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

Abstract

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 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 myofibres with 2 mm Cl^- channel blocker 9-anthracenecarboxylic acid (9AC) inhibited both effects of P_i . The biphasic dependence of Ca²⁺ release on [P_i ] is explained by a direct effect of P_i acting on the SR Ca²⁺ release channel, combined with the intra-SR precipitation of Ca²⁺ salts. The effects of 9AC demonstrate that P_i 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_i ). P_i diffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca²⁺ salts, thus contributing to the impairment of Ca²⁺ release. Information on the P_i entrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca²⁺ spark frequency is correlated with the Ca²⁺ load of the SR and can be used to monitor this variable. We studied the effects of P_i on Ca²⁺ 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 µm) 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 mm 9AC in the presence of high cytoplasmic Mg²⁺ 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 Ca²⁺ release by accumulating and precipitating with Ca²⁺ inside the SR, thus contributing to myogenic fatigue.

Keywords: 9-anthracenecarboxylic acid; chloride channel; fatigue; inorganic phosphate; sarcoplasmic reticulum; skeletal muscle.