PhD Scientific Days 2021

Budapest, 7-8 July 2021

MO_I_P: Molecular Sciences I. Posters

Understanding the Mechanism of CFTR Ion Channel Bursting and of CF Caused by Mutation R117H

Márton A. Simon, László Csanády
1 Department of Biochemistry, Semmelweis University, Budapest
2 Department of Biochemistry, Semmelweis University, Budapest

Text of the abstract

In phosphorylated CFTR chloride channels, an ABC Binding Casette protein, pore gating is linked to ATP binding/hydrolysis at two cytosolic nucleotide binding domains. CFTR exhibits bursting behavior: ‘bursts’ of openings separated by short closures are flanked by long closures, which can be described by either C(long)-O-C(short) or C(long)-C(short)-O mechanisms. CFTR mutations cause the lethal and incurable disease cystic fibrosis (CF). CF mutation R117H accelerates pore closure and decreases anion conductance. This position moves late during channel opening, and its mutations do not affect opening rate, suggesting that the R117 side chain stabilizes the open state. In open CFTR structures the R117 side chain forms a strong H-bond with the E1124 backbone carbonyl group, but this interaction is absent in closed CFTR structures.
We aim to investigate whether the R117–E1124 interaction stabilizes the open state. Single-channel and macroscopic inside-out patch recordings in a non-hydrolytic CFTR background, in which gating is reduced to reversible closed-open transitions, allow quantitation of thermodynamic mutant cycles that address gating-associated changes in interaction energy between the target positions. As perturbations of an interaction that stabilizes the O-state should differentially affect COC- or CCO-type gating behavior, our experiments might reveal the mechanism of CFTR gating.
For positions 117 and 1124, CF-causing mutation R117H and computationally suggested E1124del were chosen, respectively. As controls, mutations E1124A/P/G exerted mild or no phenotypes, however, E1124del indeed accelerated closing rate and decreased intraburst Po similarly to R117H; moreover, no additivity was observed in the double mutant R117H/E1124del, supporting a state-dependent interaction between the target positions vital for the stabilization of the open state. Based on our data, CFTR bursting can be described more efficiently by the CCO mechanism.
Supported by the ÚNKP-20-3-I New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund.

University and Doctoral School

Semmelweis University, Doctoral School of Molecular Medicine