Abstract
The Hv1 proton channel is evidently unique among voltage sensor domain proteins in mediating an intrinsic 'aqueous' H+ conductance (GAQ). Mutation of a highly conserved 'gating charge' residue in the S4 helix (R1H) confers a resting-state H+ 'shuttle' conductance (GSH) in VGCs and Ci VSP, and we now report that R1H is sufficient to reconstitute GSH in Hv1 without abrogating GAQ. Second-site mutations in S3 (D185A/H) and S4 (N4R) experimentally separate GSH and GAQ gating, which report thermodynamically distinct initial and final steps, respectively, in the Hv1 activation pathway. The effects of Hv1 mutations on GSH and GAQ are used to constrain the positions of key side chains in resting- and activated-state VS model structures, providing new insights into the structural basis of VS activation and H+ transfer mechanisms in Hv1.
Highlights
The superfamily of voltage sensor (VS) domain proteins includes tetrameric voltage-gated cation channels (VGCs), voltage-sensitive phosphatases (VSPs) and the Hv1 proton channel
As in At TPC1 DII VS X-ray structures, we find that the R1/R4.47 terminal amine is oriented toward the intracellular vestibule in Hv1 VS domain model (Hv1 D), where it is predicted to participate in a Coulombic interaction with a conserved acidic residue, D174/D3.50 (Figure 5G; Figure 5—figure supplement 4F), that is part of the intracellular electrostatic network (Ramsey et al, 2010; Long et al, 2005)
E, R1 and R1H side chains are clearly visible within the gating pores when the model structures are viewed from the extracellular space (Figure 5—figure supplement 4C; Video 3). In both Hv1 D and Hv1 model structure (Hv1 E), we find that D112/D1.51 is on the extracellular side of F2.50 and is readily accessible to solvent (Figure 5E; Figure 5—figure supplement 3C–I)
Summary
The superfamily of voltage sensor (VS) domain proteins includes tetrameric voltage-gated cation channels (VGCs), voltage-sensitive phosphatases (VSPs) and the Hv1 proton channel. Hydrophobic groups appear to limit solvent accessibility at the waist of the central crevice in both resting- and activated-state VS structures, while ionizable side chains, including conserved Arg residues in S4, appear to be solvent-exposed (Long et al, 2005; Takeshita et al, 2014; Li et al, 2014; Zhang et al, 2012; Payandeh et al, 2011; Krepkiy et al, 2009; Lacroix et al, 2014; Krepkiy et al, 2012).
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