Divalent cations permeation in a Ca non-conducting skeletal muscle dihydropyridine receptor mouse model.

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 Ca release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca channel that conducts L-type Ca 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 Ca influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα subunit abolishing Ca permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca, Mn entry and subsequent quenching did occur because Mn was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba and Ba currents were strongly blocked by external Ca. Ba permeation was smaller, current kinetics slower and Ca 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 Ca impeding in this way permeation. Because Ba binds with lower affinity to D, Ba currents occur, but with reduced amplitudes as compared to Ba 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.