Membrane electrical activity elicits inositol 1,4,5-trisphosphate-dependent slow Ca2+ signals through a G beta gamma/phosphatidylinositol 3-kinase gamma pathway in skeletal myotubes

Abstract

Tetanic electrical stimulation of myotubes evokes a ryanodine receptor-related fast calcium signal, during the stimulation, followed by a phospholipase C/inositol 1,4,5-trisphosphate-dependent slow calcium signal few seconds after stimulus end. L-type calcium channels (Cav 1.1, dihydropyridine receptors) acting as voltage sensors activate an unknown signaling pathway involved in phospholipase C activation. We demonstrated that both G protein and phosphatidylinositol 3- kinase were activated by electrical stimulation, and both the inositol 1,4,5-trisphosphate rise and slow calcium signal induced by electrical stimulation were blocked by pertussis toxin, by a G beta gamma scavenger peptide, and by phosphatidylinositol 3- kinase inhibitors. Immunofluorescence using anti-phosphatidylinositol 3- kinase gamma antibodies showed a clear location in striations within the cytoplasm, consistent with a position near the I band region of the sarcomere. The time course of phosphatidylinositol 3- kinase activation, monitored in single living cells using a pleckstrin homology domain fused to green fluorescent protein, was compatible with sequential phospholipase C gamma 1 activation as confirmed by phosphorylation assays for the enzyme. Co-transfection of a dominant negative form of phosphatidylinositol 3- kinase gamma inhibited the phosphatidylinositol 3- kinase activity as well as the slow calcium signal. We conclude that G beta gamma/phosphatidylinositol 3- kinase gamma signaling pathway is involved in phospholipase C activation and the generation of the slow calcium signal induced by tetanic stimulation. We postulate that membrane potential fluctuations in skeletal muscle cells can activate a pertussis toxin-sensitive G protein, phosphatidylinositol 3- kinase, phospholipase C pathway toward modulation of long term, activity-dependent plastic changes.