be accounted for. 6. Hypothesized Model of CFTR Function Based on All Current Experimental Data At the beginning of this project, I set out to characterize regulation of Cl' IHCO5 transport and extracellular pH by CFTR. More specifically, I wanted to distinguish CI'IHCO5 transport through CFTR as either channel mediated (mostly supported by the field) or exchanger mediated (as proposed by Choi et al., 2001b). l have used two different sets of studies, iodide efflux and microphysiometer experiments, to examine the exchanger hypothesis. Iodide efflux results were used to characterize CI‘ conductance through CFTR and examine the effectiveness of inhibitors and stimulators used. Microphysiometer studies were used in examining the exchanger hypothesis. Examining this question, I set up four possible models of CFTR function: I. CFTR as a Cl' channel ll. CFTR as an AE Ill. CFTR as both a Cl' channel and an AE IV. CFTR as a Cl' channel that allows for transport of H005 and regulates an AE Previously, I have defined three characteristics expected if CFTR was an exchanger: 1) bicarbonate transport should be Cl' dependent and stimulated with forskolin, 2) glibenclamide and DPC should stop efflux of chloride through the 100 channel, but should not prevent action of CFTR as an anion exchanger, and 3) DIDS should block bicarbonate movement thorough CFTR’s anion exchanger in expression cells. None of these characteristics were observed in my data. Glibenclamide and DPC were found to prevent the forskolin stimulated bicarbonate efflux through CFTR (as observed by increased acidification rate following the inhibitor treatment). DIDS treatment did not prevent bicarbonate efflux stimulated with forskolin; rather it was found to increase it in all cell lines used. This was indicative of inhibition of AEs found in all three cell lines, and not an AE specific to the CFTR expressing line. In addition, the decrease in acidification rate during the simultaneous DIDS and forskolin treatment was greater than with DIDS alone only in wild type cells. These results indicated that upon CFTR activation with forskolin there is an additional base efflux to compensate for increased metabolic rate. Finally, the most striking piece of evidence came from studies with Cl' free media where forskolin treatment in wild type cells caused an even larger decrease in acidification than it did in CI' containing media. H005 transport observed through CFTR was not Cl‘ dependent and therefore was most likely not mediated by an exchanger mechanism. Similarly, as shown by the data recorded in the field since the start of my Masters study, the results of Choi et al. (2001b) that were indicative of CFTR serving as a CI'/ H005 exchanger, could be explained with a very tight interaction of CFTR and other Cl‘l H005 exchangers, such as members of SL026 transporter family like DRA and A6. CFTR has been shown to regulate 101 these transporters and therefore Cl'l H005 exchange through interactions with PDZ domains (Ko et al., 2004). PDZ mediated interactions of CFTR with other transporters, however, are not an isolated event with SL026 family. CFTR also has been shown to interact with multiple PDZ proteins such as EBP50, Na"/H+ exchanger type 3 kinase A, CAP70, and so on (Kunzelmann, 2001). It has been suggested as a factor in regulation of epithelial fluid and electrolyte transport that not only functions as a 01‘ channel but also affects other exchangers and channels (Lee et al., 1999b; Ahn et al., 2001). It is thought that CFTR interacts with other cell proteins, such as ENaC, through their respective PDZ domains and scaffolding proteins (Kunzelmann, 2001). CFTR has been suggested to affect Na+ absorption through epithelial Na+ channels (ENaC), K” secretion through luminal K+ channels, and H005 salvage through NaVHCO5 exchange (Lee et al., 1999b; Ahn et al., 2001). These studies all support CFTR function as presented by model IV (CFTR as a 01' channel that also allows for transport of H005 and regulates an AE). It is important to note that our predicted "models" are just a beginning to answering the question of CFTR function. For example, we have not accounted for a model assuming that CFTR is mainly a CI' channel that also allows for a bicarbonate transport but does not regulate other transporters. This model is very similar to model IV presented in this work (CFTR as a Cl' channel that also allows for transport of H005 and regulates an AE). Essentially, a 01' channel that allows for bicarbonate transport would at times be same as model IV, the only difference would be observed when CFTR is involved in regulation of other 102 transporters and/or exchangers. Even though our research did not show the interaction of CFTR with other proteins (as it is assumed in model IV) there has been much evidence in recent literature of such occurrences. The exact influence of CFTR on these transporters and its molecular mechanism of action are still not completely characterized. Yet it seems likely that the field of CF research may be heading towards a better understanding of all of these interactions and their importance in development of the disease. It is my opinion, that the results from the current research and those of others support the idea that CFTR is a Cl' channel that allows for H005 transport and regulates Cl'l H005 exchange (model IV). My results specifically do not support the models I (CFTR as only a CI' channel), ll (CFTR as an AE) and III (CFTR as a Cl' channel and a Cl'/ H005 exchanger). 103 REFERENCES Ahn, W., Kim, K.H., Lee, J.A., Kim, J.Y., Choi, J.Y., Moe, O.W., Milgram, S.L., Muallem, S., and Lee, MG. (2001). Regulatory interaction between the cystic fibrosis transmembrane conductance regulator and H005 salvage mechanisms in model systems and the mouse pancreatic duct. J. Biol. Chem. 276: 1 7236-1 7243 Amaral, MD. (2004). CFTR and chaperones: processing and degradation. J. Mol. Neurosci. 23:41-8 Ames, G.F., and Lecar, H. (1992). ATP-dependent bacterial transporters and cystic fibrosis: analogy between channels and transporters. FASEB J. 6:2660-2666 Anderson, M.P., Rich, D.P., Gregory, R.J., Smith, AE. and Welsh, M.J. (1991a). Generation of cAMP-activated chloride currents by expression of CFTR. Science 251 :679-682 Anderson, M.P., Gregory, R.J., Thompson, S., Souza, D.W., Paul, S., Mulligan, R.C., Smith, AE., and Welsh, M.J. (1 991 b). Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 253:202-205 Ballard, S.T., Trout, L., Bebok, Z., Sorscher, E.J., and Crews, A. (1999). CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands. Am. J. Physiol. 277:L694-L699 Bear, C.E., Li, C.H., Kartner, N., Bridges, R.J., Jensen, T.J., Ramjeesingh, M., and Riordan, JR. (1992). Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell 68:809-818 Berne, R. M., Levy, M. N., Koeppen, B.M., and Stanton, BA. (2004). "Gastrointestinal secretions." Physiology 5"1 ed. Published by Mosby. Boucher, R.C., Knowles, M.R., Stutts, M.J., and Gatzy, J.T. (1983). Epithelial dysfunction in cystic fibrosis lung disease. Lung 161 :1-17 Brookes, N., and Turner, R.J. (1994). K(+)-induced alkalinization in mouse cerebral astrocytes mediated by reversal of electrogenic Na(+)-HCO5 cotransport. Am. J. Physiol. 267:01633-1640 104 Busch, R. (1990). On the history of cystic fibrosis. Acta Univ. Cami. Med. 36:13- 15 Chan, H.C., Ko, W.H., Zhao, W., Fu, W.O., and Wong, P.Y.D. (1996). Evidence for independent CI' and H005 secretion and involvement of an apical Na*- H005 cotransporter in cultured rat epididymal epithelia. Experimental Physiology 81 : 515-524 Cheng, S.H., Gregory, R.J., Marshall, J., Paul, S., Souza, D.W., White, G.A., O’Riordan, CR, and Smith, AE. (1990). Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63:827-834 Choi, J.Y., Muallem, D., Kiselyov, K., Lee, M.G., Thomas, P.J., and Muallem, S. (2001a). Aberrant CFTR-dependent H005 transport in mutations associated with cystic fibrosis. Nature 410:94-7 Choi, J.Y., Lee, M.G., Ko, 8., and Muallem, S. (2001b). Cl'-dependent H005 transport by cystic fibrosis transmembrane conductance regulator. J. Pancreas (Online) 2:243-246 Clarke, LL, and Harline, MC. (1998). Dual role of CFTR in cAMP-stimulated H005 secretion across murine duodenum. Am. J. Physiol. 274:G718—726 Coakley, RD, and Boucher, RC. (2001). Regulation and functional significance of airway surface liquid pH. J. Pancreas (Online) 2:294-300 Coakley, R.D., Grubb, B.R., Paradiso, A.M., Gatzy, J.T., Johnson, L.G., Kreda, S.M., O’Neal, W.K., and Boucher, RC. (2003). Abnormal surface liquid pH regulation by cultured cystic fibrosis bronchial epithelium. Proc. Natl. Acad. Sci. USA 26:16083-16088 Denning, G.M., Anderson, M.P., Amara, J.F., Marshall, J., Smith, AE., and Welsh, M.J. (1992). Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive [see comments]. Nature 358:761 -764 Egan, M., Flotte, T., Afione, S., Solow, R., Zeitlin, P.L., Barrie, J0, and Guggion, W.B. (1992). Defective regulation of outwardly rectifying 0l- channels by protein kinase A corrected by insertion of CFTR. Nature 358:581-584 Gilljam, H., Ellin, A., and Strandvik, B. (1989). Increased bronchial chloride concentration in cystic fibrosis. Scand. J. Clin. Lab. Invest. 49:121-124 Gray, M. A. (2004). Bicarbonate secretion: it takes two to tango. Nature Cell Biol. 6:292-294 105 Gray, M.A., Harris, A., Coleman, L., Greenwell, JR, and Argent, BE. (1989). Two types of chloride channel on duct cells cultured from human fetal pancreas. Am. J. Physiol. 257:0240-0251 Gregory, R.J., Cheng, S.H., Rich, D.P., Marshall, J., Paul, S., Hehir, K., Ostedgaard, L., Klinger, K.W., Welsh, M.J., and Smith, AE. (1990). Expression and characterization of the cystic fibrosis transmembrane conductance regulator. Nature 347:382-386 Grubb, BR, and Gabriel, SE. (1997). Intestinal physiology and pathology in gene-targeted mouse models of cystic fibrosis. Am. J. Physiol. 273:G258- G266. Hafeman, D.G., Parce, J.W., and McConell, HM. (1988). Light-addressable potentiometric sensor for biochemical systems. Science 240:1 182-1185 Haggie, P.M., Stanton, BA, and Verkman, AS. (2004). Increased diffusional mobility of CFTR at the plasma membrane after deletion of its C-terminal PDZ binding motif. J. Biol. Chem. 279:5494-5500 Humphreys, B.D., Jiang, L., Chernova, MN, and Alper, S.L. (1994). Functional characterization and regulation by pH of murine AE2 anion exchanger expressed in Xenopus oocytes. Am. J. Physiol. 267:01295—12307 Illek, B., Yankaskas, JR, and Machen, TE. (1997). cAMP and genistein stimulate H005 conductance through CFTR in human airway epithelia. Am. J. Physiol. 272:L752-L761 Illek, B., Fischer, H., and Machen, TE. (1998). Genetic disorders of membrane transport. ll. Regulation of CFTR by small molecules including H005. Am. J. Physiol. 275:G1221-G1226 Jiang, 0., Finkbeiner, W.E., Wrddicombe, J.H., McCray, P.B., Jr., and Miller, SS. (1993). Altered fluid transport across airway epithelium in cystic fibrosis. Science 262:424-427 Jones, RM, and George, AM. (2004) The ABC transporter structure and mechanism: perspectives on recent research. Cell. Mol. Life Sci. 61 :682- 699 Joris, L., Dab, l., and Quinton, PM. (1993). Elemental composition of human airway surface fluid in healthy and diseased airways. Am. Rev. Respir. Dis. 148:1633-1637 106 Kartner, N., Hanrahan, J.W., Jensen, T.J., Naismith, A.L., Sun, 82., Ackerley, C.A., Reyes, E.F., Tsui, L.-C., Rommens, J.M., Bear, CE, and Riorday, JR. (1991). Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance. Cell 64:681- 691 Knowles, M.R., and Boucher, RC. (2002). Mucus clearance as a primary innate defense mechanism for mammalian airways. J. Clin. Invest. 109:571-577 Ko. S.B.H., Shcheynikov, N., Choi, J.Y., Luo, X., lshibashi, K., Thomas, P.J., Kim, J.Y., Kim, K.H., Lee, M.G., Naruse, S., and Muallem, S. (2002). A molecular mechanism for aberrant CFTR-dependent H005 transport in cystic fibrosis. EMBO J. 21 :5662-5672 Ko, S.B., Zeng, W., Dorwart, M.R., Luo, X., Kim, K.H., Millen, L., Goto, H., Naruse, S., Soyombo, A., Thomas, P.J. and Muallem, S. (2004). Gating of CFTR by the STAS domain of SL026 transporters. Nat. Cell Biol. 6:343- 350 Kopelman, H., Corey, M. Gaskin, K., Durie, P., Weizman, Z., and Forstner, G. (1988). Impaired chloride secretion, as well as bicarbonate secretion, underlies the fluid secretory defect in the cystic fibrosis pancreas. Gastroenterology. 95:349-355 Kunzelmann, K. (1999). The cystic fibrosis transmembrane conductance regulator and its function in epithelial transport. Rev. Physiol. Biochem. Pharmacol. 137:1-70 Kunzelmann, K., and Schreiber, R. (1999). CFTR, a regulator of channels. J. Memb. Biol. 168:1-8 Kunzelmann, K. (2001). CFTR: Interacting with everything? News. Physiol. Sci. 16:167-170 Lee, lVI.C., Penland, C.M., VIfiddicombe, J.H., and Wine, J.J. (1998). Evidence That Calu-3 human airway cells secrete bicarbonate. Am. J. Physiol. 274:L450-L453 Lee, M.G., Wrgley, W.C., Zeng, W., Noel, L.E., Marino, C.R., Thomas, P.J., and Muallem, S. (1999a). Regulation of CI'IHCO5 exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cell. J. Biol. Chem. 274:3414-3421 Lee, M.G., Choi, J.Y., Luo, X., Strickland, E., Thomas, P.J., and Muallem, S. (1999b). Cystic fibrosis transmembrane conductance regulator regulates 107 luminal CI'IHCO5 exchange in mouse submandibular and pancreatic ducts. J. Biol. Chem. 274:14670-14677 Luckie, DB, and Wine, J.J. (1998). CFTR expression can change extracellular pH. Pediatric Pulmonology Suppl. 16:226A Luckie, D.B., Singh, C.N., Wine, J.J., and Wilterding, J.H. (2001). CFTR activation raises extraccellular pH of NIH/3T3 mouse fibroblasts and C127 epithelial cells. J. Memb. Biol. 179:275-284 Mall, M. Grubb, B.R., Harkema, J.R., O’Neal, W.K. and Boucher, RC. (2004). Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. Nature Med. 10:487-493 Marshall, J., Fang, S., Ostedgaard, L.S., O’Riordan, C.R., Ferrara, D., Amara, J.F., Hoppe, H., W., Scheule, R.K., Welsh, M.J., Smith, AE. and Cheng, SH. (1994). Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro. J. Biol. Chem. 269:2987-2995 Mastrocola, T., Porcelli, AM, and Rugolo, M. (1998). Role of CFTR and anion exchanger in bicarbonate fluxes in 0127 cell lines. FEBS Lett. 440:268- 272 Matsui, H., Grubb, B.R., Tarran, R., Randell, S.H., Gatzy, J.T., Davis, CW, and Boucher, RC. (1998). Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95: 1 005-1 01 5 McConnell, H. M., Owicki, J.C., Parce, J.W., Miller, D.L., Baxter, G.T., Wada, H.G., and Pitchford, S. (1992). The cytosensor microphysiometer: biological applications of silicon technology. Science 257:1906-1912 Meindl, RS. (1987). Hypothesis: a selective advantage for cystic fibrosis heterozygotes. Am. J. Physiol. Anthropol. 74:39-45 Parce, J.W., Owicki, J.C., Kercso, K.M., Sigal, G.B., Wada, H.G., Muir, V.C., Bousse, L.J., Ross, K.L., Sikic, BL, and McConnell, HM. (1989). Detection of cell-affecting agents with a silicon biosensor. Science 246:243-247 Park, M., Ko, S.B.H., Choi, J.Y., Muallem, G., Thomas, P.J., Pushkin, A., Lee, M- S., Kim, J.Y., Lee, M.G., Muallem, S., and Kurtz, l. (2002). The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the H005 salvage transporter human Na*-HCO5 cotransport isoform 3. J. Biol. Chem. 277:50503—50509 108 Pind, S., Riordan, JR. and Williams, DB. (1994). Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 269: 1 2784-1 2788 Poulsen, J. H., Fischer, H., Illek, B., and Machen, TE. (1994). Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc. Natl. Acad. Sci. USA 91:5340-5344 Poulsen, J.H., and Machen, TE. (1996). H005 dependent pH; regulation in tracheal epithelial cells. Pfluegers Arch. 432:546-554 Purves, W.K., Sadava, D., Orians, G.H., and Heller, H0 (2004 . "The mechanisms of evolution." Life: The science of biology 7 " ed. Published by W. H. Freeman & Co. Quinton, P. M. (1983). Chloride impermeability in cystic fibrosis. Nature 301 :421- 422 Quinton, PM. (1999). Physiological basis of cystic fibrosis: a historical perspective. Physiol. Rev. 79:83-822 Raghuram, V., Mak, 0.0., and Foskett, J.K. (2001). Regulation of cystic fibrosis transmembrane conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction. Proc. Natl. Acad. Sci. USA 98:1300- 1305 Raghuram, V., Hormuth, H., and Foskett, J.K. (2003). A kinase-regulated mechanism controls CFTR channel gating by disrupting bivalent PDZ domain interactions. Proc. Natl. Acad. Sci. USA 100:9620-9625 Rashid, M., Durie, P., Andrew, M., Kalnins, D., Shin, J., Corey, M., Tullis, E. (1999). Prevalence of vitamin K deficiency in cystic fibrosis. Am. J. Clin. Nutr. 70:378-382 Reddy, V.B., Garramone, A.J., Sasak, H., Wei, C.M., Watkins, P., Galli, J., and Hsiung, N. (1987). Expression of human uterine tissue-type plasminogen activator in mouse cells using BPV vectors. DNA 6:461-472 Reddy, MM, and Quinton, PM. (2002). Effect of anion transport blockers on CFTR in the human sweat duct. J. Membrane Biol. 189:15-25 Reddy, MM, and Quinton, PM. (2003). Functional interaction of CFTR and ENaC in sweat glands. Pflugers Arch. 445:499-503 109 Rich, D.P., Gregory, R.J., Anderson, M.P., Manavalan, P., Smith, AE., and Welsh, M.J. (1991). Effect of deleting the R domain on CFTR-generated chloride channels. Science 253:205-207 Riordan, J. R., Rommens, J.M, Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.L., Drumm, M.L., lannuzzi, M.C., Collins, PS, and Tsui, L.-C. (1989). Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA [published erratum appears in Science 1989 Sep 29; 245(4925):1437]. Science 245:1066-1073 Rodman, D.M., and Zamudio, S. (1991). The cystic fibrosis heterozygote- advantage in surviving cholera? Med. Hypotheses 36:253-258 Rommens, J.M., Dho, 8., Bear, C.E., Kartner, N., Kennedy, 0, Riordan, J.R., Tsui, LC, and Foskett, J.K. (1991). CAMP-inducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator. Proc. Natl. Acad. Sci. USA 88:7500-7504 Saraste, M., Sibbald, PR. and Wrttinghofer, A. (1990). The P-loop-—a common motif in ATP- and GTP-binding proteins. Trends Biochem. Sci. 15:430—434 Sato, 8., Ward, C.L., Krouse, M.E., Wine, J.J., and Kopito, RR. (1996). Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J. Biol. Chem. 271:635-638 Schwiebert, E.M., Benos, D.J., Egan, M.E., Stutts, M.J., and Guggino, W.B. (1999). CFTR is a conductance regulator as well as a chloride channel. Physiol. Rev. 79:S145-S166 Seidler, U., Blumenstein, l., Kretz, A., Viellard-Baron, D., Rossmann, H., Colledge, W.H., Evans, M., Ratcliff, R., and Gregor, M. (1997). A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca(2+)-dependent H005 secretion. J. Physiol. 505:411-423 Sferra, T.J., and Collins, PS. (1993). The molecular biology of cystic fibrosis. Annu. Rev. Med. 44:133-144 Shanna, M., Benharouga, M., Hu, W., and Lukacs, G.L. (2001). Conformational and temperature-sensitive stability defects of the AF508 cystic fibrosis transmembrane conductance regulator in post-endoplasmic reticulum compartments. J. Biol. Chem. 276:8942-8950 Shcheynikov, N., Kim, K.H., Kim, K.M., Dorwart, M.R., Ko, S.B., Goto, H., Naruse, S., Thomas, P.J., and Muallem S. (2004). Dynamic control of 110 CFTR Cl'/HCO5 selectivity by external Cl'. J. Biol. Chem. 279:21857- 21 865 Sheng, M., and Sala, C. (2001). PDZ domains and the organization of supramolecular complexes. Annu. Rev. Neurosci. 24:1-29 Sheppard, UN, and Welsh, M.J. (1999). Structure and function of the CFTR chloride channel. Physiol. Rev. 79:823-845 Shumaker, H., Amlal, H., Frizzell, R., Ulrich, CD, 2"", and Soleimani, M. (1999). CFTR drives Nai-nHCO5 cotransport in pancreatic duct cells: a basis for defective H005 secretion in CF. Am. J. Phyisol. 276:016—025 Smith, J.J., and Welsh, M.J. (1993). Fluid and electrolyte transport by cultured human airway epithelia. J. Clin. Invest. 91:1590-1597 Smith, J.J., and Welsh, M.J. (1992). cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia. J. Clin. Invest. 89:1148-1153 Smith, J.J., Karp, PH, and Welsh, M.J. (1994). Defective fluid transport by cystic fibrosis airway epithelia. J. Clin. Invest. 93:1307-1311 Smith, J.J., Travis, S.M., Greenberg, E.P., and Welsh, M.J. (1996). Cystic fibrosis airway epithelial fail to kill bacteria because of abnormal airway surface fluid. Cell 85:229-236 Tabcharani, J.A., Chang, X.B., Riordan, JR. and Hanrahan, J.W. (1991). Phosphorylation-regulated CI- channel in CHO cells stably expressing the cystic fibrosis gene. Nature 352:628-631 Tabcharani, J.A., Chang, X-B., Riordan, JR, and Hanrahan, J.W. (1992). The cystic fibrosis transmembrane conductance regulator chloride channel - Iodide block and permeation. Biophys. J. 62:14 Tate, S., MacGregor, G., Davis, M., lnnes, J.A., and Greening, AP. (2002). Airways in cystic fibrosis are acidified: detection by exhaled breath condensate. Thorax. 57:926—929 Thiagarajah, JR, and Verkman, AS. (2003). CFTR pharmacology and its role in intestinal fluid secretion. Curr. Opin. Pharmacol. 3:594-599 Timmreck, L.S., Gray, M.R., Handelin, B., Allito, B., Rohlfs, E., Davis, A.J., Gidwani, G., and Reindollar, RH. (2003). Analysis of cystic fibrosis transmembrane conductance regulator gene mutations in patients with 111 congenital absence of the uterus and vagina. Am. J. Med. Genet. 120A:72-76 Venglarik, C.J., Bridges, R.J., and Frizzell, RA. (1990). A simple assay for agonist-regulated CI and K conductances in salt-secreting epithelial cells. Am. J. Physiol. 259:0358—0364 Walker, J.E., Saraste, M., Runswick, M.J., and Gay, NJ. (1982). Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1:945-951 Wang, S., Yue, H., Derin, R.B., Guggino, W.B., and Li, M. (2000). Accessory protein facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate the chloride channel activity. Cell 103:169-179 Wang, X.F., Reddy, MM, and Quinton, PM. (2004). Effects of a new cystic fibrosis transmembrane conductance regulator inhibitor on Cl' conductance in human sweat ducts. Exp. Physiol. 89:417-425 Welsh, M.J. (1990). Abnormal regulation of ion channels in cystic fibrosis epithelia. Faseb J 4:2718-2725 Welsh, M.J., and Smith, AE. (1995). Cystic Fibrosis. Sci. Am. 273:52-59 VIfine, J. J. (1991). Basic aspects of cystic fibrosis. Clin. Rev. Allergy. 9:1-28 Zabner, J., Smith, J.J., Karp, P.H., Wrddicombe, J.H., and Welsh, M.J. (1998). Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro. Mol. Cell 2:397-403 Zhang, Y., Reenstra, WW, and Chidekel, A. (2001). Antibacterial activity of apical surface fluid from the human airway cell line Calu-3: pharmacologic alteration by corticosteroids and beta(2)-agonists. Am. J. Respir. Cell Mol. Biol. 25: 1 96-202 Zou, X., and Hwang, T.-C. (2001). ATP hydrolysis-coupled gating of CFTR chloride channels: structure and function. Biochemistry 40:5579—5586 Yoo, B. lyengar, R., and Chen, Y. (2004). Functional analysis of the interface regions involved in interactions between the central cytoplasmic loop and C-terminal tail of adenylyl cylclase. J. Biol. Chem. 279:13925-13933 112 llllllllllllllllll