Gating of Trimerized or Tetramerized Voltage-Gated H+ Channels

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VSOP/Hv1 is a dimeric voltage-gated H+ channel unlike other tetrameric voltage-gated channels. Each subunit has its own permeation pathway, and the gating of one subunit is coupled to that of the other subunit within the dimer. We previously reported that the cytoplasmic dimer coiled-coil mediated the dimeric assembly and the gating coupling, based on the crystal structure analysis of the coiled-coil domain. The crystal structure of the VSOP coiled-coil shows an I/L core packing pattern, in which well-packed Ile/Leu residues are situated at positions ‘a’/‘d’ in the heptad repeat and are periodically observed along the entire length of the coiled-coil. However, the functional significance of the patterned sequence for the channel function remains unknown. To address this issue, we changed the packing pattern to I/I, L/L and L/I-types, and analyzed the stoichiometry of the mutant coiled-coils’ assembly. Sedimentation and crystal structure analyses showed that coiled-coils with I/I and L/L-type cores formed trimers, and the version harboring the L/I core formed a tetramer. These were consistent with the results of cross-linking analysis followed by Western blotting of the full-length proteins, indicating that the assembly stoichiometry was determined by the core type. Electrophysiological analysis revealed that only the dimer types showed slow and sigmoidal activation kinetics suggestive of cooperative gating. Thus, the I/L sequence pattern of the coiled-coil core in the natural channel is optimally designed to form a dimeric channel with slow and cooperative gating.