IP3 receptor function and localization in myotubes: an unexplored Ca2+ signaling pathway in skeletal muscle

We present evidence for an unexplored inositol 1,4,5-trisphosphate-mediated Ca2+ signaling pathway in skeletal muscle. RT-PCR methods confirm expressi on of all three known isotypes of the inositol trisphosphate receptor in cu ltured rodent muscle. Confocal microscopy of cultured mouse muscle, doubly labeled for inositol receptor type 1 and proteins of known distribution, re veals that the receptors are localized to the I band of the sarcoplasmic re ticulum, and this staining is continuous with staining of the nuclear envel ope region. These results suggest that the receptors are positioned to medi ate a slowly propagating Ca2+ wave that follows the fast Ca2+ transient upo n K+ depolarization. This slow wave, imaged using fluo-3, resulted in an in crease in nucleoplasmic Ca2+ lasting tens of seconds, but not contraction; the slow wave was blocked by both the inositol trisphosphate receptor inhib itor 2-aminoethoxydiphenyl borate and the phospholipase C inhibitor U-73122 . To test the hypothesis that these slow Ca2+ signals are involved in signa l cascades leading to regulation of gene expression, we assayed for early e ffects of K+ depolarization on mitogen-activated protein kinases, specifica lly extracellular-signal related kinases 1 and 2 and the transcription fact or cAMP response element-binding protein (CREB). Within 30-60 seconds follo wing depolarization, phosphorylation of both the kinases and CREB was evide nt and could be inhibited by 2-aminoethoxydiphenyl borate. These results su ggest a signaling system mediated by Ca2+ and inositol trisphosphate that c ould regulate gene expression in muscle cells.