Biology subjects

Perozo, E.

Publications and source records attributed to Perozo, E..

3 recordsLinked to original sources

bioRxiv2022

State Dependent Anionic Pore Currents Conducted by Single Countercharge Mutants in a Voltage-Sensing Phosphatase

Mutating gating charge residues in the S4 segment of voltage-sensing domains (VSDs) can cause ionic leak currents through the VSDs. These leak currents, known as gating pore or omega currents, play important pathophysiological role in many diseases. Here, we show that mutations in a key countercharge residue, D129, in the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) facilitate conduction of unique anionic omega currents. Neutralization of D129 causes a dramatic positive shift of activation, facilitates the formation of a continuous water path through the intermediate state VSD, and creates a positive electrostatic potential landscape inside the VSD leading to anion selectivity. Increasing the population or duration of the conducting state by a high external pH or an engineered Cd2+ bridge markedly increases the current magnitude. Our findings uncover a new role of countercharge residues and could inform on the mechanisms of channelopathies linked to countercharge residue mutations.

biophysics↗

bioRxiv2022

Mechanism of Voltage Gating in the Voltage-Sensing Phosphatase Ci-VSP

The conformational changes in voltage-sensing domain (VSD) are driven by the transmembrane electric field acting on charges and countercharges. Yet, the overall energetics and detailed mechanism of this process are not fully understood. Here, we determined free energy and displacement charge landscapes, as well as major conformations corresponding to a complete functional gating cycle in the isolated voltage-sensing domain of the phosphatase Ci-VSP (Ci-VSD) comprising four transmembrane helices (segments S1-S4). Molecular dynamics simulations highlight the extent of S4 movements. In addition to the crystallographically determined activated Up and resting Down states, the simulations predict two novel Ci-VSD conformations: a deeper resting state ( Down-minus) and an extended activated ( Up-plus) state. These additional conformations were experimentally probed via systematic cysteine mutagenesis with metal-ion bridges and the engineering of proton conducting mutants at hyperpolarizing voltages. These results show that voltage activation involves sequentially populating these four states in a stepwise way, translating one arginine across the membrane electric field per step, transferring ~3 e0 charges.

biophysics↗

bioRxiv2021

Cryo-EM Structures of Prestin and the Molecular Basis of Outer Hair Cell Electromotility

The voltage-dependent motor protein, Prestin (SLC26A5) is responsible for the electromotive behavior of outer hair cells (OHCs). Here, we determined the structure of dolphin Prestin in six distinct states using single particle cryo-electron microscopy. Structural and functional data suggest that Prestin adopts a unique and complex set of states, tunable by the identity of bound anions. Complexes with the inhibitor salicylate show that it competes for the anion-binding site of Prestin. These conformations reveal a novel mechanism of area expansion that depends on the helix flexibility and conformational transitions at the membrane protein interface and putatively affects the physical state of the surrounding membrane. These observations illuminate the structural basis of Prestin electromotility, a key component of the mammalian cochlear amplifier.

neuroscience↗