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␤␥ scavenger peptide, and by phosphatidylinositol 3-kinase inhibitors. Immunofluorescence using anti-phosphatidylinositol 3-kinase ␥ 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␥1 activation as confirmed by phosphorylation assays for the enzyme. Co-transfection of a dominant negative form of phosphatidylinositol 3-kinase ␥ inhibited the phosphatidylinositol 3-kinase activity as well as the slow calcium signal. We conclude that G␤␥/phosphatidylinositol 3-kinase ␥ 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.The role of the voltage-dependent calcium channel (Cav 1.1) or dihydropyridine receptor (DHPR) 3 in excitation-contraction (EC) coupling in skeletal muscle is well known (1-4). Recently, another sequence of molecular processes activated by cell membrane depolarization involving the DHPR also acting as membrane voltage sensor has been reported. A slow calcium signal, first described in rat myotubes stimulated with K ϩ depolarization, occurs several seconds after the fast calcium signal that is due to ryanodine receptor opening and mediates EC coupling. The former is refractory to ryanodine treatment and depends on inositol 1,4,5-trisphosphate (IP 3 ) generation (5, 6). This slow calcium signal has a predominant nuclear component, and its propagation between adjacent nuclei in the myotube can be frequently recorded (5). Besides the fact that the three types of IP 3 receptors are located at different sites within rat myotubes, including the nuclear region, a functional calcium store in isolated nuclei has been recently described (7). Treatment of these isolated nuclei with IP 3 induces calcium release to the nucleoplasm, regulating phosphorylation of the transcription factor cAMP response element-binding protein (7). The slo...