VSOP Protein Lacking the C-terminal Half of S4-like Segment Retains Proton Permeation

VSOP/Hv1 is a protein that contains the voltage sensor domain but not pore domain [1, 2]. It exhibits properties of native voltage-gated proton channels reported in phagocytes and microglia. Addressing how proton permeates and how voltage-dependent gating is achieved in VSOP/Hv1 will lead to critical clues to understand mechanisms of voltage sensor operation and ion permeation. The putative fourth transmembrane segment (S4) of mouse VSOP (mVSOP) has three positively charged residues in a pattern similar to those conserved in other voltage-gated channels. We have previously shown that VSOP/Hv1 forms dimer and a version lacking the cytoplasmic region (V216X) expressing mainly as monomer exhibits robust voltage-dependent proton currents, suggesting that monomer constitutes proton permeation pathway [3]. However, V216X still contains some cytoplasmic stretch and it remained unknown whether a remaining stretch downstream of S4 segment is essential for proton channel activities. To address this, a series of deletion constructs of mVSOP were expressed in tsA201 cells and whole cell patch recording and western blot were performed. Surprisingly, voltage-gated outward currents were elicited in constructs with stop codon at sites upstream to the third arginine. Proton permeation was verified by measuring intracellular pH using the pH-sensitive fluorescent dye, simultaneously with whole-cell patch clamping. Therefore, mVSOP retains functions of voltage-gated proton channel only with a truncated S4 segment, neglecting some possible mechanisms of proton permeation. To gain more insights, we are currently trying to biochemically map the topology of S4 using the cysteine-targeting reagent.[References][1] Sasaki M. et al., (2006) Science 312:589-592.[2] Ramsey IS. et al., (2006) Nature 440:1213-1216.[3] Koch HP. et al., (2008) Proc. Natl. Acad. Sci. USA 105:9111-9116.