Abstract
Previous results from ryanodine binding and single ryanodine receptor (RyR) channel measurements suggested that RyR1 activity may be modulated by phosphatidylinositol-phosphate lipids (PtdInsPs). Possibly related, recent data showed that skeletal muscle e-c coupling is altered in several models of PtdInsPs phosphatase deficiency. We measured intracellular Ca2+ in fibers from flexor digitorum brevis (fdb)muscles microinjected with a solution containing a given PtdInsP lipid (PtdIns(3,5)P2, PtdIns(3)P, PtdIns(5)P or PtdIns) together with the calcium dye indo-1. Following equilibration, fibers were stimulated by short voltage-clamp depolarizations of increasing amplitude from −80mV. Resting [Ca2+] did not differ between control fibers and any group of PtdInsPs-injected fibers; it was though ∼1.5 larger in fibers injected with PtdIns(3,5)P2 or PtdIns(3)P than in fibers injected with PtdIns(5)P or PtdIns. Peak SR Ca2+ release was specifically depressed in fibers injected with PtdIns(3,5)P2 or PtdIns(3)P with maximum values of 34.8 ± 3 µM.ms−1 in control fibers (n=15) and of 24.4 ± 2 and 17.4 ± 2 µM.ms−1 in fibers injected with PtdIns(3,5)P2 (n=8) and PtdIns(3)P (n=8), respectively. There was no concurrent effect on the membrane current measured during the pulses and the voltage dependence of Ca2+ release inactivation was also unaffected. These results highlight the possibility that SR Ca2+ release may be physiologically regulated by certain PtdInsPs. A plasmid coding for a wild-type form of the PtdIns phosphatase MTM1 tagged with the red fluorescent protein mCherry was transfected in fdb muscles. The protein was found distributed within the entire volume of the transfected fibers and to reproducibly yield a striated transversal expression pattern consistent with localization within the triadic area. Preliminary measurements of voltage-activated Ca2+ transients in these cells indicated that under these conditions over-expression of MTM1 had limited effects on SR Ca2+ release.
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