Phosphoinositide substrates of myotubularin affect voltage-activated Ca2+ release in skeletal muscle

Skeletal muscle excitation–contraction (E–C) coupling is altered in several models of phosphatidylinositol phosphate (PtdIns P ) phosphatase deficiency and ryanodine receptor activity measured in vitro was reported to be affected by certain PtdIns P s, thus prompting investigation of the physiological role of PtdIns P s in E–C coupling. We measured intracellular Ca 2+ transients in voltage-clamped mouse muscle fibres microinjected with a solution containing a PtdIns P substrate (PtdIns(3,5) P 2 or PtdIns(3) P ) or product (PtdIns(5) P or PtdIns) of the myotubularin phosphatase MTM1. No significant change was observed in the presence of either PtdIns(5) P or PtdIns but peak SR Ca 2+ release was depressed by ~30% and 50% in fibres injected with PtdIns(3,5) P 2 and PtdIns(3) P , respectively, with no concurrent alteration in the membrane current signals associated with the DHPR function as well as in the voltage dependence of Ca 2+ release inactivation. In permeabilized muscle fibres, the frequency of spontaneous Ca 2+ release events was depressed in the presence of the three tested phosphorylated forms of PtdIns P with PtdIns(3,5) P 2 being the most effective, leading to an almost complete disappearance of Ca 2+ release events. Results support the possibility that pathological accumulation of MTM1 substrates may acutely depress ryanodine receptor-mediated Ca 2+ release. Overexpression of a mCherry-tagged form of MTM1 in muscle fibres revealed a striated pattern consistent with the triadic area. Ca 2+ release remained although unaffected by MTM1 overexpression and was also unaffected by the PtdIns-3-kinase inhibitor LY2940002, suggesting that the 3-phosphorylated PtdIns lipids active on voltage-activated Ca 2+ release are inherently maintained at a low level, inefficient on Ca 2+ release in normal conditions.