Inhibition of chloride self-exchange with stilbene disulphonates in depolarized skeletal muscle of Rana temporaria.

1. The inhibition of 36Cl efflux with stilbene disulphonates, SD, has been studied under conditions of chloride equilibrium in depolarized fibre bundles from frog semitendinosi. The chosen probes were the aminoreactive derivative SITS and the derivative DNDS with no aminoreactive group. SD were added to the medium during 36Cl efflux allowing the estimation of fractional inhibition after a single 36Cl loading. 2. Both probes inhibited chloride self-exchange reversibly within the pH range 5.5-9.5 under study. 3. At SD concentrations above the half-inhibition concentration the inhibition reached a steady level with a time lag equal to that required for extracellular fluid change. The time constant for reversibility upon the removal of SD increased with decreasing pH, but rapid reversibility always appeared upon an increase of pH to 7.2. These findings suggest that SD may enter the membrane at low pH, but that the inhibitory action is confined to superficial membrane sites. 4. The inhibitory power of both probes showed a pronounced pH dependence, pK approximately 7. The half-inhibition concentration increased about 6-7 times when pH was lowered one unit from the pK value. 5. The apparent affinity of SITS to the transport system was about 5 times higher than that of DNDS. The apparent dissociation constants at neutral pH were 8.5 x 10(-5) M (SITS) and 4.5 x 10(-4) M (DNDS). Both probes showed a maximal inhibition close to 100% at neutral pH and approximately 85% at pH 5.5. 6. The inhibition depended on the chloride concentration in a way consistent with competitive inhibition in both neutral and acid media. 7. The results are consistent with the classical model of anion transport in frog muscle, suggesting that SD and chloride may compete for binding to a site with increasing anion affinity upon protonation; the results do not, however, exclude that the conductive and the non-conductive chloride transport modes in frog muscle are mediated by separate SD-sensitive transport pathways.