Fibrosis Transmembrane Conductance Regulator Chloride Research Articles

Ractopamine, a livestock feed additive, is a full agonist at trace amine-associated receptor 1.

Ractopamine (RAC) is fed to an estimated 80% of all beef, swine, and turkey raised in the United States. It promotes muscle mass development, limits fat deposition, and reduces feed consumption. However, it has several undesirable behavioral side effects in livestock, especially pigs, including restlessness, agitation, excessive oral-facial movements, and aggressive behavior. Numerous in vitro and in vivo studies suggest RAC's physiological actions begin with its stimulation of β1- and β2-adrenergic receptor-mediated signaling in skeletal muscle and adipose tissue; however, the molecular pharmacology of RAC's psychoactive effects is poorly understood. Using human cystic fibrosis transmembrane conductance regulator (hCFTR) chloride channels as a sensor for intracellular cAMP, we found that RAC and p-tyramine (TYR) produced concentration-dependent increases in chloride conductance in oocytes coexpressing hCFTR and mouse trace amine-associated receptor 1 (mTAAR1), which was completely reversed by the trace amine-associated receptor 1 (TAAR1)-selective antagonist EPPTB [N-(3-ethoxyphenyl)-4-pyrrolidin-1-yl-3-trifluoromethylbenzamide]. Oocytes coexpressing hCFTR and the human β2-adrenergic receptor showed no response to RAC or TYR. These studies demonstrate that, contrary to expectations, RAC is not an agonist of the human β2-adrenergic receptor but rather a full agonist for mTAAR1. Since TAAR1-mediated signaling can influence cardiovascular tone and behavior in several animal models, our finding that RAC is a full mTAAR1 agonist supports the idea that this novel mechanism of action influences the physiology and behavior of pigs and other species. These findings should stimulate future studies to characterize the pharmacological, physiological, and behavioral actions of RAC in humans and other species exposed to this drug.

Anoctamin 1 induces calcium-activated chloride secretion and proliferation of renal cyst–forming epithelial cells

Polycystic kidney diseases are characterized by multiple bilateral renal cysts that gradually enlarge and lead to a decline in renal function. Cyst enlargement is driven by transepithelial chloride secretion, stimulated by enhanced levels of cyclic adenosine monophosphate, which activates apical cystic fibrosis transmembrane conductance regulator chloride channels. However, chloride secretion by calcium-dependent chloride channels, activated through stimulation of purinergic receptors, also has a major impact. To identify the molecular basis of calcium-dependent chloride secretion in cyst expansion, we determined the role of anoctamin 1 and 6, two recently discovered calcium-activated chloride channels both of which are expressed in epithelial cells. We found that anoctamin 1, which plays a role in epithelial fluid secretion and proliferation, is strongly expressed in principal-like MDCK cells (PLCs) forming cysts within a collagen matrix, in an embryonic kidney cyst model, and in human autosomal dominant polycystic kidney disease tissue. Knockdown of anoctamin 1 but not anoctamin 6 strongly diminished the calcium-dependent chloride secretion of PLCs. Moreover, two inhibitors of anoctamin ion channels, tannic acid and a more selective inhibitor of anoctamin 1, significantly inhibited PLC cyst growth and cyst enlargement in an embryonic kidney cyst model. Knockdown of ANO1 by morpholino analogs also attenuated embryonic cyst growth. Thus, calcium-activated chloride secretion by anoctamin 1 appears to be a crucial component of renal cyst growth.

Mo1675 Pharmacokinetics and Tolerability of iOWH032, an Inhibitor of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Channel, in Normal Volunteers and Cholera Patients

Mo1675 Pharmacokinetics and Tolerability of iOWH032, an Inhibitor of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Channel, in Normal Volunteers and Cholera Patients

Gastrointestinal Pathology in Juvenile and Adult CFTR-Knockout Ferrets

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.

Steviol retards renal cyst growth through reduction of CFTR expression and inhibition of epithelial cell proliferation in a mouse model of polycystic kidney disease

Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) is associated with cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel leading to renal failure for which no effective treatment is currently available. We previously reported that steviol retards Madin–Darby canine kidney (MDCK) cyst enlargement by inhibiting CFTR channel activity and promoting proteasomal-mediated CFTR degradation. It is imperative to examine the effect of steviol in animal models of ADPKD. Therefore, we examined the effect of steviol on renal cyst growth in an orthologous mouse model of human ADPKD (Pkd1flox/flox:Pkhd1-Cre). The results showed that daily treatment with both 200mg/kg BW of steviol and 1000mg/kg BW of stevioside for 14 days markedly decreased kidney weight and cystic index in these mice. However, only steviol markedly reduced blood urea nitrogen and creatinine values. Steviol also reduced cell proliferation but had no effect on cell apoptosis. In addition, steviol suppressed CFTR and mTOR/S6K expression in renal cyst-lining epithelial cells. Interestingly, steviol was found to stimulate AMP-activated protein kinase (AMPK). Our findings indicate that steviol slows cyst progression in ADPKD mouse model, in part, through the activation of AMPK which subsequently inhibits CFTR chloride channel expression and inhibits renal epithelial cell proliferation via mTOR/S6K pathway. Most importantly, steviol could markedly improve kidney function in a mouse model of ADPKD. Steviol thus has potential application for further development as a therapeutic compound for the treatment of polycystic kidney disease.

Cystic fibrosis transmembrane conductance regulator chloride channel blockers: Pharmacological, biophysical and physiological relevance

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel causes cystic fibrosis, while inappropriate activity of this channel occurs in secretory diarrhea and polycystic kidney disease. Drugs that interact directly with CFTR are therefore of interest in the treatment of a number of disease states. This review focuses on one class of small molecules that interacts directly with CFTR, namely inhibitors that act by directly blocking chloride movement through the open channel pore. In theory such compounds could be of use in the treatment of diarrhea and polycystic kidney disease, however in practice all known substances acting by this mechanism to inhibit CFTR function lack either the potency or specificity for in vivo use. Nevertheless, this theoretical pharmacological usefulness set the scene for the development of more potent, specific CFTR inhibitors. Biophysically, open channel blockers have proven most useful as experimental probes of the structure and function of the CFTR chloride channel pore. Most importantly, the use of these blockers has been fundamental in developing a functional model of the pore that includes a wide inner vestibule that uses positively charged amino acid side chains to attract both permeant and blocking anions from the cell cytoplasm. CFTR channels are also subject to this kind of blocking action by endogenous anions present in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physiological control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease states, of CFTR channel structure and function, and of how CFTR activity is controlled by its local environment.

Aqueous cigarette smoke extract induces a voltage-dependent inhibition of CFTR expressed in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel inhabits the apical membrane of airway epithelia, where its function is essential for mucus hydration, mucociliary clearance, and airway defense. Chronic obstructive pulmonary disease (COPD), most often a consequence of cigarette smoke (CS) exposure, affects 15 million persons in the US. Clinically, COPD is characterized by many of the salient features of cystic fibrosis lung disease, where CFTR is either absent or reduced in function. CS is an acidic aerosol (pH 5.3 to 6.3) reported to contain over 4,000 constituents. Acute CS exposure has been reported to decrease airway transepithelial voltage in vivo and short-circuit current in vitro; however, the mechanistic basis of these effects is uncertain. The goal of the studies described here was to develop a bioassay to characterize the effects of aqueous CS preparations on the channel function of CFTR. We studied aqueous CS extract (CSE) prepared in our laboratory, as well as commercial cigarette smoke condensate (CSC) in Xenopus oocytes expressing human CFTR. Application of CSE at pH 5.3 produced a reversible, voltage-dependent inhibition of CFTR conductance. CSE neutralized to pH 7.3 produced less inhibition of CFTR conductance. Serial dilution of CSE revealed a dose-dependent effect at acidic and neutral pH. In contrast, CSC did not inhibit CFTR conductance in oocytes. We conclude that one or more components of CSE inhibits CFTR in a manner similar to diphenylamine-2-carboxylate, a negatively charged, open-channel blocker.

CGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland

The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG.

Role of Lubiprostone on Gastrointestinal Motility

Lubiprostone is a bicyclic fatty acid derivative of prostaglandin E1 used for the treatment of chronic idiopathic constipation and constipation predominant irritable bowel syndrome.1,2 The underlying mechanism of lubiprostone is stimulation of electrogenic chloride secretion by activating chloride channel type-2 (ClC-2)3 and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels4 in the apical membrane of the intestinal epithelial cells. Activation of ClC-2 channels or CFTR chloride channels in intestinal epithelial cells produces an active secretion of chloride ions from cells into the intestinal lumen followed by a passive secretion of electrolytes and water which increases the liquidity of the luminal contents. The luminal distension by increased intestinal fluid promotes the gastrointestinal (GI) tract motility which in turn increases the intestinal and colonic transit. Lubiprostone also has an effect on cystic fibrosis tissue by independent CFTR chloride channels.5 Lubiprostone induced chloride secretory action is mediated by direct activation of ClC-2 channels as well as stimulation of these channels through activating prostaglandin receptors. Besides, lubiprostone enhances and stimulates contraction in colonic as well as gastric muscles through prostaglandin E receptors (EP1 or EP4),6 suggesting the modulatory effects of lubiprostone on GI motility through the activation of prostaglandin receptors. In this issue of Journal of Neurogastroenterology and Motility, Chan and Mashimo7 investigated the effects of lubiprostone on intestinal smooth muscle contractions and pyloric sphincter tones from murine. They found that lubiprostone increased electrical field stimulation-induced contractions of intestinal circular smooth muscles and pyloric sphincter basal tone but not intestinal longitudinal smooth muscles. The excitatory effects of lubiprostone on intestinal circular smooth muscle and pyloric sphincter basal tone were blocked by EP1 antagonist. They speculated that lubiprostone can change the GI motility by prostaglandin receptor activation and the excitatory action of lubiprostone may be another mechanism for the increased intestinal transit and the delayed gastric emptying by lubiprostone.8 Although this study provides that lubiprostone has a role as a prokinetic agent in GI tract, the underlying mechanisms of modulating contractions were not evaluated. Many cells of GI tract express E-type prostanoid receptors including smooth muscle, neuron, macrophage and interstitial cells of Cajal that contribute to modulation of GI motility.9-11 Therefore, to evaluate the clinical effects of lubiprostone for modulating GI motility, further basic studies should be carried out to find underlying mechanisms.

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

Conformational changes in the catalytically inactive nucleotide-binding site of CFTR

A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C→O1→O2→C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1.Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N6-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2×) transition O1→O2 and decreases the nonhydrolytic closing rate (transition O1→C) of CFTR mutants K1250A (∼4×) and E1371S (∼3×). Mutation H1348A also slowed (∼3×) the O1→O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A (∼3×) and E1371S (∼3×) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1→C and O1→O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.

New Pulmonary Therapies Directed at Targets Other than CFTR

Our current understanding of the pathogenesis of cystic fibrosis (CF) lung disease stresses the importance of the physical and chemical properties of the airway surface liquid (ASL). In particular, the loss of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel function in CF reduces the volume and fluidity of the ASL, thus impairing mucociliary clearance and innate antimicrobial mechanisms. Besides direct approaches to restoring mutant CFTR function, alternative therapeutic strategies may also be considered to correct the basic defect of impaired salt and water transport. Such alternative strategies are focused on the restoration of mucociliary transport by (1) reducing sodium and fluid absorption by inhibiting the ENaC channel; (2) activating alternative chloride channels; and (3) increasing airway surface hydration with osmotic agents. Therapeutic approaches directed at targets other than CFTR are attractive because they are potentially useful to all patients irrespective of their genotype. Clinical trials are underway to test the efficacy of these approaches.

48 Reserpine inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in Caco-2 monolayers

CFTR is a protein widely distributed in a variety of cell types. We have recently shown that human monocytes express CFTR and we developed a single-cell fluorescence imaging assay to evaluate CFTR activity in isolated cells. In the present work, we characterized the electrophysiological properties of CFTR, by patch clamp technique, in human monocytes from healthy and CF subjects and related it to single cell fluorescence imaging. cAMP-dependent chloride currents were detected only in cells from healthy subjects with a concordance between the two methods. These data further support previous observations from our group showing that monocytes could be useful for diagnosing CF and possibly to assess the effects of drugs active on the basic defect.

Molecular Motors and Apical CFTR Traffic in Epithelia

Intracellular protein traffic plays an important role in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channels. Microtubule and actin-based motor proteins direct CFTR movement along trafficking pathways. As shown for other regulatory proteins such as adaptors, the involvement of protein motors in CFTR traffic is cell-type specific. Understanding motor specificity provides insight into the biology of the channel and opens opportunity for discovery of organ-specific drug targets for treating CFTR-mediated diseases.

Absolute Configuration and Biological Properties of Enantiomers of CFTR Inhibitor BPO-27

We previously reported benzopyrimido-pyrrolo-oxazinedione (BPO) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in a model of polycystic kidney disease. Here, we separated the enantiomers of lead compound BPO-27, (1), which contains a single chiral center, and determined their absolute configuration, activity and metabolic stability. Following separation by chiral supercritical fluid chromatography, the R enantiomer, as determined by x-ray crystallography, inhibited CFTR chloride conductance with IC50 ~ 4 nM, while S enantiomer was inactive. In vitro metabolic stability in hepatic microsomes showed both enantiomers as stable, with <5 % metabolism in 4 h. Following bolus interperitoneal administration in mice, serum (R)-1 decayed with t1/2 ~ 1.6 h and gave sustained therapeutic concentrations in kidney.

CFTR and V‐ATPase trafficking and function in the intestinal brush border membrane

The multi‐subunit proton pump, vacuolar ATPase (V‐ATPase) and the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are both regulated by cAMP‐dependent traffic from endosomes to the plasma membrane in ion transporting epithelia. We hypothesized that V‐ATPase traffic and function are linked to CFTR in the intestine. The distribution of V‐ATPase Voa1, Voa2 and V1E subunits and CFTR were examined in wild‐type (WT), CFTR−/− mouse intestine and polarized intestinal CaCo‐2 BBe cells following cAMP stimulation. Functional dependence of V‐ATPase on CFTR was determined in CaCo‐2 BBe cells using the pH sensitive dye BCECF. Co‐immunoprecipitation confirmed Voa1, Voa2 and V1E interaction with CFTR. V‐ATPase Voa1 and Voa2 subunits were localized to the apical and basolateral domains in CaCo‐2 BBe cells. V‐ATPase Voa1 and Voa2 subunits were localized to distinct domains in untreated and treated enterocytes from WT and CFTR−/−intestine. Following cAMP activation, both transporters co‐localized in the apical membrane. Inhibition of V‐ATPase or CFTR significantly decreased proton efflux in CaCo2 BBe cells. These studies suggest functional synergy between both transporters in maintaining intracellular pH; and localization in the intestinal brush border membrane. NIH Grants: R01‐DK‐077065 to N. Ameen and P30‐DK‐34989 to the Yale Liver Center.

Steviol reduces MDCK Cyst formation and growth by inhibiting CFTR channel activity and promoting proteasome-mediated CFTR degradation.

Cyst enlargement in polycystic kidney disease (PKD) involves cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. This study aimed to investigate an inhibitory effect and detailed mechanisms of steviol and its derivatives on cyst growth using a cyst model in Madin-Darby canine kidney (MDCK) cells. Among 4 steviol-related compounds tested, steviol was found to be the most potent at inhibiting MDCK cyst growth. Steviol inhibition of cyst growth was dose-dependent; steviol (100 microM) reversibly inhibited cyst formation and cyst growth by 72.53.6% and 38.2±8.5%, respectively. Steviol at doses up to 200 microM had no effect on MDCK cell viability, proliferation and apoptosis. However, steviol acutely inhibited forskolin-stimulated apical chloride current in MDCK epithelia, measured with the Ussing chamber technique, in a dose-dependent manner. Prolonged treatment (24 h) with steviol (100 microM) also strongly inhibited forskolin-stimulated apical chloride current, in part by reducing CFTR protein expression in MDCK cells. Interestingly, proteasome inhibitor, MG-132, abolished the effect of steviol on CFTR protein expression. Immunofluorescence studies demonstrated that prolonged treatment (24 h) with steviol (100 microM) markedly reduced CFTR expression at the plasma membrane. Taken together, the data suggest that steviol retards MDCK cyst progression in two ways: first by directly inhibiting CFTR chloride channel activity and second by reducing CFTR expression, in part, by promoting proteasomal degradation of CFTR. Steviol and related compounds therefore represent drug candidates for treatment of polycystic kidney disease.

Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle.

Vx-770 (Ivacaftor), a Food and Drug Administration (FDA)-approved drug for clinical application to patients with cystic fibrosis (CF), shifts the paradigm from conventional symptomatic treatments to therapeutics directly tackling the root of the disease: functional defects of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel caused by pathogenic mutations. The underlying mechanism for the action of Vx-770 remains elusive partly because this compound not only increases the activity of wild-type (WT) channels whose gating is primarily controlled by ATP binding/hydrolysis, but also improves the function of G551D-CFTR, a disease-associated mutation that abolishes CFTR's responsiveness to ATP. Here we provide a unified theory to account for this dual effect of Vx-770. We found that Vx-770 enhances spontaneous, ATP-independent activity of WT-CFTR to a similar magnitude as its effects on G551D channels, a result essentially explaining Vx-770's effect on G551D-CFTR. Furthermore, Vx-770 increases the open time of WT-CFTR in an [ATP]-dependent manner. This distinct kinetic effect is accountable with a newly proposed CFTR gating model depicting an [ATP]-dependent "reentry" mechanism that allows CFTR shuffling among different open states by undergoing multiple rounds of ATP hydrolysis. We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Our data corroborate that Vx-770 increases the open time of WT-CFTR by stabilizing a posthydrolytic open state and thereby fosters decoupling between the gating cycle and ATP hydrolysis cycle. The current study also suggests that this unique mechanism of drug action can be further exploited to develop strategies that enhance the function of CFTR.

Cysteine Scanning of CFTR’s First Transmembrane Segment Reveals Its Plausible Roles in Gating and Permeation

Previous cysteine scanning studies of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have identified several transmembrane segments (TMs), including TM1, 3, 6, 9, and 12, as structural components of the pore. Some of these TMs such as TM6 and 12 may also be involved in gating conformational changes. However, recent results on TM1 seem puzzling in that the observed reactive pattern was quite different from those seen with TM6 and 12. In addition, whether TM1 also plays a role in gating motions remains largely unknown. Here, we investigated CFTR’s TM1 by applying methanethiosulfonate (MTS) reagents from both cytoplasmic and extracellular sides of the membrane. Our experiments identified four positive positions, E92, K95, Q98, and L102, when the negatively charged MTSES was applied from the cytoplasmic side. Intriguingly, these four residues reside in the extracellular half of TM1 in previously defined CFTR topology; we thus extended our scanning to residues located extracellularly to L102. We found that cysteines introduced into positions 106, 107, and 109 indeed react with extracellularly applied MTS probes, but not to intracellularly applied reagents. Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. These findings lead us to propose a revised topology for CFTR’s TM1 that spans at least from E92 to Y109. Additionally, side-dependent modifications of these positions indicate a narrow region (L102-I106) that prevents MTS reagents from penetrating the pore, a picture similar to what has been reported for TM6. Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR’s gating cycle. The structural implications of these findings are discussed in light of the crystal structures of ABC transporters and homology models of CFTR.

Conformational Changes in the Catalytically Inactive Nucleotide Binding Site of CFTR

A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C-->O1-->O2-->C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1.Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N6-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2x) transition O1-->O2 and decreases the nonhydrolytic closing rate (transition O1-->C) of CFTR mutants K1250A ( approximately 4x) and E1371S ( approximately 3x). Mutation H1348A also slowed ( approximately 3x) the O1-->O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A ( approximately 3x) and E1371S ( approximately 3x) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1-->C and O1-->O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.