Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of cortical, brain stem and spinal motor neurons that leads to muscle weakness and death. A previous study implicated CACN A1H encoding for Ca v 3.2 calcium channels as a susceptibility gene in ALS. In the present study, two heterozygous CACNA1H variants were identified by whole genome sequencing in a small cohort of ALS patients. These variants were functionally characterized using patch clamp electrophysiology, biochemistry assays, and molecular modeling. A previously unreported c.454GTAC > G variant produced an inframe deletion of a highly conserved isoleucine residue in Ca v 3.2 (p.ΔI153) and caused a complete loss-of-function of the channel, with an additional dominantnegative effect on the wild-type channel when expressed in trans. In contrast, the c.3629C > T variant caused a missense substitution of a proline with a leucine (p.P1210L) and produced a comparatively mild alteration of Ca v 3.2 channel activity. The newly identified ΔI153 variant is the first to be reported to cause a complete loss of Ca v 3.2 channel function. These findings add to the notion that loss-of-function of Ca v 3.2 channels associated with rare CACNA1H variants may be risk factors in the complex etiology of ALS.

The zebrafish has emerged as a very relevant animal model for probing the pathophysiology of human skeletal muscle disorders. This vertebrate animal model displays a startle response characterized by high-frequency swimming activity powered by contraction of fast skeletal muscle fibers excited at extremely high frequencies, critical for escaping predators and capturing prey. Such intense muscle performance requires extremely fast properties of the contractile machinery but also of excitation–contraction coupling, the process by which an action potential spreading along the sarcolemma induces a change in configuration of the dihydropyridine receptors, resulting in intramembrane charge movements, which in turn triggers the release of Ca2+ from the sarcoplasmic reticulum. However, thus far, the fastest Ca2+ transients evoked by vertebrate muscle fibers has been described in muscles used to produce sounds, such as those in the toadfish swim bladder, but not in muscles used for locomotion. By performing intracellular Ca2+ measurements under voltage control in isolated fast skeletal muscle fibers from adult zebrafish and mouse, we demonstrate that fish fast muscle fibers display superfast kinetics of action potentials, intramembrane charge movements, and action potential–evoked Ca2+ transient, allowing fusion and fused sustained Ca2+ transients at frequencies of excitation much higher than in mouse fast skeletal muscle fibers and comparable to those recorded in muscles producing sounds. The present study is the first demonstration of superfast kinetics of excitation–contraction coupling in skeletal muscle allowing superfast locomotor behaviors in a vertebrate.