Molecular Mechanism of Store Operated Ca2+ Entry in Adult Mammalian Skeletal Muscle

Abstract

In skeletal muscle, store-operated Ca2+ entry (SOCE) is a trans-sarcolemmal Ca2+ influx pathway activated when sarcoplasmic reticulum (SR) Ca2+ stores are depleted. However, the mechanism of activation and physiological role of SOCE in adult skeletal muscle remains largely unknown. We recently reported that both STIM1 and Orai1 proteins are required for SOCE in skeletal myotubes. Here we investigated the mechanism of SOCE in adult skeletal muscles using mouse FDB fibers following electroporation of either cherry-tagged dnOrai1 cDNA or STIM1 siRNAs. Using a Mn2+ quench assay, we found that thapsigargin-induced SR Ca2+ store depletion activates a Ca2+ influx pathway in adult FDB fibers that is inhibited by: 1. multiple SOCE channel blockers (La3+, BTP-2 or SK&F96365) (3321±598 counts/sec, n=16 and 477±274 counts/sec, n=4 in the absence and presence of La3+, respectively), 2. expression of cherry-tagged dnOrai1 (571±130 counts/sec, n=12), or 3. STIM1 knockdown (7565±1590 counts/sec, n=3 and 217±247 counts/sec, n=5 in control and after STIM1 knockdown, respectively). To further assess the role of SOCE in muscle, we generated skeletal muscle-specific HA-tagged dominant negative Orai1 transgenic mice (HSAdnOrai) using a transgene driven by the human skeletal muscle actin (HSA) promoter (provided by Dr. J. Molkentin). HSAdnOrai mice survive beyond weaning and develop/breed normally. Western blot analysis using an HA antibody confirmed dnOrai1 transgene expression in skeletal muscle, but not in heart, lung, brain, spleen kidney, or liver. Primary myotubes derived from HSAdnOrai mice show significantly decreased SOCE following store depletion as assessed in Mn2+ quench (>92%) and Ca2+ influx (>95%) assays These results demonstrate that STIM1-Orai1 coupling mediates SOCE in adult skeletal muscle and that HSAdnOrai transgenic mice are a valuable tool for future studies of the physiological role of SOCE in skeletal muscle.