JGP_201711834 1139..1148

Propagation of action potentials along the muscle cell membrane induces Ca release from the SR and subsequent contraction of the cell. In the sequence of events that occur from excitation to contraction, the Cav1.1 protein anchored in the t-tubule membrane plays the dual role of voltage sensor of SR Ca release and of L-type voltage-gated Ca channel (Ríos and Pizarro, 1991; Schneider, 1994; Melzer et al., 1995). Given these pivotal roles, the absence of Cav1.1 is lethal, and any mutation occurring in the gene encoding Cav1.1 and producing changes in the functional properties of the protein leads to severe neuromuscular diseases. Among them, type 1 hypokalemic periodic paralysis (HypoPP1) is one of the least understood genetic disorders, characterized by transient failure of muscle excitability occurring in association with hypokalemia and triggered by rest after strenuous exercise or carbohydrate load (Lehmann-Horn et al., 2004; Cannon, 2015). Cav1.1 is composed of 4 homologous domains (I to IV), each of which contains 6 membrane-spanning segments (S1 to S6; Catterall, 2011). S1 to S4 segments of each domain constitute the voltage-sensing domain, within which S4 segments enriched with positively charged amino acids are able to translocate through a “gating pore” pathway formed by the S1, S2, and S3 segments upon depolarization (Gandhi and Isacoff, 2002). This concerted translocation of the S4 segments controls the gating of the L-type Ca channel and Ca release from the SR. The vast majority of HypoPP1 mutations identified so far were shown to consist in the replacement of the outermost arginine residues by less basic residues in S4 segments of domains II, III, or IV (Cannon, 2010; Matthews and Hanna, 2010; Jurkat-Rott et al., 2012; Moreau et al., 2014). Site-directed mutagenesis experiments in the closely structurally related voltage-dependent K or Na channels have revealed that such substitutions generated an inward gating pore current at hyperpolarized potentials, also called the ω current, through the voltage-sensing domain (Starace and Bezanilla, 2004; Sokolov et al., 2005; Tombola et al., 2005). In addition, experiments performed in fibers from muscle biopsies 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 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 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. Na leak with gating pore properties in hypokalemic periodic paralysis V876E mutant muscle Ca channel