The Permeation Pathway Mechanism in Ciona Intestinalis Hv Channel
Voltage-gated proton (HV) channels are expressed by different cells type including immune cells, microglia, among others. In other voltage-gated cation channels, the pore-forming domains are S5, S6, and the P loop connecting S5 and S6. Since Hv channels do not contain a S5-S6 region, other parts of the channel must form the pore domain. Interestingly, Hv channel present three gating charges in the S4 followed by an asparagine, N264, highly conserved among all Hv channel. Using non-stationary fluctuation analysis we establish the conductance for the dimer and the monomer in 200 and 100 fS, respectively. Mutations at position S191 (S2) and N264 (S4) modified the unitary conductance of Hv channel. Furthermore, mutants S191E/D and N264R removed the H+ current remaining the S4 functioning unaltered, according to voltage-clamp fluorometry experiments. The introduction of an arginine or lysine at position 264 or negative residue at position S191, drastically reduced or abolished the proton currents. Interestingly, mutations of the analogue position S191 of Ciona VSOP, in the voltage sensor domain of a non-conductor Shaker K+ channel produce fully functional voltage gated H+ channels. In the same way, mutation resembling the position N264 of Ciona Hv in the VSD of the voltage-dependent phosphate Ci-VSP spontaneously entail the formation of a voltage-gated H+ channel. Thus, S191 and N264 form the molecular determinants of permeation pathway in Hv channel. Supported by Fondecyt grants ACT 1104 and Fondecyt 1120802 to CG and 1110430 to R.L.