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

The rate, magnitude and pharmacology of inorganic phosphate (Pi) 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 Pi entry into the sarcoplasmic reticulum increased with increasing myoplasmic [P(i)] 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 μMs-1while 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, 1mM) 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.