Differential Role of Calsequestrin Isoforms on Calcium Entry in Skeletal Muscle FDB Fibres

Abstract

JP45 is a sarcoplasmic reticulum membrane protein interacting with Cav1.1 and calsequestrin (CASQ). The JP45/CASQ complex enhances Cav1.1 channel calcium activity to promote calcium entry to maintain an adequate level of releasable SR calcium during tetanic contraction. In this study, we investigated FDB fibres from JP45/CASQ1 double KO (DKO), JP45/CASQ2 double KO (DKO) and JP45/CASQ1/CASQ2 triple KO (TKO) mice to assess the role, if any, of JP45 and CASQ2 on calcium entry. Mag-Fluo-4 was used to monitor rapid Ca2+ transients evoked by a 0.5 ms pulse at supramaximal field stimulation. In the presence of 1.8 mM Ca2+ in the extracellular solution, the peak calcium signal in FDB fibers from WT and JP45/CASQ2 DKO was similar, in JP45/CASQ1 DKO and JP45/CASQ1/CASQ2 TKO FDB fibres, peak calcium was lower compared to WT. The half time of the decay of the Ca2+ transient in FDB fibres from JP45/CASQ1 DKO, JP45/CASQ2 DKO and JP45/CASQ1/CASQ2 TKO was significantly slower compared to WT. Such a difference disappeared when Ca2+ was substituted by 100 μMLa3+ in the extracellular medium. The effect of La3+ was more evident upon stimulation of FDB fibres with repetitive 0.5 ms pulses at 100 Hz. The calcium signal evoked by a train of pulses at 100 Hz for 300 ms was strongly inhibited by La3+ in fibers from JP45/CASQ1 DKO and JP45/CASQ1/CASQ2 TKO, but negligibly affected in WT and in JP45/CASQ2 DKO. Mn2+ quenching experiments in fura-2 loaded FDB fibers from JP45/CASQ1 DKO and JP45/CASQ1/CASQ2 TKO show that calcium influx during tetanic stimulation is blocked by 50 µM nifedipine an inhibitor of Cav1.1 calcium currents. These results show that ablation of CASQ2 in a JP45 KO background does not affect calcium entry via Cav1.1.