APPS November 2002 Meeting Abstract 2408

There are many uncertainties about the precise effects of acidic cytoplasmic conditions on the various steps in the excitation-contraction (E-C) coupling sequence in skeletal muscle fibres. In this study we used the mechanically-skinned fibre technique¹ to examine the effects of low pH on the contractile apparatus, Ca²⁺ movements into and out of the sarcoplasmic reticulum (SR), and voltage-sensor controlled Ca²⁺ release in rat EDL fibres. Individual fibres were skinned under paraffin oil, mounted on a force transducer and bathed in a solution mimicking the normal intracellular environment (high [K⁺], 8 mM total ATP, 1 mM free Mg²⁺, pH 7.10). The properties of the contractile apparatus were examined by measuring the force response by direct activation in heavily-buffered Ca²⁺ solutions¹. Ca²⁺ uptake was assessed by measuring the area of the force response obtained when fully emptying the SR with caffeine/low [Mg²⁺] after loading the SR for a set time at the different pHs and at different pCa (6.7, 6.4 and 6.2). The sensitivity of the calcium release channels to Ca²⁺-induced Ca²⁺ release was examined by loading the SR to a set level (at pH 7.1) and then applying a submaximal caffeine stimulus (8 mM) at different pHs. Voltage-sensor induced responses were elicited by both ion substitution (Na⁺ for K⁺) and electric field stimulation². Lowering the pH from 7.1 to 6.2 reduced maximum Ca²⁺-activated force by ~50% and the Ca²⁺-sensitivity of the contractile apparatus by ~0.7 pCa units, and reduced the Ca²⁺ uptake rate >10 fold at pC 6.7, with smaller relative effect at pCa 6.2. Caffeine-induced Ca²⁺ release was evidently still substantial at pH 6.2. Finally, taking into account the direct effects of pH on the contractile apparatus, the size of the depolarization-induced responses to ion substitution and the twitch and tetanic responses, all showed that the voltage-sensors were able to elicit very substantial Ca²⁺ release even at pH 6.2.