Type 1 hypokalemic periodic paralysis (HypoPP1) is a poorly understood genetic neuromuscular disease characterized by episodic attacks of paralysis associated with low blood K + . The vast majority of HypoPP1 mutations involve the replacement of an arginine by a neutral residue in one of the S4 segments of the α1 subunit of the skeletal muscle voltage-gated Ca 2+ channel, which is thought to generate a pathogenic gating pore current. The V876E HypoPP1 mutation has the peculiarity of being located in the S3 segment of domain III, rather than an S4 segment, raising the question of whether such a mutation induces a gating pore current. Here we successfully transfer cDNAs encoding GFP-tagged human wild-type (WT) and V876E HypoPP1 mutant α1 subunits into mouse muscles by electroporation. The expression profile of these WT and V876E channels shows a regular striated pattern, indicative of their localization in the t-tubule membrane. In addition, L-type Ca 2+ current properties are the same in V876E and WT fibers. However, in the presence of an external solution containing low-Cl − and lacking Na + and K + , V876E fibers display an elevated leak current at negative voltages that is increased by external acidification to a higher extent in V876E fibers, suggesting that the leak current is carried by H + ions. However, in the presence of Tyrode’s solution, the rate of change in intracellular pH produced by external acidification was not significantly different in V876E and WT fibers. Simultaneous measurement of intracellular Na + and current in response to Na + readmission in the external solution reveals a rate of Na + influx associated with an inward current, which are both significantly larger in V876E fibers. These data suggest that the V876E mutation generates a gating pore current that carries strong resting Na + inward currents in physiological conditions that are likely responsible for the severe HypoPP1 symptoms associated with this mutation.

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.

Voltage-gated Ca 2+ channels (VGCC) play a key role in many physiological functions by their high selectivity for Ca 2+ over other divalent and monovalent cations in physiological situations. Divalent/monovalent selection is shared by all VGCC and is satisfactorily explained by the existence, within the pore, of a set of four conserved glutamate/aspartate residues (EEEE locus) coordinating Ca 2+ ions. This locus however does not explain either the choice of Ca 2+ among other divalent cations or the specific conductances encountered in the different VGCC. Our systematic analysis of high- and low-threshold VGCC currents in the presence of Ca 2+ and Ba 2+ reveals highly specific selectivity profiles. Sequence analysis, molecular modeling, and mutational studies identify a set of nonconserved charged residues responsible for these profiles. In HVA (high voltage activated) channels, mutations of this set modify divalent cation selectivity and channel conductance without change in divalent/monovalent selection, activation, inactivation, and kinetics properties. The Ca V 2.1 selectivity profile is transferred to Ca V 2.3 when exchanging their residues at this location. Numerical simulations suggest modification in an external Ca 2+ binding site in the channel pore directly involved in the choice of Ca 2+ , among other divalent physiological cations, as the main permeant cation for VGCC. In LVA (low voltage activated) channels, this locus (called DCS for divalent cation selectivity) also influences divalent cation selection, but our results suggest the existence of additional determinants to fully recapitulate all the differences encountered among LVA channels. These data therefore attribute to the DCS a unique role in the specific shaping of the Ca 2+ influx between the different HVA channels.