RESUMO
The Transient Receptor Potential Melastatin 2 (TRPM2) channel is a homotetrameric ligand-gated cation channel opened by the binding of cytosolic ADP ribose (ADPR) and Ca2+. In addition, strong temperature dependence of its activity has lately become a center of attention for both physiological and biophysical studies. TRPM2 temperature sensitivity has been affirmed to play a role in central and peripheral thermosensation, pancreatic insulin secretion, and immune cell function. On the other hand, a number of different underlying mechanisms have been proposed from studies in intact cells. This review summarizes available information on TRPM2 temperature sensitivity, with a focus on recent mechanistic insight obtained in a cell-free system. Those biophysical results outline TRPM2 as a channel with an intrinsically endothermic opening transition, a temperature threshold strongly modulated by cytosolic agonist concentrations, and a response steepness greatly enhanced through a positive feedback loop generated by Ca2+ influx through the channel's pore. Complex observations in intact cells and apparent discrepancies between studies using in vivo and in vitro models are discussed and interpreted in light of the intrinsic biophysical properties of the channel protein.
Assuntos
Cálcio , Canais de Cátion TRPM , Cálcio/metabolismo , Canais de Cátion TRPM/metabolismo , Adenosina Difosfato Ribose/metabolismo , Insulina/metabolismo , Secreção de InsulinaRESUMO
CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle is observable at single-molecule level in patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization of its two nucleotide-binding domains (NBDs) and in wild-type (WT) channels are mostly terminated by ATP hydrolysis. The slow rate of non-hydrolytic closure - which determines how tightly bursts and ATP hydrolysis are coupled - is unknown, as burst durations of catalytic site mutants span a range of ~200-fold. Here, we show that Walker A mutation K1250A, Walker B mutation D1370N, and catalytic glutamate mutations E1371S and E1371Q all completely disrupt ATP hydrolysis. True non-hydrolytic closing rate of WT CFTR approximates that of K1250A and E1371S. That rate is slowed ~15-fold in E1371Q by a non-native inter-NBD H-bond, and accelerated ~15-fold in D1370N. These findings uncover unique features of the NBD interface in human CFTR.
Assuntos
Trifosfato de Adenosina , Regulador de Condutância Transmembrana em Fibrose Cística , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico , Mutação , Domínio CatalíticoRESUMO
Light-controlled availability for phosphorylation reveals dominant roles of select R-domain serines in CFTR channel activation.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Ativação do Canal Iônico , Fosfosserina , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ativação do Canal Iônico/fisiologia , Fosforilação , Serina/metabolismoRESUMO
CFTR chloride channel mutations cause the lethal and incurable disease cystic fibrosis (CF). CFTR is activated by phosphorylation, and phosphorylated channels exhibit "bursting" behavior-"bursts" of openings separated by short "flickery" closures and flanked by long "interburst" closures-driven by ATP binding/hydrolysis at two nucleotide-binding domains. The human channel (hCFTR) and the distant zebrafish ortholog (zCFTR) display differences both in their gating properties and structures. In phosphorylated ATP-bound hCFTR, the hR117 side chain, conserved across evolution, forms an H-bond that stabilizes the open state. Lack of that bond in the hR117H mutant causes CF. In the phosphorylated ATP-bound zCFTR structure that H-bond is not observable. Here, we show that the zR118H mutation does not affect the function of zCFTR. Instead, we identify an H-bond between the zS109 and zS120 side chains of phosphorylated ATP-bound, but not of unphosphorylated apo-, zCFTR. We investigate the role of that interaction using thermodynamic mutant cycles built on gating parameters determined in inside-out patch clamp recordings. We find that zS109 indeed forms an H-bond with zN120 in the flickery closed state, but not in the open or interburst closed states. Although in hCFTR an isoleucine (hI119) replaces the asparagine, mutation hS108A produces a strong hR117H-like phenotype. Since the effects of the latter two mutations are not additive, we conclude that in hCFTR these two positions interact, and the hS108-hR117 and hR117-hE1124 H-bonds cooperate to stabilize the open state. These findings highlight an example of how the gating mechanism was optimized during CFTR molecular evolution.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Animais , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/química , Trifosfato de Adenosina , Peixe-Zebra , Canais de Cloreto , Proteínas de Peixe-ZebraRESUMO
The Ca2+ and ADP ribose (ADPR)-activated cation channel TRPM2 is the closest homolog of the cold sensor TRPM8 but serves as a deep-brain warmth sensor. To unravel the molecular mechanism of heat sensing by the TRPM2 protein, we study here temperature dependence of TRPM2 currents in cell-free membrane patches across ranges of agonist concentrations. We find that channel gating remains strictly agonist-dependent even at 40°C: heating alone or in combination with just Ca2+, just ADPR, Ca2+ + cyclic ADPR, or H2O2 pretreatment only marginally activates TRPM2. For fully liganded TRPM2, pore opening is intrinsically endothermic, due to ~10-fold larger activation enthalpy for opening (~200 kJ/mol) than for closure (~20 kJ/mol). However, the temperature threshold is too high (>40°C) for unliganded but too low (<15°C) for fully liganded channels. Thus, warmth sensitivity around 37°C is restricted to narrow ranges of agonist concentrations. For ADPR, that range matches, but for Ca2+, it exceeds bulk cytosolic values. The supraphysiological [Ca2+] needed for TRPM2 warmth sensitivity is provided by Ca2+ entering through the channel's pore. That positive feedback provides further strong amplification to the TRPM2 temperature response (Q10 ~ 1,000), enabling the TRPM2 protein to autonomously respond to tiny temperature fluctuations around 37°C. These functional data together with published structures suggest a molecular mechanism for opposite temperature dependences of two closely related channel proteins.
Assuntos
Canais de Cátion TRPM , Canais de Cátion TRPM/metabolismo , Temperatura Alta , Peróxido de Hidrogênio/metabolismo , Cálcio/metabolismo , Adenosina Difosfato Ribose/metabolismoRESUMO
The phosphorylation-activated anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is gated by an ATP hydrolysis cycle at its two cytosolic nucleotide-binding domains, and is essential for epithelial salt-water transport. A large number of CFTR mutations cause cystic fibrosis. Since recent breakthrough in targeted pharmacotherapy, CFTR mutants with impaired gating are candidates for stimulation by potentiator drugs. Thus, understanding the molecular pathology of individual mutations has become important. The relatively common R117H mutation affects an extracellular loop, but nevertheless causes a strong gating defect. Here, we identify a hydrogen bond between the side chain of arginine 117 and the backbone carbonyl group of glutamate 1124 in the cryo-electronmicroscopic structure of phosphorylated, ATP-bound CFTR. We address the functional relevance of that interaction for CFTR gating using macroscopic and microscopic inside-out patch-clamp recordings. Employing thermodynamic double-mutant cycles, we systematically track gating-state-dependent changes in the strength of the R117-E1124 interaction. We find that the H-bond is formed only in the open state, but neither in the short-lived 'flickery' nor in the long-lived 'interburst' closed state. Loss of this H-bond explains the strong gating phenotype of the R117H mutant, including robustly shortened burst durations and strongly reduced intraburst open probability. The findings may help targeted potentiator design.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Ligação de Hidrogênio , Patologia Molecular , Humanos , MutaçãoRESUMO
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel is essential for epithelial salt-water balance. CFTR mutations cause cystic fibrosis, a lethal incurable disease. In cells CFTR is activated through the cAMP signaling pathway, overstimulation of which during cholera leads to CFTR-mediated intestinal salt-water loss. Channel activation is achieved by phosphorylation of its regulatory (R) domain by cAMP-dependent protein kinase catalytic subunit (PKA). Here we show using two independent approaches--an ATP analog that can drive CFTR channel gating but is unsuitable for phosphotransfer by PKA, and CFTR mutants lacking phosphorylatable serines--that PKA efficiently opens CFTR channels through simple binding, under conditions that preclude phosphorylation. Unlike when phosphorylation happens, CFTR activation by PKA binding is completely reversible. Thus, PKA binding promotes release of the unphosphorylated R domain from its inhibitory position, causing full channel activation, whereas phosphorylation serves only to maintain channel activity beyond termination of the PKA signal. The results suggest two levels of CFTR regulation in cells: irreversible through phosphorylation, and reversible through R-domain binding to PKA--and possibly also to other members of a large network of proteins known to interact with the channel.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ânions/metabolismo , Fenômenos Biofísicos , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Ativação do Canal Iônico/fisiologia , Mutagênese Sítio-Dirigida , Nucleotídeos/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Fosforilação , Ligação Proteica/fisiologia , Serina/metabolismo , Canais de Ânion Dependentes de Voltagem/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
Transient receptor potential melastatin 2 (TRPM2) is a homotetrameric Ca2+-permeable cation channel important for the immune response, body temperature regulation, and insulin secretion, and is activated by cytosolic Ca2+ and ADP ribose (ADPR). ADPR binds to two distinct locations, formed by large N- and C-terminal cytosolic domains, respectively, of the channel protein. In invertebrate TRPM2 channels, the C-terminal site is not required for channel activity but acts as an active ADPR phosphohydrolase that cleaves the activating ligand. In vertebrate TRPM2 channels, the C-terminal site is catalytically inactive but cooperates with the N-terminal site in channel activation. The precise functional contributions to channel gating and the nucleotide selectivities of the two sites in various species have not yet been deciphered. For TRPM2 of the sea anemone Nematostella vectensis (nvTRPM2), catalytic activity is solely attributable to the C-terminal site. Here, we show that nvTRPM2 channel gating properties remain unaltered upon deletion of the C-terminal domain, indicating that the N-terminal site is single-handedly responsible for channel gating. Exploiting such functional independence of the N- and C-terminal sites, we selectively measure their affinity profiles for a series of ADPR analogues, as reflected by apparent affinities for channel activation and catalysis, respectively. Using site-directed mutagenesis, we confirm that the same N-terminal site observed in vertebrate TRPM2 channels was already present in ancient cnidarians. Finally, by characterizing the functional effects of six amino acid side chain truncations in the N-terminal site, we provide first insights into the mechanistic contributions of those side chains to TRPM2 channel gating.
Assuntos
Sítios de Ligação/fisiologia , Nucleotídeos/metabolismo , Canais de Cátion TRPM/metabolismo , Adenosina Difosfato Ribose/genética , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Sítios de Ligação/genética , Cálcio/metabolismo , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Ligantes , Mutagênese Sítio-Dirigida/métodos , Mutação/genética , Nucleotídeos/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Xenopus laevisRESUMO
The devastating inherited disease cystic fibrosis (CF) is caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel. The recent approval of the CFTR potentiator drug ivacaftor (Vx-770) for the treatment of CF patients has marked the advent of causative CF therapy. Currently, thousands of patients are being treated with the drug, and its molecular mechanism of action is under intensive investigation. Here we determine the solubility profile and true stimulatory potency of Vx-770 towards wild-type (WT) and mutant human CFTR channels in cell-free patches of membrane. We find that its aqueous solubility is ~200 fold lower (~60 nanomolar), whereas the potency of its stimulatory effect is >100 fold higher, than reported, and is unexpectedly fully reversible. Strong, but greatly delayed, channel activation by picomolar Vx-770 identifies multiple sequential slow steps in the activation pathway. These findings provide solid guidelines for the design of in vitro studies using Vx-770.
Assuntos
Aminofenóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Fibrose Cística/tratamento farmacológico , Ativação do Canal Iônico/efeitos dos fármacos , Quinolonas/farmacologia , Aminofenóis/química , Aminofenóis/uso terapêutico , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Humanos , Ativação do Canal Iônico/genética , Transporte de Íons/efeitos dos fármacos , Mutação , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Quinolonas/química , Quinolonas/uso terapêutico , Solubilidade , Solventes/química , Xenopus laevisRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the ATP binding cassette (ABC) transporter superfamily but functions as an anion channel crucial for salt and water transport across epithelial cells. CFTR dysfunction, because of mutations, causes cystic fibrosis (CF). The anion-selective pore of the CFTR protein is formed by its two transmembrane domains (TMDs) and regulated by its cytosolic domains: two nucleotide binding domains (NBDs) and a regulatory (R) domain. Channel activation requires phosphorylation of the R domain by cAMP-dependent protein kinase (PKA), and pore opening and closing (gating) of phosphorylated channels is driven by ATP binding and hydrolysis at the NBDs. This review summarizes available information on structure and mechanism of the CFTR protein, with a particular focus on atomic-level insight gained from recent cryo-electron microscopic structures and on the molecular mechanisms of channel gating and its regulation. The pharmacological mechanisms of small molecules targeting CFTR's ion channel function, aimed at treating patients suffering from CF and other diseases, are briefly discussed.
Assuntos
Trifosfato de Adenosina/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ativação do Canal Iônico/fisiologia , Animais , Ânions/metabolismo , Humanos , Mutação/genética , Fosforilação/fisiologiaRESUMO
Lung health relies on effective mucociliary clearance and innate immune defence mechanisms. In cystic fibrosis (CF), an imbalance in ion transport due to an absence of chloride ion secretion, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) and a concomitant sodium hyperabsorption, caused by dyregulation of the epithelial sodium channel (ENaC), results in mucus stasis which predisposes the lungs to cycles of chronic infection and inflammation leading to lung function decline. An increased understanding of CFTR structure and function has provided opportunity for the development of a number of novel modulators targeting mutant CFTR however, it is important to also consider other ion channels and transporters present in the airways as putative targets for drug development. In this review, we discuss recent advances in CFTR biology which will contribute to further drug discovery in the field. We also examine developments to inhibit the epithelial sodium channel (ENaC) and potentially activate alternative chloride channels and transporters as a multi-tracked strategy to hydrate CF airways and restore normal mucociliary clearance mechanisms in a manner independent of CFTR mutation.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Depuração Mucociliar , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Descoberta de Drogas , Humanos , Transporte de Íons/fisiologia , Depuração Mucociliar/efeitos dos fármacos , Depuração Mucociliar/fisiologiaRESUMO
CFTR, the chloride channel mutated in cystic fibrosis (CF) patients, is opened by ATP binding to two cytosolic nucleotide binding domains (NBDs), but pore-domain mutations may also impair gating. ATP-bound NBDs dimerize occluding two nucleotides at interfacial binding sites; one site hydrolyzes ATP, the other is inactive. The pore opens upon tightening, and closes upon disengagement, of the catalytic site following ATP hydrolysis. Extent, timing, and role of non-catalytic-site movements are unknown. Here we exploit equilibrium gating of a hydrolysis-deficient mutant and apply Φ value analysis to compare timing of opening-associated movements at multiple locations, from the cytoplasmic ATP sites to the extracellular surface. Marked asynchrony of motion in the two ATP sites reveals their distinct roles in channel gating. The results clarify the molecular mechanisms of functional cross-talk between canonical and degenerate ATP sites in asymmetric ABC proteins, and of the gating defects caused by two common CF mutations.
Assuntos
Trifosfato de Adenosina/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Multimerização Proteica , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Hidrólise , Oócitos , Técnicas de Patch-Clamp , Ligação Proteica , Conformação Proteica , Xenopus laevisRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that uniquely functions as an ion channel. Here, we present a 3.9 Å structure of dephosphorylated human CFTR without nucleotides, determined by electron cryomicroscopy (cryo-EM). Close resemblance of this human CFTR structure to zebrafish CFTR under identical conditions reinforces its relevance for understanding CFTR function. The human CFTR structure reveals a previously unresolved helix belonging to the R domain docked inside the intracellular vestibule, precluding channel opening. By analyzing the sigmoid time course of CFTR current activation, we propose that PKA phosphorylation of the R domain is enabled by its infrequent spontaneous disengagement, which also explains residual ATPase and gating activity of dephosphorylated CFTR. From comparison with MRP1, a feature distinguishing CFTR from all other ABC transporters is the helix-loop transition in transmembrane helix 8, which likely forms the structural basis for CFTR's channel function.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Transportadores de Cassetes de Ligação de ATP/química , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Microscopia Crioeletrônica , Humanos , Hidrólise , Modelos Moleculares , Domínios Proteicos , Xenopus laevis , Peixe-Zebra , Proteínas de Peixe-Zebra/químicaRESUMO
Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9. When expressed in E.coli, chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified. In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity (kcat~4-9s(-1)), that of NUDT9H did not support catalysis. Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating (K1/2~1-5 µM), confirming functionality of chimeric domains. Thus, TRPM2 is not a 'chanzyme'. Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Hidrolases/metabolismo , Canais de Cátion TRPM/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Hidrolases/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPM/genéticaRESUMO
In CFTR, the chloride channel mutated in cystic fibrosis (CF) patients, ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) opens the pore, and dimer disruption following ATP hydrolysis closes it. Spontaneous openings without ATP are rare in wild-type CFTR, but in certain CF mutants constitute the only gating mechanism, stimulated by ivacaftor, a clinically approved CFTR potentiator. The molecular motions underlying spontaneous gating are unclear. Here we correlate energetic coupling between residues across the dimer interface with spontaneous pore opening/closure in single CFTR channels. We show that spontaneous openings are also strictly coupled to NBD dimerization, which may therefore occur even without ATP. Coordinated NBD/pore movements are therefore intrinsic to CFTR: ATP alters the stability, but not the fundamental structural architecture, of open- and closed-pore conformations. This explains correlated effects of phosphorylation, mutations, and drugs on ATP-driven and spontaneous activity, providing insights for understanding CF mutation and drug mechanisms.
Assuntos
Trifosfato de Adenosina/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dimerização , Domínios Proteicos , Animais , Conformação Proteica , Xenopus , Zigoto/metabolismoRESUMO
P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate, and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico Ativo , Domínio Catalítico , Animais , Células Cultivadas , Cães , Camundongos , Ligação Proteica , Conformação ProteicaRESUMO
In CFTR, the chloride ion channel mutated in cystic fibrosis (CF) patients, pore opening is coupled to ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) and closure to dimer disruption following ATP hydrolysis. CFTR opening rate, unusually slow because of its high-energy transition state, is further slowed by CF mutation ΔF508. Here, we exploit equilibrium gating of hydrolysis-deficient CFTR mutant D1370N and apply rate-equilibrium free-energy relationship analysis to estimate relative timing of opening movements in distinct protein regions. We find clear directionality of motion along the longitudinal protein axis and identify an opening transition-state structure with the NBD dimer formed but the pore still closed. Thus, strain at the NBD/pore-domain interface, the ΔF508 mutation locus, underlies the energetic barrier for opening. Our findings suggest a therapeutic opportunity to stabilize this transition-state structure pharmacologically in ΔF508-CFTR to correct its opening defect, an essential step toward restoring CFTR function.