Cl− transport in basolateral renal medullary vesicles: I. Cl− transport in intact vesicles

Abstract

This paper provides the results of studies which characterized conductive 36Cl- flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive 36Cl- uptake was studied under two different experimental conditions. In the first, 36Cl- flux was driven by an inside positive voltage created with oppositely directed Cl- and gluconate gradients. In the second, an inwardly direct K+ gradient was used to drive 36Cl- uptake. By these two methods, voltage-sensitive 36Cl- uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total 36Cl- flux. Separate paired studies demonstrated that the conductive 36Cl- uptake was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (DPC) with an IC50 for DPC of 154 microM. The voltage-dependent 36Cl- uptake had an activation energy of 6.4 kcal/mole. This 36Cl- conductance had an anion selectivity sequence of I- greater than Cl- greater than or equal to NO3- much greater than gluconate.

Cl− transport in basolateral renal medullary vesicles: II. Cl− channels in planar lipid bilayers

“…We note in this connection that, as pointed out in the preceding paper [1], the vesicles used in these experiments, while basolaterally enriched, also contain an admixture of apical membranes. However, in the mTALH, C1-conductances are limited to basolateral membranes [11].…”

“…As indicated in the preceding paper [1], the C1-channel blocker diphenylamine-2-carboxylate (DPC) suppressed C1-conductance in basolateral renal medullary vesicles. To test the effects of DPC on C1-channels, we incorporated the latter into bilayers according to the protocol shown in Figs.…”

“…In the preceding paper [1], we described some of the characteristics of conductive C1-flux in basolaterally enriched renal medullary vesicles. The present experiments, involving vesicles fused with planar bilayers, indicate that at least one of the C1--conductive pathways in these basolaterally enriched renal medullary vesicles is a C1-channel.…”

“…The procedure for preparing basolaterally enriched vesicles from rabbit renal outer medulla, and the enzymatic characteristics of these vesicles, are described in the preceding paper [1]. For the present studies, these vesicles were suspended in 300 mM mannitol, 10 mM imidazole, 1 mM Mg gluconate and 0.1 mM Ca gluconate (pH 7.4) at a protein concentration of 10 mg/ml.…”

“…In the preceding paper [1], we provided evidence that a conductive pathway accounted for about half of 36C1-uptake into basolaterally enriched renal medullary vesicles. The energetics of diffusion through this conductive 36C1-pathway were similar to those in free solution.…”