Impaired Orai1-mediated resting Ca²⁺ entry reduces the cytosolic [Ca²⁺] and sarcoplasmic reticulum Ca²⁺ loading in quiescent junctophilin 1 knock-out myotubes

In the absence of store depletion, plasmalemmal Ca²⁺ permeability in resting muscle is very low, and its contribution in the maintenance of Ca²⁺ homeostasis at rest has not been studied in detail. Junctophilin 1 knock-out myotubes (JP1 KO) have a severe reduction in store-operated Ca²⁺ entry, presumably caused by physical alteration of the sarcoplasmic reticulum (SR) and T-tubule junction, leading to disruption of the SR signal sent by Stim1 to activate Orai1. Using JP1 KO myotubes as a model, we assessed the contribution of the Orai1-mediated Ca²⁺ entry pathway on overall Ca²⁺ homeostasis at rest with no store depletion. JP1 KO myotubes have decreased Ca²⁺ entry, [Ca²⁺] _rest, and intracellular Ca²⁺ content compared with WT myotubes and unlike WT myotubes, are refractory to BTP2, a Ca²⁺ entry blocker. JP1 KO myotubes show down-regulation of Orai1 and Stim1 proteins, suggesting that this pathway may be important in the control of resting Ca ²⁺ homeostasis. WT myotubes stably transduced with Orai1(E190Q) had similar alterations in their resting Ca²⁺ homeostasis as JP1 KO myotubes and were also unresponsive to BTP2. JP1 KO cells show decreased expression of TRPC1 and -3 but overexpress TRPC4 and -6; on the other hand, the TRPC expression profile in Orai1(E190Q) myotubes was comparable with WT. These data suggest that an important fraction of resting plasmalemmal Ca²⁺ permeability is mediated by the Orai1 pathway, which contributes to the control of [Ca²⁺]_rest and resting Ca²⁺ stores and that this pathway is defective in JP1 KO myotubes.