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

Key Points: Accumulation of inorganic phosphate (P ) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of P into SR are fully established.  We showed that P promoted Ca release at concentrations below 10 mm and decreased it at higher concentrations. This decrease correlated well with that of [Ca ] .  Pre-treatment of permeabilized myofibres with 2 mm Cl channel blocker 9-anthracenecarboxylic acid (9AC) inhibited both effects of P .  The biphasic dependence of Ca release on [P ] is explained by a direct effect of P acting on the SR Ca release channel, combined with the intra-SR precipitation of Ca salts. The effects of 9AC demonstrate that P enters the SR via a Cl pathway of an as-yet-undefined molecular nature.

Abstract: Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (P ). P diffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca salts, thus contributing to the impairment of Ca release. Information on the P entrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca spark frequency is correlated with the Ca load of the SR and can be used to monitor this variable. We studied the effects of P on Ca sparks in permeabilized fibres of the frog. Relative event frequency (f/f ) rose with increasing [P ], reaching 2.54 ± 1.6 at 5 mm, and then decreased monotonically, reaching 0.09 ± 0.03 at [P ] = 80 mm. Measurement of [Ca ] confirmed a decrease correlated with spark frequency at high [P ]. A large [Ca ] surge was observed upon P removal. Anion channels are a putative path for P into the SR. We tested the effect of the chloride channel blocker 9-anthracenecarboxylic acid (9AC) on P entrance. 9AC (400 µm) applied to the cytoplasm produced a non-significant increase in spark frequency and reduced the P effects on this parameter. Fibre treatment with 2 mm 9AC in the presence of high cytoplasmic Mg suppressed the effects of P on [Ca ] and spark frequency up to 55 mm [P ]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that P impairs Ca release by accumulating and precipitating with Ca inside the SR, thus contributing to myogenic fatigue.