Response of chloride efflux from skeletal muscle of Rana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment

Abstract

Efflux of 36Cl- from frog sartorius muscles equilibrated in two 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. For temperatures between 0 and 20 degrees C, the measured activation energy is 7.5 kcal/mol for Cl- efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl- efflux. The pH-dependent Cl-efflux can be described by the relation mu = 1/(1 + 10n(pK alpha-pH], where mu is the Cl- efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150 mM KCl plus 120 mM NaCl (internal potential about -15 mV), the apparent pK alpha is 6.5 at both 0 and 20 degrees C, and n = 2.5 for 0 degrees C and 1.5 for 20 degrees C. For muscles equilibrated in solutions containing 7.5 mM KCl plus 120 mM NaCl (internal potential about -65 mV), the apparent pK alpha at 0 degrees C is 6.9 and n is 1.5. The voltage dependence of the apparent pK alpha suggests that the critical pH-sensitive moiety producing the pH-dependent Cl- efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact that n is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl- efflux increment on raising external pH. The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl- efflux according to the relation, efflux = exp(-k.[DEPC].t), where t is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mM). At 17 degrees C, k-1 = 188 mM . min. For temperatures between 10 and 23 degrees C, k has an apparent Q10 of 2.5. The Cl- efflux inhibitor SCN- at a concentration of 20 mM substantially retards the reduction of the pH-dependent Cl- efflux by DEPC. The findings that the apparent pK alpha is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl- efflux, and that this action is retarded by SCN- supports the notion that protonation of histidine groups associated with Cl- channels is the controlling reaction for the pH-dependent Cl- efflux.