Electrostatic Networks and Mechanisms of ΔpH-Dependent Gating in the Human Voltage-Gated Proton Channel Hv1

Author

Ashley L. Bennett, Virginia Commonwealth UniversityFollow

DOI

https://doi.org/10.25772/QT3Z-G356

Author ORCID Identifier

https://orcid.org/0000-0001-7927-4511

Defense Date

2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Physiology and Biophysics

First Advisor

Dr. I. Scott Ramsey

Second Advisor

Dr. John Hackett

Third Advisor

Dr. Lei Zhou

Fourth Advisor

Dr. Bill Barton

Fifth Advisor

Dr. Phil Mosier

Abstract

The structure of the voltage-gated proton (H⁺) channel Hv1 is homologous to the voltage sensor domain (VSD) of tetrameric voltage-gated Na⁺, K⁺ and Ca²⁺ channels (VGCs), but lacks a pore domain and instead forms a homodimer. Similar to other VSD proteins, Hv1 is gated by changes in membrane potential (V), but unlike VGCs, voltage-dependent gating in Hv1 is modulated by changes in the transmembrane pH gradient (DpH = pH_o - pH_i). In Hv1, pH_o or pH_i changes shift the open probability (P_OPEN)-V relation by ~40 mV per pH unit. To better understand the structural basis of pH_o-dependent gating in Hv1, we constructed new resting- and activated-state Hv1 VSD homology models using physical constraints determined from experimental data measured under voltage clamp and conducted all-atom molecular dynamics (MD) simulations. Analyses of salt bridges and calculated pK_as at conserved side chains suggests the existence of intracellular and extracellular electrostatic networks (ICEN and ECEN, respectively) that stabilize resting- or activated-state conformations of the Hv1 VSD. Structural analyses led to a novel hypothesis: two ECEN residues (E119 and D185) with coupled pK_as coordinately interact with two S4 ‘gating charge’ Arg residues to modulate activated-state pH_o sensitivity. Experimental data confirm that pH-dependent gating is compromised at acidic pH_o in Hv1 E119A-D185A mutants, indicating that specific ECEN residue interactions are critical components of the ∆pH-dependent gating mechanism. E119 and D185 are known to participate in extracellular Zn²⁺ coordination, suggesting that H⁺ and Zn²⁺ utilize similar mechanisms to allosterically modulate the activated/resting state equilibrium in Hv1.

Rights

© Ashley L. Bennett

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

11-18-2019