Biophysical Society Conference | Tahoe 2023

Proton Reactions: From Basic Science to Biomedical Applications

Monday Speaker Abstracts

MOLECULAR DETERMINANTS OF VOLTAGE SENSOR IN CIHV1 CHANNEL Carlos L. Gonzalez 1,2,3 ; Miguel Fernandez 2,3 ; Juan Alvear-Arias 2,3 ; Emerson M Carmona 4 ; Josè A Garate 3,5,6 ; 1 University of Miami, Department of Physiology and Biophysics, Miller School of Medicine, Miami, FL, USA 2 Universidad de Valparaíso, Centro Interdisciplinario de Neurociencia de Valparaíso, Valparaiso, Chile 3 Universidad de Valparaíso, Millennium Nucleus in NanoBioPhysics, Valparaiso, Chile 4 Texas Tech University , Health Sciences Center, Lubbock, TX, USA 5 Universidad San Sebastián, acultad de Ingeniería y Tecnología,, Santiago, Chile 6 Centro Científico y Tecnológico de Excelencia Ciencia y Vida, Santiago, Chile The Ciona intestinalis proton channel (Hv1) is a membrane protein with the voltage sensing, pH, and permeation pathway located in the same structural region. In the mutant ΔNΔC -N264R, we observed a reduction of conductance and a fast activation kinetics accompanied by a robust ON gating current and diminished macroscopic proton current. We named the decrease of the OFF gating-charge component upon repolarization as trapping. We used patch-clamp gating currents and molecular dynamics experiments to elucidate the mechanism of voltage sensor movement during channel activation. The trapped charge disappears when selectivity filter (D160) is mutated (D160N) on the ΔNΔC monomeric background, indicating that trapping is not an intrinsic featur e of the channel. However, the double mutant (ΔNΔC -D160N N264R) showed only partial trapping, revealing that electrostatic effects are not the only driving force. Increasing the D160 hydrophobicity increases the trapping phenomenon. Molecular dynamics simulations showed that this effect causes electrostatic repulsion towards the arginines (258 and 261) of the voltage sensor, allowing them to move upwards, which facilitates salt bridge formation with D160, trapping the voltage sensor in its active state. Gating charge trapping can be mimicked by blocking a specific inhibitor (2GBI) or increasing proton concentration (positive ΔpH), indicating that changes in the electrostatic-hydrophobicity environment can induce a delay in the voltage sensor returning to the resting position. In conclusion, the OFF-gating charges delay on the N264R mutant is due to a trapped voltage sensor in the active state during channel activation, where residues D160 and N264 are essential in Hv1 voltage sensor displacement molecular mechanism.

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