A chloride channel blocker prevents the suppression by inorganic phosphate of the cytosolic calcium signals that control muscle contraction.

Ferreira, Juan J; Pequera, Germán; Launikonis, Bradley S; Ríos, Eduardo; Brum, Gustavo

KEY POINTS Accumulation of inorganic phosphate, Pi , may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum, SR. Direct demonstration of this process nor definition of the entry pathway of Pi into SR are fully established. We show that Pi promoted Ca2+ release at concentrations below 10 mm and decreased it at higher concentrations. This decrease correlated well with that of [Ca2+ ]SR . Pre-treatment of permeabilized myofibers with 2 mm Cl- channel blocker 9-anthracenecarboxylic acid (9AC) inhibited both effects of Pi . The biphasic dependence of Ca2+ release on [Pi ] is explained by a direct effect of Pi acting on the SR Ca2+ release channel, combined with the intra-SR precipitation of Ca2+ salts. 9AC effects demonstrates that Pi enters the SR via a Cl- pathway of as yet undefined molecular nature. ABSTRACT Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (Pi ). Pi diffuses into the sarcoplasmic reticulum (SR) where it is believed to forms insoluble Ca2+ salts, thus contributing to the impairment of Ca2+ release. Information on the Pi entrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca2+ spark frequency is correlated with Ca2+ load of the SR and can be used to monitor this variable. We studied the effects of Pi on Ca2+ sparks in permeabilized fibres of the frog. Relative event frequency (f/fref ) rose with increasing [Pi ] reaching 2.54±1.6 at 5 mm and then decreased monotonically, reaching 0.09±0.03 at [Pi ] = 80 mm. Measurement of [Ca2+ ]SR confirmed a decrease correlated with spark frequency at high [Pi ]. A large [Ca2+ ]SR surge was observed upon Pi removal. Anion channels are a putative path for Pi into the SR. We tested the effect of chloride channel blocker 9-anthracenecarboxylic acid (9AC) on Pi entrance. 400 μm 9AC applied to the cytoplasm produced a non-significant increase in spark frequency and reduced the Pi effects on this parameter. Fibre treatment with 2 mm 9AC in the presence of high cytoplasmic Mg2+ suppressed Pi effects on [Ca2+ ]SR and spark frequency up to 55 mm Pi . These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that it impairs Ca2+ release by accumulating and precipitating with Ca2+ inside the SR, thus contributing to myogenic fatigue. This article is protected by copyright. All rights reserved.