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Free keywords:
chloride channel; endocytotic vesicles; rat kidney; H+-ATPase; vacuolar acidification; vesicle fusion
Abstract:
Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H+ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl− channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of −60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than −50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl−=Br−=I−>SO2−4 ≈F−. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl−-flux measurements and studies on the H+ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H+ uptake into the endosomes.
