scholarly journals Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Márton A Simon ◽  
László Csanády
Keyword(s):  
Cystic Fibrosis ◽  
Hydrogen Bond ◽  
Molecular Pathology ◽  
Atp Hydrolysis ◽  
Salt Water ◽  
Anion Channel ◽  
Open Probability ◽  
Binding Domains ◽  
State Dependent ◽  
Cftr Mutations

The phosphorylation-activated anion channel 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.

scholarly journals Molecular pathology of a cystic fibrosis mutation is explained by loss of a hydrogen bond

2021 ◽  
Author(s):  
Márton A Simon ◽  
LászlÓ Csanády
Keyword(s):  
Cystic Fibrosis ◽  
Hydrogen Bond ◽  
Molecular Pathology ◽  
Atp Hydrolysis ◽  
Salt Water ◽  
Anion Channel ◽  
Open Probability ◽  
Binding Domains ◽  
State Dependent ◽  
Cftr Mutations

The phosphorylation-activated anion channel 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 entire gating phenotype of the R117H mutant, including robustly shortened burst durations and strongly reduced intraburst open probability. The findings may help targeted potentiator design.

scholarly journals Simple binding of protein kinase A prior to phosphorylation allows CFTR anion channels to be opened by nucleotides

2020 ◽  
Vol 117 (35) ◽  
pp. 21740-21746
Author(s):  
Csaba Mihályi ◽  
Iordan Iordanov ◽  
Beáta Töröcsik ◽  
László Csanády
Keyword(s):  
Cystic Fibrosis ◽  
Protein Kinase ◽  
Salt Water ◽  
Anion Channel ◽  
Large Network ◽  
Channel Activation ◽  
Incurable Disease ◽  
Cftr Mutations ◽  
In Cells ◽  
R Domain

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.

scholarly journals A common mechanism for CFTR potentiators

2017 ◽  
Vol 149 (12) ◽  
pp. 1105-1118 ◽  
Author(s):  
Han-I Yeh ◽  
Yoshiro Sohma ◽  
Katja Conrath ◽  
Tzyh-Chang Hwang
Keyword(s):  
Cystic Fibrosis ◽  
Atp Hydrolysis ◽  
Single Channel ◽  
Underlying Mechanism ◽  
Common Mechanism ◽  
Loss Of Function ◽  
Open Probability ◽  
High Throughput Drug Screening ◽  
State Dependent ◽  
Opening And Closing

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.

scholarly journals Structure and Function of the CFTR Chloride Channel

1999 ◽  
Vol 79 (1) ◽  
pp. S23-S45 ◽  
Author(s):  
DAVID N. SHEPPARD ◽  
MICHAEL J. WELSH
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Atp Hydrolysis ◽  
Current Knowledge ◽  
Structure And Function ◽  
Binding Domains ◽  
Transporter Family ◽  
Membrane Spanning ◽  
And Function ◽  
R Domain

Sheppard, David N., and Michael J. Welsh. Structure and Function of the CFTR Chloride Channel. Physiol. Rev. 79 , Suppl.: S23–S45, 1999. — The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ABC transporter family that forms a novel Cl− channel. It is located predominantly in the apical membrane of epithelia where it mediates transepithelial salt and liquid movement. Dysfunction of CFTR causes the genetic disease cystic fibrosis. The CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. Here we review the structure and function of this unique channel, with a focus on how the various domains contribute to channel function. The MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls channel gating. Current knowledge of CFTR structure and function may help us understand better its mechanism of action, its role in electrolyte transport, its dysfunction in cystic fibrosis, and its relationship to other ABC transporters.

scholarly journals Cystic Fibrosis Gene Therapy: Looking Back, Looking Forward

Genes ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 538 ◽  
Author(s):  
Ashley Cooney ◽  
Paul McCray ◽  
Patrick Sinn
Keyword(s):  
Cystic Fibrosis ◽  
Gene Therapy ◽  
Anion Channel ◽  
Gene Replacement ◽  
Cystic Fibrosis Gene ◽  
Cftr Gene ◽  
System Disease ◽  
Cftr Mutations ◽  
Infection And Inflammation

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a cAMP-regulated anion channel. Although CF is a multi-organ system disease, most people with CF die of progressive lung disease that begins early in childhood and is characterized by chronic bacterial infection and inflammation. Nearly 90% of people with CF have at least one copy of the ΔF508 mutation, but there are hundreds of CFTR mutations that result in a range of disease severities. A CFTR gene replacement approach would be efficacious regardless of the disease-causing mutation. After the discovery of the CFTR gene in 1989, the in vitro proof-of-concept for gene therapy for CF was quickly established in 1990. In 1993, the first of many gene therapy clinical trials attempted to rescue the CF defect in airway epithelia. Despite the initial enthusiasm, there is still no FDA-approved gene therapy for CF. Here we discuss the history of CF gene therapy, from the discovery of the CFTR gene to current state-of-the-art gene delivery vector designs. While implementation of CF gene therapy has proven more challenging than initially envisioned; thanks to continued innovation, it may yet become a reality.

scholarly journals Progress Toward The Synthesis Of Labeled Derivatives Of The Cystic Fibrosis Drug Ivacaftor

2021 ◽  
Author(s):  
Salma Elmallah
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Genetic Diseases ◽  
Early Death ◽  
Open Probability ◽  
Gene Encoding ◽  
Short Period ◽  
Cftr Mutations ◽  
Cftr Protein ◽  
Orally Bioavailable

Cystic fibrosis (CF) is one of the most common genetic diseases, affecting approximately 70,000 people worldwide causing severe complications and often leading to early death. CF is caused by a mutation in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein which is responsible for fluid and ion transport through epithelial membranes maintaining the formation of a thin slippery mucous layer. CFTR mutations either lead to a trafficking defect where the CFTR protein does not reach the plasma membrane or a gating defect where CFTR protein at the plasma membrane does not function properly. Treatment of cystic fibrosis usually addresses the symptoms to overcome the complications of the disease such as pneumonia, lung infections, pancreatitis, maldigestion and infertility. Vertex pharmaceuticals has been interested in developing small molecules that have the ability to interact with mutated CFTR proteins, aiding in their delivery to the cell membrane and/or restoring their channel function. VX-770 is an orally bioavailable potentiator that has the ability to improve the gating activity and increasing the open probability of CFTR protein in patients carrying the G551D mutation. VX770, Ivacaftor, was recently approved by the US FDA after showing very good improvements in the lung function in CF patients with good safety profile. Our research is focusing on the synthesis of VX770 under mild conditions and formation of labeled derivatives to help in the understanding of its exact mode of action. Different methods were developed toward the synthesis of the two main components, LHS and RHS, of VX770 by using less harsh conditions for a short period of time. We were successfully able to make two photoaffinity labeled derivatives, aryl azide and benzophenone derivatives, which will be beneficial in tracking the drug molecule and revealing the exact site of interaction between the drug and the protein. Synthesis of VX770 fragments was is another focus of interest in our research as they will provide us with information about the best positions for further derivatization.

scholarly journals Structural comparative modeling of multi-domain ΔF508 CFTR

2021 ◽  
Author(s):  
Eli Fritz McDonald ◽  
Hope Woods ◽  
Shannon Smith ◽  
Minsoo Kim ◽  
Clara T. Schoeder ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Rational Design ◽  
Anion Channel ◽  
Comparative Modeling ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Δf508 Cftr ◽  
Domain Models ◽  
Cystic Fibrosis Transmembrane

Cystic Fibrosis (CF) is a common genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), an epithelial anion channel expressed in several vital organs. Absence of functional CFTR results in imbalanced osmotic equilibrium and subsequent mucus build up in the lungs - which increases the risk of infection and eventually causes death. CFTR is an ATP binding cassette (ABC) transporter composed of two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and an unstructured regulatory domain. The most prevalent patient mutation is the deletion of F508 (ΔF508), making ΔF508 CFTR the primary target for current FDA approved CF therapies. However, no experimental multi-domain ΔF508 CFTR structure has been determined and few studies have modeled ΔF508 using multi-domain WT CFTR structures. Here, we used cryo-EM density data and Rosetta comparative modeling (RosettaCM) to compare a ΔF508 model with published experimental data on CFTR NBD1 thermodynamics. We then apply this modeling method to generate multi-domain WT and ΔF508 CFTR structural models. These models demonstrate the destabilizing effects of ΔF508 on NBD1 and the NBD1/TMD interface in both the closed and open conformation of CFTR. Furthermore, we modeled ΔF508/R1070W and ΔF508 bound to a the CFTR corrector VX-809. Our models reveal the stabilizing effects of R1070W and VX-809 on multi-domain models of ΔF508 CFTR and pave the way for rational design of additional drugs that target ΔF508 CFTR for treatment of CF.

scholarly journals Modulation of CFTR gating by permeant ions

2014 ◽  
Vol 145 (1) ◽  
pp. 47-60 ◽  
Author(s):  
Han-I Yeh ◽  
Jiunn-Tyng Yeh ◽  
Tzyh-Chang Hwang
Keyword(s):  
Atp Hydrolysis ◽  
Single Channel ◽  
Common Mechanism ◽  
Transmembrane Conductance Regulator ◽  
Open Probability ◽  
Binding Domains ◽  
Gating Model ◽  
Sites Of Action ◽  
Opening And Closing ◽  
Closing Rate

Cystic fibrosis transmembrane conductance regulator (CFTR) is unique among ion channels in that after its phosphorylation by protein kinase A (PKA), its ATP-dependent gating violates microscopic reversibility caused by the intimate involvement of ATP hydrolysis in controlling channel closure. Recent studies suggest a gating model featuring an energetic coupling between opening and closing of the gate in CFTR’s transmembrane domains and association and dissociation of its two nucleotide-binding domains (NBDs). We found that permeant ions such as nitrate can increase the open probability (Po) of wild-type (WT) CFTR by increasing the opening rate and decreasing the closing rate. Nearly identical effects were seen with a construct in which activity does not require phosphorylation of the regulatory domain, indicating that nitrate primarily affects ATP-dependent gating steps rather than PKA-dependent phosphorylation. Surprisingly, the effects of nitrate on CFTR gating are remarkably similar to those of VX-770 (N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide), a potent CFTR potentiator used in clinics. These include effects on single-channel kinetics of WT CFTR, deceleration of the nonhydrolytic closing rate, and potentiation of the Po of the disease-associated mutant G551D. In addition, both VX-770 and nitrate increased the activity of a CFTR construct lacking NBD2 (ΔNBD2), indicating that these gating effects are independent of NBD dimerization. Nonetheless, whereas VX-770 is equally effective when applied from either side of the membrane, nitrate potentiates gating mainly from the cytoplasmic side, implicating a common mechanism for gating modulation mediated through two separate sites of action.

scholarly journals ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

2008 ◽  
Vol 364 (1514) ◽  
pp. 247-255 ◽  
Author(s):  
Daniella Muallem ◽  
Paola Vergani
Keyword(s):  
Cystic Fibrosis ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Functional Difference ◽  
Transmembrane Conductance Regulator ◽  
Cellular Functions ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and γ-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway.

scholarly journals Progress Toward The Synthesis Of Labeled Derivatives Of The Cystic Fibrosis Drug Ivacaftor

2021 ◽  
Author(s):  
Salma Elmallah
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Genetic Diseases ◽  
Early Death ◽  
Open Probability ◽  
Gene Encoding ◽  
Short Period ◽  
Cftr Mutations ◽  
Cftr Protein ◽  
Orally Bioavailable

Cystic fibrosis (CF) is one of the most common genetic diseases, affecting approximately 70,000 people worldwide causing severe complications and often leading to early death. CF is caused by a mutation in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein which is responsible for fluid and ion transport through epithelial membranes maintaining the formation of a thin slippery mucous layer. CFTR mutations either lead to a trafficking defect where the CFTR protein does not reach the plasma membrane or a gating defect where CFTR protein at the plasma membrane does not function properly. Treatment of cystic fibrosis usually addresses the symptoms to overcome the complications of the disease such as pneumonia, lung infections, pancreatitis, maldigestion and infertility. Vertex pharmaceuticals has been interested in developing small molecules that have the ability to interact with mutated CFTR proteins, aiding in their delivery to the cell membrane and/or restoring their channel function. VX-770 is an orally bioavailable potentiator that has the ability to improve the gating activity and increasing the open probability of CFTR protein in patients carrying the G551D mutation. VX770, Ivacaftor, was recently approved by the US FDA after showing very good improvements in the lung function in CF patients with good safety profile. Our research is focusing on the synthesis of VX770 under mild conditions and formation of labeled derivatives to help in the understanding of its exact mode of action. Different methods were developed toward the synthesis of the two main components, LHS and RHS, of VX770 by using less harsh conditions for a short period of time. We were successfully able to make two photoaffinity labeled derivatives, aryl azide and benzophenone derivatives, which will be beneficial in tracking the drug molecule and revealing the exact site of interaction between the drug and the protein. Synthesis of VX770 fragments was is another focus of interest in our research as they will provide us with information about the best positions for further derivatization.

Sign in / Sign up

Export Citation Format

Share Document