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-i) 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(i)into SR are fully established. We showed that P(i)promoted Ca(2+)release at concentrations below 10 mmand decreased it at higher concentrations. This decrease correlated well with that of [Ca2+](SR). Pre-treatment of permeabilized myofibres with 2 mmCl(-)channel blocker 9-anthracenecarboxylic acid (9AC) inhibited both effects of P-i. The biphasic dependence of Ca(2+)release on [P-i] is explained by a direct effect of P(i)acting on the SR Ca(2+)release channel, combined with the intra-SR precipitation of Ca(2+)salts. The effects of 9AC demonstrate that P(i)enters the SR via a Cl(-)pathway of an as-yet-undefined molecular nature. Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (P-i). P(i)diffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca(2+)salts, thus contributing to the impairment of Ca(2+)release. Information on the P(i)entrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca(2+)spark frequency is correlated with the Ca(2+)load of the SR and can be used to monitor this variable. We studied the effects of P(i)on Ca(2+)sparks in permeabilized fibres of the frog. Relative event frequency (f/f(ref)) rose with increasing [P-i], reaching 2.54 +/- 1.6 at 5 mm,and then decreased monotonically, reaching 0.09 +/- 0.03 at [P-i] = 80 mm. Measurement of [Ca2+](SR)confirmed a decrease correlated with spark frequency at high [P-i]. A large [Ca2+](SR)surge was observed upon P(i)removal. Anion channels are a putative path for P(i)into the SR. We tested the effect of the chloride channel blocker 9-anthracenecarboxylic acid (9AC) on P(i)entrance. 9AC (400 mu m)applied to the cytoplasm produced a non-significant increase in spark frequency and reduced the P(i)effects on this parameter. Fibre treatment with 2 mm9AC in the presence of high cytoplasmic Mg(2+)suppressed the effects of P(i)on [Ca2+](SR)and spark frequency up to 55 mm[P-i]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that P(i)impairs Ca(2+)release by accumulating and precipitating with Ca(2+)inside the SR, thus contributing to myogenic fatigue.