Trans-potassium effects on the chloride/proton symporter activity of guinea-pig ileal brush-border membrane vesicles

Abstract

To investigate the inhibitory effect of trans-potassium on the Cl −/H + symporter activity of brush-border membrane vesicles from guinea pig ileum, we measured both 36Cl uptake and, by the pyranine fluorescence method, proton fluxes, in the presence of appropriate H + and K + gradients. In the absence of valinomycin, a time-dependent inhibitory effect of chloride uptake by trans K + was demonstrated. This inhibition was independent of the presence or absence of any K + gradient. Electrical effects cannot be invoked to explain these inhibitions because the intrinsic permeability of these vesicles to Cl −1 and K + is negligibly small. Rather, our results show that, in the absence of valinomycin, the inhibitory effects of intravesicular K + involves an acceleration of the rate of dissipation of the proton gradient through an electroneutral exchange of trans K + for cis H +, catalyzed by the K +/H + antiporter also present in these membranes. Valinomycin can further accelerate the rate of pH gradient dissipation by facilitating an electrically-coupled exchange between K + and H +. To evaluate the apparent rate of pH-dissipating, downhill proton influx, we measured chloride uptake by vesicles preincubated in the presence of alkaline-inside pH gradients (pH out/pH in = 5.0/7.5), charged or not with K +. In the absence of intravesicular K +, proton influx exhibited monoexponential kinetics with a time constant k = 11 s −1. Presence of 100 mM K + within the vesicles significantly increased the rate of pH gradient dissipation which, furthermore, became bi-exponential and revealed the appearance of an additional, faster proton influx component with k = 71 s −1. This new component we interpret as representing the sum of the electroneutral and the electrically-coupled exchange of trans K + for cis H +, mentioned above. Finally, by using the pH-sensitive fluorophore, pyranine, we demonstrate that, independent of the absence or presence of a pH gradient, either vesicle acidification or alkalinisation can be generated by adding, respectively, Cl −1 or K + to the extravesicular medium. Such results confirm the independent existence of both Cl −1/H + symporter and K +/H + antiporter activities in our vesicle preparations, the relative activity of the former being larger under the conditions of the present experiments. The possible interplay of these two proton-transfer mechanisms in the regulation of the intracellular pH is discussed.