Accessibility of the S4 Arginines in the Human Voltage Gated Proton Channel, hHV1

Abstract

The voltage gated proton channel, HV1, has four transmembrane segments and bears a striking resemblance to the voltage-sensing domain (VSD) of other voltage gated ion channels. One difference is that the human HV1, hHV1, has only three Arg residues in its S4 helix, Arg205 (R1), Arg208 (R2), and Arg211 (R3). Cys scanning indicates that S4 of hHV1 moves outward during channel opening (Gonzalez et al, 2010. Nat. Struct. Mol. Biol. 17:51-56). To explore the extent of this movement, we replaced each Arg individually with His, and probed accessibility by applying Zn2+ externally or internally. All mutations were carried out in a background lacking the two His residues (H140 & H193) that are responsible for inhibition of WT channels by Zn2+ (Ramsey et al., 2006. Nature 440:1213-1216). Both R1 and R2 were accessible to Zn2+ from the external solution. R3 was not accessible from the external solution, but was accessible from the internal solution, and surprisingly, remained accessible in the open state. In contrast with analogous studies of the Shaker K channel VSD (Starace & Bezanilla, 2004. Nature 427:548-553), none of the Arg→His mutants was a hyperpolarization-activated proton channel. Molecular dynamics simulations produced a similar picture of the open hHV1 channel. At the selectivity filter, Asp112 forms a salt bridge with Arg208. R1 and R3 participate in external and internal charge clusters, respectively, that stabilize the open state. Existing evidence thus suggests that the S4 helix of hHV1 moves outward during channel opening, qualitatively like S4 in K+ channels, but the extent of this movement is substantially less. This difference may reflect the fact that the conduction pathway in other channels resides in a physically separate domain (S5-S6), whereas in hHV1 it is within the VSD itself.