ATP-dependent proton transport by isolated brain clathrin-coated vesicles. Role of clathrin and other determinants of acidification

Abstract

We have systematically investigated certain characteristics of the ATP-dependent proton transport mechanism of bovine brain clathrin-coated vesicles. H + transport specific activity was shown by column chromatography to co-purify with coated vesicles, however, the clathrin coat is not required for vesicle acidification as H + transport was not altered by prior removal of the clathrin coat. Acidification of the vesicle interior, measured by fluorescence quenching of acridine orange, displayed considerable anion selectively (CI − > Br −⪢NO 3 −⪢ gluconate, SO 4 2-, HPO 4 2-, mannitol; K m for Cl - ≈- 15 mM), but was relatively insensitive to cation replacement as long as Cl - was present. Acidification was unaffected by ouabain or vanadate but was inhibited by N-ethylmaleimide (IC 50 < 10 μM), dicyclohexylcarbodiimide (DCCD) (lC 50 ≈- 10 μM), chlorpromazine (lC 50 ≈- 15 μM), and oligomycin (lC 50 ≈- 3 μM). In contrast to N-ethylmaleimide, chlorpromazine rapidly dissipated preformed pH gradients. Valinomycin stimulated H + transport in the presence of potassium salts (gluconate ⪢ NO 3 − > Cl −), and the membrane-potential-sensitive dye Oxonol V demonstrated an ATP-dependent interior-positive vesicle membrane potential which was greater in the absence of permeant anions (mannitol > potassium gluconate > KCI) and was abolished by N-ethylmaleimide, protonophores or detergent. Total vesicle-associated ouabain-insensitive ATPase activity was inhibited 64% by 1 mM N-ethylmaleimide, and correlated poorly with H + transport, however N-ethylmaleimide-sensitive ATPase activity correlated well with proton transport ( r = 0.95) in the presence of various Cl - salts and KNO 3- Finally, vesicles prepared from bovine brain synaptic membranes exhibited H + transport activity similar to that of the coated vesicles. Collectively these findings indicate that: (1) the H + transport mechanism of bovine brain clathrin-coated vesicles is not dependent upon the clathrin coat, and closely resembles that of rat liver clathrin-coated vesicles, (2) the H + transport mechanism is intrinsically electrogenic and pH and electrical gradients established by the proton transport mechanism vary inversely in the presence of permeable or impermeable anions, (3) at concentrations above 3 μM, oligomycin inhibits vesicle acidification, (4) chlorpromazine inhibits coated vesicle acidification, at least in part, by increasing proton conductance, and (5) other smooth membranes from bovine brain, including synaptic membranes, exhibit a similar H + transport mechanism.