Phosphate transport into the sarcoplasmic reticulum of skinned fibres from rat skeletal muscle

The rate, magnitude and pharmacology of inorganic phosphate (P-i) transport into the sarcoplasmic reticulum were estimated in single, mechanically skinned skeletal muscle fibres of the rat. This was done, indirectly, by using a technique that measured the total Ca2+ content of the sarcoplasmic reticulum and by taking advantage of the 1:1 stoichiometry of Ca2+ and P-i transport into the sarcoplasmic reticulum lumen during Ca-P-i precipitation-induced Ca2+ loading. The apparent rate of P, entry into the sarcoplasmic reticulum increased with increasing myoplasmic [P,1 in the 10 mM-50 mM range at a fixed, resting myoplasmic pCa of 7.15, as judged by the increase in the rate of Ca-P-i precipitation-induced sarcoplasmic reticulum Ca2+ uptake. At 20 mM myoplasmic [P-i] the rate of P-i entry was calculated to be at least 51 mu Ms(-1) while the amount of P-i loaded appeared to saturate at around 3.5 mM (per fibre volume). These values are approximations due to the complex kinetics of formation of different species of Ca-P-i precipitate formed under physiological conditions. Phenylphosphonic acid (PhPA, 2.5 mM) inhibited P-i transport by 37% at myoplasmic pCa 6.5 and also had a small, direct inhibitory effect on the sarcoplasmic reticulum Ca2+ pump (16%). In contrast, phosphonoformic acid (PFA, 1 mM) appeared to enhance both the degree of P-i entry and the activity of the sarcoplasmic reticulum Ca2+ pump, results that were attributed to transport of PFA into the sarcoplasmic reticulum lumen and its subsequent complexation with Ca2+ Thus, results from these studies indicate the presence of a P-i transporter in the sarcoplasmic reticulum membrane of mammalian skeletal muscle fibres that is (1) active at physiological concentrations of myoplasmic P-i and Ca2+ and (2) partially inhibited by PhPA. This P-i transporter represents a link between changes in myoplasmic [P-i] and subsequent changes in sarcoplasmic reticulum luminal [P-i]. It might therefore play a role in the delayed metabolic impairment of sarcoplasmic reticulum Ca2+ release seen during muscle fatigue, which should occur abruptly once the Ca-P-i solubility product is exceeded in the sarcoplasmic reticulum lumen.