Passive Activation of Store-Operated Ca2+ Entry in Myotubes Depends on the Rate of RyR1-Dependent Ca2+ Leak

Abstract

In spite of extensive studies of store operated calcium entry (SOCE), the detailed mechanism of SOCE activation in skeletal muscle remains largely unknown. We recently reported that STIM1-Orai1 coupling is required for SOCE activation in myotubes. However, other proteins that control sarcoplasmic reticulum (SR) Ca2+ content may also contribute to SOCE activation. We hypothesized that passive SOCE activation in skeletal muscle depends on the rate of SR Ca2+ leak through the type 1 ryanodine receptor (RyR1). To test this hypothesis, we conducted a series of whole-cell patch-clamp measurements of SOCE current (ISOC) in myotubes obtained from normal and RyR1-null (dyspedic) mice. Myotubes were bathed in external solution containing (in mM): 138 TEA-methanesulfonate, 10 CaCl2, 10 HEPES, 1 MgCl2, 0.1 nifedipine, pH 7.4. The internal patch pipette solution contained (in mM): 140 mM Cs-methanesulfonate, 10 HEPES, 20 Na-EGTA, 4 MgCl2, pH7.4. SOCE was activated by passive SR Ca2+ depletion following intracellular dialysis with 20 mM EGTA. ISOC in myotubes exhibited many hallmarks of SOCE including strong inward rectification and inhibition by La3+, Gd3+, BTP-2, and 2-APB. ISOC current density at −80 mV was significantly (p<0.01) larger in normal myotubes (1.05 ± 0.09 pA/pF, n = 33) compared to that from dyspedic myotubes (0.74 ± 0.07 pA/pF, n = 18). Moreover, the speed of ISOC activation was slower in dyspedic myotubes. Specifically, the time to 10%, 50%, and 90% maximal activation were 4.95 ± 0.65 s, 15.3 ± 1.4 s, and 53.9 ± 7.5 s (n = 19), respectively, in normal myotubes and 67.5 ± 11.1 s, 114 ± 13 s, and 159 ± 17 s (n = 18), respectively, in dyspedic myotubes. These results indicate that RyR1 Ca2+ leak promotes passive SOCE activation.