Zinc inhibition of chloride efflux from skeletal muscle ofRana pipiens and its modification by external pH and chloride activity

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Abstract
Efflux of36Cl− from frog sartorius muscles equilibrated in depolarizing solutions was measured. Cl− efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases. In depolarized muscles fromRana pipiens, the pH-dependent Cl− efflux has an apparent pK a near 6.4. The reduction of Cl− efflux by external Zn2+ was determined at different external pHs and chloride activities. The effect of external chloride activity on the pH-dependent Cl− efflux was also examined. At pH 6.5 and a membrane potential of −22 mV, increasing external Cl− activity from 0.108 to 0.28m decreased inhibition of the pH-dependent Cl− efflux at all activities of Zn2+. The Zn2+ activity needed to reduce Cl− efflux by half increased from 0.39×10−3 to 2.09×10−3 m. By contrast, external Cl− activity had no measurable effect on the apparent pK a of the pH-dependent efflux. At constant Cl− activity less than 0.21m, increasing external pH from 6.5 to 7.5 decreased inhibition by low Zn2+ activities with either a slight increase or no change in the Zn2+ activity producing half-inhibition. In other words, for relatively low Cl− activities, protection against inhibition of Cl− efflux by low Zn2+ activities was obtained by raising, not lowering, external pH; this is not what is expected if H+ and Zn2+ ions compete at the same site to produce inhibition of Cl− efflux. We conclude that Zn2+ and low pH inhibit Cl− efflux by separate and distinct mechanisms. By contrast, the protection against Zn2+ inhibition produced by high external Cl− activity (0.28m) was partially reversed by raising external pH from 6.5 to 7.5 at all Zn2+ activities. The half-inhibition Zn2+ activity decreased from 2.09×10−3 to 0.68×10−3 m. The results can be simulated quantitatively by a model in which single Cl− channel elements are in equilibrium with sextets of associated single-channel elements, each sextet having a conductance six times that of a single-channel element. The association into sextets is promoted by OH− or Cl− binding to a control site on the single-channel elements. Both the single Cl− channel element and the sextet of Cl− channel elements are closed when this same control site instead binds ZnOH+. The sextet has a much higher affinity for ZnOH+ than does the single Cl− channel element.