Rheogenic Cl- conductance and Cl(-)-Cl(-)-exchange activities in guinea pig jejunal basolateral membrane vesicles

Abstract

We describe a method for the simultaneous purification of apical (brush-border membrane) and basolateral membrane (BLM) vesicles from the same sample of guinea pig jejunum. We applied functional tests to demonstrate the absence of reciprocal cross contamination between the two vesicle preparations. By using the BLM vesicles and a rapid filtration technique, we quantified 36Cl uptake under conditions of equilibrated pH (pHout = pHin = 7.5). The presence of 200 mM cis of either Na+ or K+, or an equimolar mixture of both, significantly increased the initial Cl- entry rate. In the presence of K+, valinomycin further increased Cl- uptake, but no Cl- uphill transport was ever observed under any of the conditions. All the increases were abolished by voltage clamping, indicating that the alkali-metal ions act by creating an inside-positive membrane potential capable of stimulating a Cl(-)-conductance pathway. In the absence of K+, BLM vesicle preloading to obtain a [Cl-]out/[Cl-]in = 16/200 mM gradient (delta Cl-) resulted in a 500% increase in the initial 36Cl- entry rate, accompanied by a transient Cl- accumulation, with an overshoot at approximately 5 min. In the presence of both a positive-inside electrical gradient (delta psi) and a delta Cl-, the initial Cl- uptake rate was increased by 800%, indicating that the effects of delta psi and of delta Cl- are additive. The delta Cl- effect was blocked, but only partially, by short-circuiting the membrane potential with equilibrated K+ and valinomycin, thus indicating that it has both rheogenic and electroneutral components. We conclude that Cl- influx across the guinea pig intestinal BLM involves a Cl(-)-conductance pathway plus a distinct Cl(-)-Cl(-)-exchange system, exhibiting both electroneutral and rheogenic components. Alternatively, the possibility can also be entertained that the conductance and the exchange pathways share a common molecular basis, e.g., a nonobligatory Cl(-)-Cl- exchanger or rheogenic uniport.