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 involved to explain these inhibitions because the intrinsic permeability of these vesicles to Cl- and K+ is negligibly small. Rather, our results show that, in the absence of valinomycin, the inhibitory effect 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 (pHout/pHin = 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- or K+ to the extravesicular medium. Such results confirm the independent existence of both Cl-/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.