Divalent cations permeation in a Ca2+ non-conducting skeletal muscle dihydropyridine receptor mouse model

Abstract

In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca²⁺ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca²⁺ channel that conducts L-type Ca²⁺ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca²⁺ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα_1S subunit abolishing Ca²⁺ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn²⁺ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca²⁺ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca²⁺ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca²⁺, Mn²⁺ entry and subsequent quenching did occur because Mn²⁺ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba²⁺ and Ba²⁺ currents were strongly blocked by external Ca²⁺. Ba²⁺ permeation was smaller, current kinetics slower and Ca²⁺ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca²⁺ impeding in this way permeation. Because Ba²⁺ binds with lower affinity to D, Ba²⁺ currents occur, but with reduced amplitudes as compared to Ba²⁺ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment.

Keywords: Ca(V)1.1; Dihydropyridine receptor; Skeletal muscle fiber; Voltage clamp; Voltage-gated Ca(2+)channel.