Abstract
1. The effect of pH on excitation-contraction coupling in skeletal muscle of the toad was examined using a skinned fibre preparation which gives ready access to the intracellular environment while still allowing stimulation of Ca2+ release by the normal voltage-sensor mechanism. 2. In each fibre, depolarization-induced responses (produced by changing the ions in the bathing solution) were examined first at pH 7.1, and then at another pH between 6.1 and 8.0. At all pH levels examined, the first depolarization elicited a large response which was slightly greater (pH 7.6 and 8.0) or smaller (pH 6.6 and 6.1) than that at pH 7.1. The size of the first depolarization-induced response varied with pH in almost exactly the same manner as did the maximum Ca(2+)-activated response. The duration of the depolarization-induced response at all other pH levels was longer than at pH 7.1. 3. Repeated depolarizations (30 s or more apart) produced similar responses at pH 7.1, but at all other pH levels examined the second and subsequent responses became progressively smaller. The reasons for this decrease were different at low and high pH. 4. Examination of the size of the depolarization-induced response after reloading the depleted sarcoplasmic reticulum (SR) with Ca2+ in solutions at various pH levels, indicated that the SR Ca2+ pump operated more poorly at pH 6.6 and 6.1 than at pH 7.1. This can account for the wide and successively smaller depolarization-induced responses at acidic pH. 5. Examination of the size of the depolarization-induced response after 5 min bathing without stimulation in a very low [Ca2+] solution (-log10[Ca2+] (pCa) greater than 8.0, 1 mM-BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), indicated that the sarcoplasmic reticulum (SR) lost far more Ca2+ at pH 7.6 and 8.0 than at pH 7.1 or 6.1. This indicates that SR is 'leakier' at alkaline pH. 6. Exposure of fibres heavily loaded with Ca2+ to solutions at pH 8.0 invariably induced a large response within 1-6 s. This response was unaffected by inactivation of the voltage sensors and was blocked completely by 2 microM-Ruthenium Red. This response and the 'leak' at alkaline pH are consistent with previous studies of the pH dependence of Ca(2+)-activated opening of single Ca2+ release channels in lipid bilayers. 7. These findings indicate that depolarization can induce massive Ca2+ release at both acidic and alkaline pH, provided that the SR is normally loaded with Ca2+, and give further insight into the intracellular events underlying fatigue in skeletal muscle.