Abstract
Skeletal muscle excitation-contraction (EC) coupling roots in Ca2+-influx-independent inter-channel signaling between the sarcolemmal dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) in the sarcoplasmic reticulum. Although DHPR Ca2+ influx is irrelevant for EC coupling, its putative role in other muscle-physiological and developmental pathways was recently examined using two distinct genetically engineered mouse models carrying Ca2+ non-conducting DHPRs: DHPR(N617D) (Dayal et al., 2017) and DHPR(E1014K) (Lee et al., 2015). Surprisingly, despite complete block of DHPR Ca2+-conductance, histological, biochemical, and physiological results obtained from these two models were contradictory. Here, we characterize the permeability and selectivity properties and henceforth the mechanism of Ca2+ non-conductance of DHPR(N617). Our results reveal that only mutant DHPR(N617D) with atypical high-affinity Ca2+ pore-binding is tight for physiologically relevant monovalent cations like Na+ and K+. Consequently, we propose a molecular model of cooperativity between two ion selectivity rings formed by negatively charged residues in the DHPR pore region.
Highlights
Excitation-contraction (EC) coupling in skeletal muscle does not require Ca2+ influx through the sarcolemmal L-type voltage-gated Ca2+ channel CaV1.1 or dihydropyridine receptor (DHPR), as was convincingly demonstrated in influential studies nearly half a century ago (Armstrong et al, 1972; Schneider and Chandler, 1973)
We demonstrate that the mutant DHPR(N617D) remains Ca2+ impermeant even under conditions known to augment L-type Ca2+ currents
Based on our recent findings, we propose a molecular model of cooperativity between the divalent cation selectivity (DCS) locus in the outer DHPR pore region (Cens et al, 2007) and the EEEE locus in the central pore (Sather and McCleskey, 2003)
Summary
Excitation-contraction (EC) coupling in skeletal muscle does not require Ca2+ influx through the sarcolemmal L-type voltage-gated Ca2+ channel CaV1.1 or dihydropyridine receptor (DHPR), as was convincingly demonstrated in influential studies nearly half a century ago (Armstrong et al, 1972; Schneider and Chandler, 1973). Our results explicitly show that the DHPR pore mutation N617D leads to an increase in Ca2+ pore binding affinity from ~ 1 μM (characteristic for wt DHPR) to nM range This more than four-fold enhanced Ca2+ binding affinity is sufficient to completely block Ca2+ conductance through the mutant DHPR(N617D) and does not allow permeation of monovalent cations like Cs+, Li+, and Na+ under physiological Ca2+ concentrations. Based on our recent findings, we propose a molecular model of cooperativity between the divalent cation selectivity (DCS) locus in the outer DHPR pore region (Cens et al, 2007) and the EEEE locus in the central pore (Sather and McCleskey, 2003) With this model, we can convincingly explain the divergent impacts of both DHPR pore mutations, N617D and E1014K, on Ca2+ selectivity and Ca2+ conductance and provide an explanation for the incongruences in muscle performance and functioning between the two distinct pore-mutant mouse models. Conductance helps us in understanding the Ca2+ non-conductance mechanism in previously identified (Schredelseker et al, 2010) additional DHPR pore mutations, E Q and D K (in the EEEE locus and DCS locus, respectively) that emerged during evolution of other Ca2+ non-conducting DHPR isoforms in skeletal muscle of bony fish
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