Characterization of chloride channels in membrane vesicles from the kidney outer medulla

Summary

The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is highly enriched in Na⁺/K⁺ ATPase and has been shown by electrophysiological methods to be highly conductive to Cl⁻. In order to study the Cl⁻ conductive pathways, membrane vesicles were isolated from the TALH-containing region of the porcine kidney, the red outer medulla, and Cl⁻ channel activity was determined by a³⁶Cl uptake assay where the uptake of the radioactive tracer is driven by the membrane potential (positive inside) generated by an outward Cl⁻ gradient. The accumulation of³⁶Cl⁻ inside the vesicles was found to be dependent on the intravesicular Cl⁻ concentration and was abolished by clamping the membrane potential with valinomycin. The latter finding indicated the involvement of conductive pathways. Cl⁻ channel activity was also observed using a fluorescent potential-sensitive carbocyanine dye, which detected a diffusion potential induced by an imposed inward Cl⁻ gradient. The anion selectivity of the channels was Cl⁻>NO ⁻₃ =I⁻≫ gluconate. Among the Cl⁻ transport inhibitors tested, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPAB), 4,4′-diisothiocyano-stilbene-2,2′-disulfonate (DIDS), and diphenylamine-2-carboxylate (DPC) showed IC₅₀ of 110, 200 and 550 μm, respectively. Inhibition of³⁶Cl uptake by NPPAB and two other structural analogues was fully reversible, whereas that by DIDS was not. The nonreactive analogue of DIDS, 4,4′-dinitrostilbene-2,2′-disulfonate (DNDS), was considerably less inhibitory than DIDS (25% inhibition at 200 μm). The irreversible inhibition by DIDS was prevented by NPPAB, whereas DPC was ineffective, consistent with its low inhibitory potency. It is proposed that NPPAB and DIDS bind to the same or functionally related site on the Cl⁻ channel protein.