Biophysical characterization of the honeybee DSC1 orthologue reveals a novel voltage-dependent Ca(2+) channel subfamily: Ca(V)4

Bilaterian voltage-gated Na(+) channels (Na(V)) evolved from voltage-gated Ca(2+) channels (Ca(V)). The Drosophila melanogaster Na(+) channel 1 (DSC1), which features a D-E-E-A selectivity filter sequence that is intermediate between Ca(V) and Na(V) channels, is evidence of this evolution. Phylogenetic analysis has classified DSC1 as a Ca(2+)-permeable Na(+) channel belonging to the Na(V)2 family because of its sequence similarity with Na(V) channels. This is despite insect Na(V)2 channels (DSC1 and its orthologue in Blatella germanica, BSC1) being more permeable to Ca(2+) than Na(+). In this study, we report the cloning and molecular characterization of the honeybee (Apis mellifera) DSC1 orthologue. We reveal several sequence variations caused by alternative splicing, RNA editing, and genomic variations. Using the Xenopus oocyte heterologous expression system and the two-microelectrode voltage-clamp technique, we find that the channel exhibits slow activation and inactivation kinetics, insensitivity to tetrodotoxin, and block by Cd(2+) and Zn(2+). These characteristics are reminiscent of Ca(V) channels. We also show a strong selectivity for Ca(2+) and Ba(2+) ions, marginal permeability to Li(+), and impermeability to Mg(2+) and Na(+) ions. Based on current ion channel nomenclature, the D-E-E-A selectivity filter, and the properties we have uncovered, we propose that DSC1 homologues should be classified as Ca(V)4 rather than Na(V)2. Indeed, channels that contain the D-E-E-A selectivity sequence are likely to feature the same properties as the honeybee’s channel, namely slow activation and inactivation kinetics and strong selectivity for Ca(2+) ions.