Response of chloride efflux from skeletal muscle ofRana 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.

Chloride current in toad skeletal muscle and its modification by the histidine-modifying reagent diethylpyrocarbonate

“…A protonation of the imidazole group of histidine associated with CI-channels could be the controlling reaction for pH-dependent CI-movements. These results are in agreement with the C1-fluxes measurements by Spalding et al (1991), who have found that DEPC affects the increment in CI-flux when the external pH is raised.…”

“…To minimize the loss of reagent by hydrolysis, the DEPC was added to solution immediately before the experiment. Since DEPC hydrolyzes spontaneously to ethanol and CO2 with a half-time of about 69 min at 20 ° C (Spalding et al 1991), the concentration of DEPC to which the fibres were exposed was somewhat less than the initial prepared concentration. As with the control fibres, currents in the treated fibres were measured in solution B at different pH values.…”

“…The imidazol group is the only one that has a pK of 6 (for tabulations of pK values see Dugas and Penney, 1981). A valuable approach for testing this hypothesis is to examine the effects upon CI-currents of diethylpyrocarbonate (DEPC), a reagent which carbetboxylates specifically histidil residues in proteins (Mfihlrfid et al 1969;Ovfidi et al 1967;Spalding et al 1991).…”

Modification of Cl− transport in skeletal muscle of Rana temporaria with the arginine‐binding reagent phenylglyoxal

“…The relatively high steady‐state chloride conductance, g Cl , of frog skeletal muscle fibre membranes, which is important for stabilization of the resting membrane potential, is strongly dependent on external pH. Cl − exchange under conditions of Cl − equilibrium in depolarized fibres (Skydsgaard, 1987) and g Cl in both normal (∼120 mM Cl − ) and high‐KCl Ringer solution (Hutter & Warner, 1967 a ) are all inactivated in acid medium with an apparent p K of ∼7 in Rana temporaria , possibly due to the protonation of histidines in the Cl − channels (Hutter & Warner, 1967 a,b ; Spalding, Taber, Swift & Horowicz, 1991). The acidification reduces g Cl more than it reduces the Cl − flux and there is evidence that a considerable fraction of the residual flux at low pH may be non‐conductive, possibly taking place via exchange diffusion (Harris, 1965; Hutter & Warner, 1967 b ; Skydsgaard, 1987).…”