Dimeric subunit stoichiometry of the human voltage-dependent proton channel Hv1

Abstract

In voltage-gated Na ؉ , K ؉ , and Ca 2؉ channels, four voltage-sensor domains operate on a central pore domain in response to membrane voltage. In contrast, the voltage-gated proton channel (Hv) contains only a voltage-sensor domain, lacking a separate pore domain. The subunit stoichiometry and organization of Hv has been unknown. Here, we show that human Hv1 forms a dimer in the membrane and define regions that are close to the dimer interface by using cysteine cross-linking. Two dimeric interfaces appear to exist in Hv1, one mediated by S1 and the adjacent extracellular loop, and the other mediated by a putative intracellular coiled-coil domain. It may be significant that Hv1 uses for its dimer interface a surface that corresponds to the interface between the voltage sensor and pore in Kv channels.membrane protein ͉ voltage-dependent ion channel ͉ voltage sensor V oltage-gated six-transmembrane cation channels (Na ϩ , K ϩ , and Ca 2ϩ ) contain voltage-sensor and pore domains (1). In this class of ion channels voltage-sensor and pore domains carry out voltage sensing and cation conduction, respectively. Four voltage-sensor domains surround a single, centrally located ion conduction pathway. Each voltage sensor is attached to the pore in a specific manner so that conformational changes within the voltage sensors are transmitted to the pore's gate (2). It was originally thought that voltage-sensor domains existed only in the context of voltage-gated cation channels. However, the cloning of voltage-gated proton (Hv) channels and voltagesensor phosphatase enzymes revealed that voltage-sensor domains also exist in other contexts, apparently as independent (of a cation pore) membrane proteins (3-5), corroborating the studies of MacKinnon and coworkers (2, 6, 7) and Lu and coworkers (8) that supported the idea that voltage sensors, even in the context of voltage-dependent cation channels, are rather loosely attached to the central pore.The long-sought molecular identity of Hv1 (9) showed that it contains only a voltage-sensor domain without a separate pore domain in the membrane (3, 4). This observation suggested that in contrast to canonical voltage-dependent cation channels, the voltage-sensor domain of Hv1 is responsible for both H ϩ conduction and voltage sensing. In this study we evaluate the subunit stoichiometry of the Hv1 channel in cell membranes. ResultsHv1 Is a Dimer in the Membrane. To probe the oligomeric state of Hv1, cell membranes isolated from tsA201 cells (HEK293 derivatives) transfected with human Hv1 cDNAs were subjected to the amino-group specific bifunctional cross-linker disuccinimidyl suberate (DSS) and visualized by Western blot analysis using antibodies directed against the Hv1 channel. Amino groupspecific cross-linkers have been successful in defining the oligomeric status of several membrane proteins (10-12). Recombinant Hv1 makes functional channels in HEK293 cells (3, 4). In Fig. 1a, human Hv1 migrates at Ϸ35 kDa in SDS/PAGE under reducing conditions, which is consistent with the molecula...

The cytoplasmic coiled-coil mediates cooperative gating temperature sensitivity in the voltage-gated H+ channel Hv1

“…Thus, rigid structure of the linker region enables to mediate the stability of the coiled-coil to the transmembrane domain, and to mediate the cooperative gating within the dimer. Considering the mechanical interaction between two channel subunits, a certain level of physical intersubunit contact between transmembrane regions within a dimer has been reported 9,17 . These make it plausible that two transmembrane regions in close proximity in a dimer physically interact with one another, and the coiled-coil domain facilitates the close positioning of the two transmembrane regions (Supplementary Fig.…”

“…It is reported that the cytoplasmic end of S4 helix is close to each other within a dimer, where Cys introduced into the position of Thr222 in hHv1/VSOP make a disulfide bond between two monomers 9 . To examine whether this spatial proximity was disarranged by the GGG mutation, we performed crosslinking experiments by oxidation with the T218C mHv1/VSOP mutants that corresponds toT222C in hHv1/VSOP.…”

“…Hv1/VSOP has fourtransmembrane segments that correspond to the voltage-sensor domain (S1-S4) of other voltage-gated channels 1,2 , it does not have a canonical pore domain, and the voltage-sensor domain is thought to act as both the voltage-sensor and the H + -permeation pathway 7 . The composition of the functional unit by the minimally designed voltage-gated channel subunit is dimeric [8][9][10] .…”

“…However, it remains unclear through which part of the channel the conformational change is transmitted from one subunit to the other during channel gating. A certain level of physical intersubunit contact between transmembrane regions has been shown by biochemical approaches 9 , raising the possibility that the cooperative gating in the dimer is mediated via interaction between the tramsmembrane domains. Meanwhile, Hv1/VSOP contains a conserved coiled-coil motif in the cytoplasmic region.…”

Do Hv1 proton channels regulate the ionic and redox homeostasis of phagosomes?

“…Multiple functions were proposed for proton channels, but these remained speculative until the cloning of the Hv1 protein by two separate laboratories (Ramsey et al, 2006;Sasaki et al, 2006) and the generation of Hv1 knockout mice offered the possibility to test the physiological role of proton channels. Structure-function studies revealed that Hv1 channels are dimers that gate cooperatively (Gonzalez et al, 2010;Musset et al, 2010;Tombola et al, 2010), with each monomer containing a separate conduction pathway and voltage sensor (Koch et al, 2008;Lee et al, 2008;Tombola et al, 2008). Hv1 channels are perfectly selective for protons and are activated by depolarizing voltages and by intracellular acidification, a combination that ensures that the channels only open in conditions that favor proton extrusion from cells.…”