Clinical and genetic features of cystic fibrosis in Japan.

Cystic fibrosis (CF) is an autosomal recessive disease caused by pathogenic variants in CF transmembrane conductance regulator (CFTR). While CF is the most common hereditary disease in Caucasians, it is rare in East Asia. In the present study, we have examined clinical features and the spectrum of CFTR variants of CF patients in Japan. Clinical data of 132 CF patients were obtained from the national epidemiological survey since 1994 and CF registry. From 2007 to 2022, 46 patients with definite CF were analyzed for CFTR variants. All exons, their boundaries, and part of promoter region of CFTR were sequenced and the presence of large deletion and duplications were examined by multiplex ligation-dependent probe amplification. CF patients in Japan were found to have chronic sinopulmonary disease (85.6%), exocrine pancreatic insufficiency (66.7%), meconium ileus (35.6%), electrolyte imbalance (21.2%), CF-associated liver disease (14.4%), and CF-related diabetes (6.1%). The median survival age was 25.0 years. The mean BMI percentile was 30.3%ile in definite CF patients aged < 18 years whose CFTR genotypes were known. In 70 CF alleles of East Asia/Japan origin, CFTR-dele16-17a-17b was detected in 24 alleles, the other variants were novel or very rare, and no pathogenic variants were detected in 8 alleles. In 22 CF alleles of Europe origin, F508del was detected in 11 alleles. In summary, clinical phenotype of Japanese CF patients is similar to European patients, but the prognosis is worse. The spectrum of CFTR variants in Japanese CF alleles is entirely different from that in European CF alleles.

Identifying the molecular target sites for CFTR potentiators GLPG1837 and VX-770.

The past two decades have witnessed major breakthroughs in developing compounds that target the chloride channel CFTR for the treatment of patients with cystic fibrosis. However, further improvement in affinity and efficacy for these CFTR modulators will require insights into the molecular interactions between CFTR modulators and their binding targets. In this study, we use in silico molecular docking to identify potential binding sites for GLPG1837, a CFTR potentiator that may share a common mechanism and binding site with VX-770, the FDA-approved drug for patients carrying mutations with gating defects. Among the five binding sites predicted by docking, the two top-scoring sites are located at the interface between CFTR's two transmembrane domains: site I consists of D924, N1138, and S1141, and site IIN includes F229, F236, Y304, F312, and F931. Using mutagenesis to probe the importance of these sites for GLPG187 binding, we find that disruption of predicted hydrogen-bonding interactions by mutation of D924 decreases apparent affinity, while hydrophobic amino acids substitutions at N1138 and introduction of positively charged amino acids at S1141 improve the apparent affinity for GLPG1837. Alanine substitutions at Y304, F312, and F931 (site IIN) decrease the affinity for GLPG1837, whereas alanine substitutions at F229 and F236 (also site IIN), or at residues in the other three lower-scoring sites, have little effect. In addition, current relaxation analysis to assess the apparent dissociation rate of VX-770 yields results consistent with the dose-response experiments for GLPG8137, with the dissociation rate of VX-770 accelerated by D924N, F236A, Y304A, and F312A, but decelerated by N1138L and S1141K mutations. Collectively, these data identify two potential binding sites for GLPG1837 and VX-770 in CFTR. We discuss the pros and cons of evidence for these two loci and the implications for future drug design.

Characterization of Δ(G970-T1122)-CFTR, the most frequent CFTR mutant identified in Japanese cystic fibrosis patients.

A massive deletion over three exons 16-17b in the CFTR gene was identified in Japanese CF patients with the highest frequency (about 70% of Japanese CF patients definitely diagnosed). This pathogenic mutation results in a deletion of 153 amino acids from glycine at position 970 (G970) to threonine at 1122 (T1122) in the CFTR protein without a frameshift. We name it Δ(G970-T1122)-CFTR. In the present study, we characterized the Δ(G970-T1122)-CFTR expressed in CHO cells using immunoblots and a super resolution microscopy. Δ(G970-T1122)-CFTR proteins were synthesized and core-glycosylated but not complex-glycosylated. This observation suggests that the Δ(G970-T1122) mutation can be categorized into the class II mutation like ΔF508. However, VX-809 a CFTR corrector that can help maturation of ΔF508, had no effect on Δ(G970-T1122). Interestingly C-terminal FLAG tag seems to partially rescue the trafficking defect of Δ(G970-T1122)-CFTR; however the rescued Δ(G970-T1122)-CFTR proteins do not assume channel function. Japanese, and perhaps people in other Asian nations, carry a class II mutation Δ(G970-T1122) with a higher frequency than previously appreciated. Further study of the Δ(G970-T1122)-CFTR is essential for understanding CF and CFTR-related diseases particularly in Asian countries.

Molecular dynamics simulation study on the structural instability of the most common cystic fibrosis-associated mutant ΔF508-CFTR.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that belongs to the ATP binding cassette protein superfamily. Deletion of phenylalanine at position 508 (ΔF508) is the most common CF-associated mutation and is present in nearly 90% of CF patients. Currently, atomistic level studies are insufficient for understanding the mechanism by which the deletion of a single amino acid causes greatly reduced folding as well as trafficking and gating defects. To clarify this mechanism, we first constructed an atomic model of the inward-facing ΔF508-CFTR and performed allatom molecular dynamics (MD) simulations of the protein in a membrane environment. All of the computational methodologies used are based on those developed in our previous study for wild-type CFTR. Two important findings were obtained. First, consistent with several previous computational results, the deletion of F508 causes a disruption of a hydrophobic cluster located at the interface between the nucleotide binding domain 1 (NBD1) and intracellular loop 4 (ICL4). This exerts unfavorable influences on the correlated motion between ICLs and transmembrane domains (TMDs), likely resulting in gating defects. Second, the F508 deletion affected the NBD1-NBD2 interface via allosteric communication originating from the correlated motion between NBDs and ICLs. As a result, several unusual inter-residue interactions are caused at the NBD1-NBD2 interface. In other words, their correct dimerization is impaired. This study provided insight into the atomic-level details of structural and dynamics changes caused by the ΔF508 mutation and thus provides good insight for drug design.

CFTR potentiators: from bench to bedside.

One major breakthrough in cystic fibrosis research in the past decade is the development of drugs that target the root cause of the disease-dysfunctional CFTR protein. One of the compounds, Ivacaftor or Kalydeco, which has been approved for clinical use since 2012, acts by promoting the gating function of CFTR. Our recent studies have led to a gating model that features energetic coupling between nucleotide-binding domain (NBD) dimerization and gate opening/closing in CFTR's transmembrane domains (TMDs). Based on this model, we showed that ATP analogs can enhance CFTR gating by facilitating NBD dimerization, whereas Ivacaftor works by stabilizing the open channel conformation of the TMDs. This latter idea also explains the near omnipotence of Ivacaftor. Furthermore, this model identifies multiple approaches to synergistically boost the open probability of CFTR by influencing distinct molecular events that control gating conformational changes.

A common mechanism for CFTR potentiators.

Cystic fibrosis (CF) is a channelopathy caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a phosphorylation-activated and adenosine triphosphate (ATP)-gated chloride channel. In the past few years, high-throughput drug screening has successfully realized the first US Food and Drug Administration-approved therapy for CF, called ivacaftor (or VX-770). A more recent CFTR potentiator, GLPG1837 (N-(3-carbamoyl-5,5,7,7-tetramethyl-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-1H-pyrazole-3-carboxamide), has been shown to exhibit a higher efficacy than ivacaftor for the G551D mutation, yet the underlying mechanism of GLPG1837 remains unclear. Here we find that despite their differences in potency and efficacy, GLPG1837 and VX-770 potentiate CFTR gating in a remarkably similar manner. Specifically, they share similar effects on single-channel kinetics of wild-type CFTR. Their actions are independent of nucleotide-binding domain (NBD) dimerization and ATP hydrolysis, critical steps controlling CFTR's gate opening and closing, respectively. By applying the two reagents together, we provide evidence that GLPG1837 and VX-770 likely compete for the same site, whereas GLPG1837 and the high-affinity ATP analogue 2'-deoxy-N6-(2-phenylethyl)-adenosine-5'-O-triphosphate (dPATP) work synergistically through two different sites. We also find that the apparent affinity for GLPG1837 is dependent on the open probability of the channel, suggesting a state-dependent binding of the drug to CFTR (higher binding affinity for the open state than the closed state), which is consistent with the classic mechanism for allosteric modulation. We propose a simple four-state kinetic model featuring an energetic coupling between CFTR gating and potentiator binding to explain our experimental results.

Bicarbonate-rich fluid secretion predicted by a computational model of guinea-pig pancreatic duct epithelium.

KEY POINTS: The ductal system of the pancreas secretes large volumes of alkaline fluid containing HCO3- concentrations as high as 140 mm during hormonal stimulation. A computational model has been constructed to explore the underlying ion transport mechanisms. Parameters were estimated by fitting the model to experimental data from guinea-pig pancreatic ducts. The model was readily able to secrete 140 mm HCO3- . Its capacity to do so was not dependent upon special properties of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels and solute carrier family 26 member A6 (SLC26A6) anion exchangers. We conclude that the main requirement for secreting high HCO3- concentrations is to minimize the secretion of Cl- ions. These findings help to clarify the mechanism responsible for pancreatic HCO3- secretion, a vital process that prevents the formation of protein plugs and viscous mucus in the ducts, which could otherwise lead to pancreatic disease. ABSTRACT: A computational model of guinea-pig pancreatic duct epithelium was developed to determine the transport mechanism by which HCO3- ions are secreted at concentrations in excess of 140 mm. Parameters defining the contributions of the individual ion channels and transporters were estimated by least-squares fitting of the model predictions to experimental data obtained from isolated ducts and intact pancreas under a range of experimental conditions. The effects of cAMP-stimulated secretion were well replicated by increasing the activities of the basolateral Na+ -HCO3- cotransporter (NBC1) and apical Cl- /HCO3- exchanger (solute carrier family 26 member A6; SLC26A6), increasing the basolateral K+ permeability and apical Cl- and HCO3- permeabilities (CFTR), and reducing the activity of the basolateral Cl- /HCO3- exchanger (anion exchanger 2; AE2). Under these conditions, the model secreted ∼140 mm HCO3- at a rate of ∼3 nl min-1  mm-2 , which is consistent with experimental observations. Alternative 1:2 and 1:1 stoichiometries for Cl- /HCO3- exchange via SLC26A6 at the apical membrane were able to support a HCO3- -rich secretion. Raising the HCO3- /Cl- permeability ratio of CFTR from 0.4 to 1.0 had little impact upon either the secreted HCO3- concentration or the volume flow. However, modelling showed that a reduction in basolateral AE2 activity by ∼80% was essential in minimizing the intracellular Cl- concentration following cAMP stimulation and thereby maximizing the secreted HCO3- concentration. The addition of a basolateral Na+ -K+ -2Cl- cotransporter (NKCC1), assumed to be present in rat and mouse ducts, raised intracellular Cl- and resulted in a lower secreted HCO3- concentration, as is characteristic of those species. We conclude therefore that minimizing the driving force for Cl- secretion is the main requirement for secreting 140 mm HCO3- .

Synergistic Potentiation of Cystic Fibrosis Transmembrane Conductance Regulator Gating by Two Chemically Distinct Potentiators, Ivacaftor (VX-770) and 5-Nitro-2-(3-Phenylpropylamino) Benzoate.

Cystic fibrosis (CF) is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation-activated but ATP-gated chloride channel. Previous studies suggested that VX-770 [ivacaftor, N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide], a CFTR potentiator now used in clinics, increases the open probability of CFTR by shifting the gating conformational changes to favor the open channel configuration. Recently the chloride channel blocker and CFTR potentiator 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB) has been reported to enhance CFTR activity by a mechanism that exploits the ATP hydrolysis-driven, nonequilibrium gating mechanism unique to CFTR. Surprisingly however, NPPB increased the activity of nonhydrolytic G551D-CFTR, the third most common disease-associated mutation. Here, we further investigated the mechanism of NPPB's effects on CFTR gating by assessing its interaction with well-studied VX-770. Interestingly, once G551D-CFTR was maximally potentiated by VX-770, NPPB further increased its activity. However, quantitative analysis of this drug-drug interaction suggests that this pharmacologic synergism is not due to independent actions of NPPB and VX-770 on CFTR gating; instead, our data support a dependent mechanism involving two distinct binding sites. This latter idea is further supported by the observation that the locked-open time of a hydrolysis-deficient mutant K1250A was shortened by NPPB but prolonged by VX-770. In addition, the effectiveness of NPPB, but not of VX-770, was greatly diminished in a mutant whose second nucleotide-binding domain was completely removed. Interpreting these results under the framework of current understanding of CFTR gating not only reveals insights into the mechanism of action for different CFTR potentiators but also brings us one step forward to a more complete schematic for CFTR gating.

On the mechanism of gating defects caused by the R117H mutation in cystic fibrosis transmembrane conductance regulator.

KEY POINTS: Two functional abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR), a 25% reduction of the single-channel conductance (g) and a ∼13-fold lower open probability (Po ), were found with the R117H mutation that is associated with mild forms of cystic fibrosis. Characterizations of the gating defects of R117H-CFTR led to the conclusion that the mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains (TMDs) without altering the function of the nucleotide binding domains (NBDs). Nonetheless, gating of the R117H-CFTR can be improved by a variety of pharmacological reagents supposedly acting on NBDs such as ATP analogues, or TMDs (e.g. VX-770 or nitrate). These reagents potentiate synergistically R117H-CFTR gating to a level that allows accurate assessments of its gating deficits. Our studies not only elucidate the mechanism underpinning gating dysfunction of R117H-CFTR, but also provide a mechanistic understanding of how VX-770 ameliorates the gating defects in the R117H mutant. ABSTRACT: Cystic fibrosis (CF) is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation-activated, but ATP-gated chloride channel. In the current study, we investigated the mechanism responsible for the gating defects manifested in R117H-CFTR, an arginine-to-histidine substitution at position 117 of CFTR that is associated with mild forms of CF. We confirmed previous findings of a 25% decrease of the single-channel conductance (g) in R117H-CFTR, but found a ∼13-fold lower open probability (Po ). This dramatic gating deficit is not due to dysfunctional nucleotide binding domains (NBDs) as the mutation does not alter the apparent affinity for ATP, and the mutant channels respond to ATP analogues in a similar manner as wild-type CFTR. Furthermore, once ATP hydrolysis is abolished, the R117H mutant can be trapped in a prolonged 'burst opening' conformation that is proposed to be equipped with a stable NBD dimer. On the other hand, our results support the conclusion that the R117H mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains as even when NBDs are kept at a dimerized configuration, Po is reduced by ∼10-fold. Moreover, our data demonstrate that a synergistic improvement of R117H-CFTR function can be accomplished with a combined regiment of VX-770 (Ivacaftor), nitrate ion (NO3 (-) ) and N(6) -(2-phenylethyl)-2'-deoxy-ATP (d-PATP), which almost completely rectifies the gating defect of R117H-CFTR. Clinical implications of our results are discussed.

Expression and localization of aquaporin-4 in sensory ganglia.

Aquaporin-4 (AQP4) is a water channel protein that is predominantly expressed in astrocytes in the CNS. The rapid water flux through AQP4 may contribute to electrolyte/water homeostasis and may support neuronal activities in the CNS. On the other hand, little is known about the expression of AQP4 in the peripheral nervous system (PNS). Using AQP4(-/-) mice as a negative control, we demonstrated that AQP4 is also expressed in sensory ganglia, such as trigeminal ganglia and dorsal root ganglia in the PNS. Immunohistochemistry revealed that AQP4 is exclusively localized to satellite glial cells (SGCs) surrounding the cell bodies of the primary afferent sensory neurons in the sensory ganglia. Biochemical analyses revealed that the expression levels of AQP4 in sensory ganglia were considerably lower than those in astrocytes in the CNS. Consistently, behavioral analyses did not show any significant difference in terms of mechanical and cold sensitivity between wild type and AQP4(-/-) mice. Overall, although the pathophysiological relevance of AQP4 in somatosensory perception remains unclear, our findings provide new insight into the involvement of water homeostasis in the peripheral sensory system.

Glucose-induced electrical activities and insulin secretion in pancreatic islet ß-cells are modulated by CFTR.

The cause of insulin insufficiency remains unknown in many diabetic cases. Up to 50% adult patients with cystic fibrosis (CF), a disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), develop CF-related diabetes (CFRD) with most patients exhibiting insulin insufficiency. Here we show that CFTR is a regulator of glucose-dependent electrical acitivities and insulin secretion in ß-cells. We demonstrate that glucose elicited whole-cell currents, membrane depolarization, electrical bursts or action potentials, Ca(2+) oscillations and insulin secretion are abolished or reduced by inhibitors or knockdown of CFTR in primary mouse ß-cells or RINm5F ß-cell line, or significantly attenuated in CFTR mutant (DF508) mice compared with wild-type mice. VX-809, a newly discovered corrector of DF508 mutation, successfully rescues the defects in DF508 ß-cells. Our results reveal a role of CFTR in glucose-induced electrical activities and insulin secretion in ß-cells, shed light on the pathogenesis of CFRD and possibly other idiopathic diabetes, and present a potential treatment strategy.

Direct visualization and quantitative analysis of water diffusion in complex biological tissues using CARS microscopy.

To date, it has not been possible to measure microscopic diffusive water movements in epithelia and in the interstitial space of complex tissues and organs. Diffusive water movements are essential for life because they convey physiologically important small molecules, e.g. nutrients and signaling ligands throughout the extracellular space of complex tissues. Here we report the development of a novel method for the direct observation and quantitative analysis of water diffusion dynamics in a biologically organized tissue using Coherent Anti-Stokes Raman Scattering (CARS) microscopy. Using a computer simulation model to analyze the CARS O-H bond vibration data during H2O/D2O exchange in a 3D epithelial cyst, we succeeded in measuring the diffusive water permeability of the individual luminal and basolateral water pathways and also their response to hormonal stimulation. Our technique will be applicable to the measurement of diffusive water movements in other structurally complex and medically important tissues and organs.

Amiodarone inhibits apamin-sensitive potassium currents.

BACKGROUND: Apamin sensitive potassium current (I KAS), carried by the type 2 small conductance Ca(2+)-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles. OBJECTIVE: To test the hypothesis that amiodarone inhibits I KAS in human embryonic kidney 293 (HEK-293) cells. METHODS: We used the patch-clamp technique to study I KAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration. RESULTS: Amiodarone inhibited IKAS in a dose-dependent manner (IC50, 2.67 ± 0.25 µM with 1 µM intrapipette Ca(2+)). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6 ± 3.1% of IKAS induced with 1 µM intrapipette Ca(2+) (n = 3). IKAS inhibition by amiodarone was not voltage-dependent, but was Ca(2+)-dependent: 30 µM amiodarone inhibited 81.5±1.9% of I KAS induced with 1 µM Ca(2+) (n = 4), and 16.4±4.9% with 250 nM Ca(2+) (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca(2+) dependent inhibition of I KAS. CONCLUSION: Both amiodarone and desethylamiodarone inhibit I KAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca(2+) concentration. SK2 current inhibition may in part underlie amiodarone's effects in preventing electrical storm in failing ventricles.

Mechanisms underlying regional differences in the Ca2+ sensitivity of BK(Ca) current in arteriolar smooth muscle.

Abstract ß1-Subunits enhance the gating properties of large-conductance Ca(2+)-activated K(+) channels (BKCa) formed by α-subunits. In arterial vascular smooth muscle cells (VSMCs), ß1-subunits are vital in coupling SR-generated Ca(2+) sparks to BKCa activation, affecting contractility and blood pressure. Studies in cremaster and cerebral VSMCs show heterogeneity of BKCa activity due to apparent differences in the functional ß1-subunit:α-subunit ratio. To define these differences, studies were conducted at the single-channel level while siRNA was used to manipulate specific subunit expression. ß1 modulation of the α-subunit Ca(2+) sensitivity was studied using patch-clamp techniques. BKCa channel normalized open probability (NPo) versus membrane potential (Vm) curves were more left-shifted in cerebral versus cremaster VSMCs as cytoplasmic Ca(2+) was raised from 0.5 to 100 µm. Calculated V1/2 values of channel activation decreased from 72.0 ± 6.1 at 0.5 µm Ca(2+)i to -89 ± 9 mV at 100 µm Ca(2+)i in cerebral compared with 101 ± 10 to -63 ± 7 mV in cremaster VSMCs. Cremaster BKCa channels thus demonstrated an ∼2.5-fold weaker apparent Ca(2+) sensitivity such that at a value of Vm of -30 mV, a mean value of [Ca(2+)]i of 39 µm was required to open half of the channels in cremaster versus 16 µm [Ca(2+)]i in cerebral VSMCs. Further, shortened mean open and longer mean closed times were evident in BKCa channel events from cremaster VSMCs at either -30 or 30 mV at any given [Ca(2+)]. ß1-Subunit-directed siRNA decreased both the apparent Ca(2+) sensitivity of BKCa in cerebral VSMCs and the appearance of spontaneous transient outward currents. The data are consistent with a higher ratio of ß1-subunit:α-subunit of BKCa channels in cerebral compared with cremaster VSMCs. Functionally, this leads both to higher Ca(2+) sensitivity and NPo for BKCa channels in the cerebral vasculature relative to that of skeletal muscle.

Curcumin and genistein: the combined effects on disease-associated CFTR mutants and their clinical implications.

Genistein and curcumin are major components of Asian foods, soybean and curry turmeric respectively. These compounds have been intensively investigated for their chemical and biological features conferring their anti-cancer activity. Genistein and curcumin have also been investigated for their potentiation effects on disease-associated CFTR mutants such as ΔF508 and G551D. Recently, we investigated the combined effect of genistein and curcumin on G551D-CFTR, which exhibits gating defects without abnormalities in protein synthesis or trafficking using the patch-clamp technique. We found that genistein and curcumin showed additive effects on their potentiation of G551D-CFTR in high concentration range and also, more importantly, showed a significant synergistic effect in their minimum concentration ranges. These results are consistent with the idea that multiple mechanisms are involved in the action of these CFTR potentiators. In this review, we revisit the pharmacology of genistein and curcumin on CFTR and also propose new pharmaceutical implications of combined use of these compounds in the development of drugs for CF pharmacotherapy.

The power stroke driven by ATP binding in CFTR as studied by molecular dynamics simulations.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP binding cassette (ABC) protein superfamily. Currently, it remains unclear how ATP binding causes the opening of the channel gate at the molecular level. To clarify this mechanism, we first constructed an atomic model of the inward-facing CFTR using the X-ray structures of other ABC proteins. Molecular dynamics (MD) simulations were then performed to explore the structure and dynamics of the inward-facing CFTR in a membrane environment. In the MgATP-bound state, two nucleotide-binding domains (NBDs) formed a head-to-tail type of dimer, in which the ATP molecules were sandwiched between the Walker A and signature motifs. Alternatively, one of the final MD structures in the apo state was similar to that of a "closed-apo" conformation found in the X-ray analysis of ATP-free MsbA. Principal component analysis for the MD trajectory indicated that NBD dimerization causes significant structural and dynamical changes in the transmembrane domains (TMDs), which is likely indicative of the formation of a chloride ion access path. This study suggests that the free energy gain from ATP binding acts as a driving force not only for NBD dimerization but also for NBD-TMD concerted motions.

Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) protein superfamily. Unlike most other ABC proteins that function as active transporters, CFTR is an ATP-gated chloride channel. The opening of CFTR's gate is associated with ATP-induced dimerization of its two nucleotide-binding domains (NBD1 and NBD2), whereas gate closure is facilitated by ATP hydrolysis-triggered partial separation of the NBDs. This generally held theme of CFTR gating-a strict coupling between the ATP hydrolysis cycle and the gating cycle-is put to the test by our recent finding of a short-lived, post-hydrolytic state that can bind ATP and reenter the ATP-induced original open state. We accidentally found a mutant CFTR channel that exhibits two distinct open conductance states, the smaller O1 state and the larger O2 state. In the presence of ATP, the transition between the two states follows a preferred O1→O2 order, a telltale sign of a violation of microscopic reversibility, hence demanding an external energy input likely from ATP hydrolysis, as such preferred gating transition was abolished in a hydrolysis-deficient mutant. Interestingly, we also observed a considerable amount of opening events that contain more than one O1→O2 transition, indicating that more than one ATP molecule may be hydrolyzed within an opening burst. We thus conclude a nonintegral stoichiometry between the gating cycle and ATP consumption. Our results lead to a six-state gating model conforming to the classical allosteric mechanism: both NBDs and transmembrane domains hold a certain degree of autonomy, whereas the conformational change in one domain will facilitate the conformational change in the other domain.

Identification of a novel post-hydrolytic state in CFTR gating.

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, ubiquitous proteins found in all kingdoms of life, catalyze substrates translocation across biological membranes using the free energy of ATP hydrolysis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of this superfamily in that it functions as an ATP-gated chloride channel. Despite difference in function, recent studies suggest that the CFTR chloride channel and the exporter members of the ABC protein family may share an evolutionary origin. Although ABC exporters harness the free energy of ATP hydrolysis to fuel a transport cycle, for CFTR, ATP-induced dimerization of its nucleotide-binding domains (NBDs) and subsequent hydrolysis-triggered dimer separation are proposed to be coupled, respectively, to the opening and closing of the gate in its transmembrane domains. In this study, by using nonhydrolyzable ATP analogues, such as pyrophosphate or adenylyl-imidodiphosphate as baits, we captured a short-lived state (state X), which distinguishes itself from the previously identified long-lived C2 closed state by its fast response to these nonhydrolyzable ligands. As state X is caught during the decay phase of channel closing upon washout of the ligand ATP but before the channel sojourns to the C2 closed state, it likely emerges after the bound ATP in the catalysis-competent site has been hydrolyzed and the hydrolytic products have been released. Thus, this newly identified post-hydrolytic state may share a similar conformation of NBDs as the C2 closed state (i.e., a partially separated NBD and a vacated ATP-binding pocket). The significance of this novel state in understanding the structural basis of CFTR gating is discussed.