A large family of unusual voltage‐sensing proton channels (Hv3) in mollusks

Mollusks exhibit an unusually high diversity of genes encoding voltage-gated proton channel homologs. In addition to the conventional proton channels Hv1, Hv2, and Hv4, a structurally and functionally atypical proton channel was previously identified in Aplysia californica (AcHv3, subsequently renamed AcHv3.1). A comprehensive analysis of cDNA, transcriptome shotgun assembly (TSA) and genomic databases revealed numerous orthologous and paralogous Hv3-like sequences in Aplysia and across all other molluscan species analyzed. These Hv3-like channels share canonical features of voltage-gated proton channels, including four transmembrane spanning segments (S1-S4), a negatively charged residue in S1 responsible for proton selectivity, a conserved voltage-sensing motif in S4 containing positively charged arginine residues and a fully conserved tryptophan residue. However, Hv3-like channels differ markedly from conventional Hv channels in possessing much larger intracellular N- and C-terminal domains, an elongated extracellular loop between S1 and S2 and a reduced number of gating charges in S4, with only two arginine residues (or a single arginine in the case of Hv3.6 channels) compared to three in canonical Hv channels. In addition, Hv3-like channels contain a unique proline residue within S4, forming a characteristic PWRxxR motif. Based on structural differences, including putative PDZ-Binding motifs and spacing of transmembrane cysteine residues, together with molecular distance tree analyses, a classification into seven subfamilies (Hv3.1-Hv3.7) is proposed. Some of these subfamilies were found only in Gastropoda or Bivalvia, while others were identified in all molluscan classes. Although GFP-tagged AcHv3.1, AcHv3.2, and AcHv3.5 localized to the plasma membrane, only AcHv3.1 generated detectable proton currents.