RESPONSE OF CHLORIDE EFFLUX FROM SKELETAL-MUSCLE OF RANA-PIPIENS TO CHANGES OF TEMPERATURE AND MEMBRANE-POTENTIAL AND DIETHYLPYROCARBONATE TREATMENT

Efflux of Cl-36(-) 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 u = 1/(1 + 10n(pK(a)-pH), where u 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(a) 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(a) at 0-degrees-C is 6.9 and n is 1.5. The voltage dependence of the apparent pK(a) 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(a) 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.