Calcium modulation of phosphoinositide kinases in transverse tubule vesicles from frog skeletal muscle

Highly purified transverse tubule membranes isolated from frog skeletal muscle phosphorylate phosphatidylinositol to phosphatidylinositol 4-phosphate and phosphatidylinositol (4,5)-bisphosphate. The two phosphorylation reactions have different calcium requirements. Phosphorylation of phosphatidylinositol to phosphatidylinositol 4-phosphate, which takes place in both isolated transverse tubules and sarcoplasmic reticulum membranes, is independent of calcium in a range of concentrations from 10-9 to 10-6m, and is progressively inhibited to 10% of the maximal values by increasing calcium to 10-4m or higher (K0.5 = 5 × 10-6M). In contrast, phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol (4,5)-bisphosphate, a reaction exclusively present in transverse tubule membranes, is maximal at calcium concentrations higher than 2 × 10-6m and decreases to 30% of maximal values at calcium concentrations of 2 × 10-7m or lower (K0.5 = 10-6M). Unlike frog membranes, transverse

Phosphorylation of phosphatidylinositol by transverse tubule vesicles and its possible role in excitation-contraction coupling

Phosphorylation of phosphatidylinositol to phosphatidylinositol 4-monophosphate and to phosphatidyl-inositol 4,5-bisphosphate was demonstrated in transverse-tubule membranes isolated from frog skeletal muscle using [γ-32P]ATP as substrate. At millimolar concentrations of Mg2+ both phosphorylation reactions were completed within 15 s at 25°C. Isolated sarcoplasmic reticulum vesicles phosphorylated phosphatidyl-inositol to phosphatidylinositol 4-phosphate with a lower specific activity than the transverse tubules, and lacked the ability to produce phosphatidylinositol 4,5-bisphosphate. These findings show, for the first time, that isolated transverse-tubule membranes carry out one of the steps required to sustain a role for inositol trisphosphate as the physiological messenger in excitation-contraction coupling in skeletal muscle. The finding that 0.5 mM tetracaine apparently inhibits the phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-bisphosphate also s