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

Márton A Simon,László Csanády

doi:10.7554/elife.74693

Abstract

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.

Highlights

Loss-of-function mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel disrupt transepithelial salt-water transport in the lung, intestine, pancreatic duct, and sweat duct, and cause cystic fibrosis (CF), the most common inherited lethal disease among caucasians (O'Sullivan and Freedman, 2009)
CFTR is an Adenosine 5′triphosphoribose magnesium (ATP) Binding Cassette (ABC) protein which contains two transmembrane domains (TMD1, 2; Fig. 1A, gray) and two cytosolic nucleotide binding domains (NBD1, 2; Fig. 1A, blue and green). These two ABC-typical TMD-NBD halves are linked by a cytosolic regulatory (R) domain (Fig. 1A, magenta) which is unique to CFTR and contains multiple serines that must be phosphorylated by cAMP-dependent protein kinase (PKA) to allow channel activity (Riordan et al, 1989)
Our analysis provides a mechanistic explanation for the strong energetic role that the conserved arginine at position 117 plays in CFTR channel gating, as well as for the molecular pathology caused by its mutations

Summary

Introduction

Loss-of-function mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel disrupt transepithelial salt-water transport in the lung, intestine, pancreatic duct, and sweat duct, and cause cystic fibrosis (CF), the most common inherited lethal disease among caucasians (O'Sullivan and Freedman, 2009). Based on their molecular consequences the several hundred identified CF mutations have been categorized into classes, such as those that impair synthesis of the full-length CFTR polypeptide (Class I), processing and trafficking of the CFTR potein (Class II), channel gating (Class III), or anion permeation through the open pore (Class IV) (De Boeck and Amaral, 2016).

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