Differential dependence of store-operated and excitation-coupled Ca2+ entry in skeletal muscle on STIM1 and Orai1

In non-excitable cells, agonist-induced depletion of intracellular Ca2+ stores triggers Ca2+ influx via a process termed store-operated Ca2+ entry (SOCE). In T-lymphocytes, stromal interaction molecule 1 (STIM1) acts as the intra-store Ca2+ sensor and Orai1 functions as the Ca2+-permeable SOCE channel activated by STIM1 following store depletion. Two functionally distinct Ca2+ entry pathways exist in skeletal muscle; one activated by store depletion (SOCE) and a second by sustained/repetitive depolarization that does not require store depletion (excitation-coupled Ca2+ entry, ECCE). However, the role of STIM1 and Orai1 in coordinating SOCE and ECCE activity in skeletal muscle and whether these two Ca2+ entry pathways represent distinct molecular entities or two different activation mechanisms of the same channel complex is unknown. Here we address these issues using siRNA-mediated STIM1 knockdown, dominant-negative Orai1, and permeation-defective Orai1 to determine the role of STIM1 and Orai1 in store-operated and excitation-coupled Ca2+ entry in skeletal myotubes. SOCE and ECCE activity were quantified from both intracellular Ca2+ measurements and Mn2+ quench assays. We found that STIM1 siRNA reduced STIM1 protein by more than 90% and abolished SOCE activity, while expression of siRNA-resistant hSTIM1 fully restored SOCE. SOCE was also abolished by dominant-negative Orai1 (E106Q) and markedly reduced by expression of a permeation-defective Orai1 (E190Q). In contrast, ECCE was unaffected by STIM1 knockdown, E106Q expression or E190Q expression. These results are the first to demonstrate that SOCE in skeletal muscle requires both STIM1 and Orai1 and that SOCE and ECCE represent two distinct molecular entities.