Membrane electrical activity elicits inositol 1,4,5-trisphosphate-dependent slow Ca2+ signals through a Gbetagamma/phosphatidylinositol 3-kinase gamma pathway in skeletal myotubes.
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 Gbetagamma 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 Cgamma1 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 Gbetagamma/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.
Eltit
José M
JM
Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultades de Medicina y Ciencias Químicas y Farmacéuticas, Universidad de Chile, Independecia 1027, Santiago 7, Chile.
García
Alejandra A
AA
Hidalgo
Jorge
J
Liberona
José L
JL
Chiong
Mario
M
Lavandero
Sergio
S
Maldonado
Edio
E
Jaimovich
Enrique
E
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D058544:Class Ib Phosphatidylinositol 3-Kinase; D003593:Cytoplasm / Q000378:metabolism; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
D004791:Enzyme Inhibitors; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007527:Isoenzymes; D010446:Peptide Fragments; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D058544:Class Ib Phosphatidylinositol 3-Kinase; D010738:Type C Phospholipases; D002118:Calcium