Zn2+ Modulates Hv1 Proton Channel Gating via Conformational Coupling to an Intracellular Coulombic Network

Abstract

Gating of the intrinsic ‘aqueous’ H+ conductance (GAQ) in the Hv1 proton channel is sensitive to extracellular Zn2+ and other divalent cations in the micromolar range. Previous studies showed that the extracellularly-accessible residues H140 and H193 are required for Zn2+ to positively shift the GAQ-V relation. However, the molecular mechanism by which extracellular Zn2+ occupancy is coupled to voltage-dependent GAQ gating remains known. We used site-directed mutagenesis and electrophysiology to identify Zn2+-interacting residues and used the experimental data to build new models of the Hv1 voltage sensor (VS) domain in Zn2+-liganded (Hv1F·Zn2+) and unliganded (Hv1F + Zn2+) conformations. Although imidazole nitrogen atoms (H140-Nδ1 and H193-Nδ1) are close enough to simultaneously coordinate a single Zn2+ ion, we did not observe a stable Zn2+ coordination sphere during all-atom molecular dynamics (MD) simulations using the CHARMM36 force field. To simulate electronic interactions between Zn2+ and H140-Nδ1 and H193-Nδ1, we imposed harmonic constraints (5 kcal/mol/Å) and equilibrated the Hv1F·Zn2+ model during a 50 ns MD simulation. 20 ns after releasing constraints, ZN13 spontaneously dissociates from H140-Nδ1 and H193-Nδ1 but remains associated with the carboxyl group of E119. The VS also undergoes backbone reorganization involving the S1, S3 and S4 helices after ZN13 dissociation that suggest Zn2+-dependent conformational changes. Concomitant changes in the organization of an electrostatic network involving intracellular acidic residues (E153 and D174) and S4 ‘gating charge’ Arg side chains may reveal how voltage-dependent gating is altered by Zn2+. In support of this hypothesis, we find that E153 neutralization also alters Zn2+ potency. In summary, extracellular Zn2+ occupancy is coupled to reorganization of an intracellular Coulombic network that is required for voltage-dependent gating in Hv1.