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

Rebecca W. Van Dyke, Bruce F. Scharschmidt, Clifford J. Steer

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18 Scopus citations


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-≫NO3-≫ gluconate, SO42-, HPO42-, mannitol; Km 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 (IC50 < 10 μM), dicyclohexylcarbodiimide (DCCD) (lC50 ≈- 10 μM), chlorpromazine (lC50 ≈- 15 μM), and oligomycin (lC50 ≈- 3 μM). In contrast to N-ethylmaleimide, chlorpromazine rapidly dissipated preformed pH gradients. Valinomycin stimulated H+ transport in the presence of potassium salts (gluconate ≫ NO3- > 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 KNO3- 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.

Original languageEnglish (US)
Pages (from-to)423-436
Number of pages14
JournalBBA - Biomembranes
Issue number2
StatePublished - Jan 25 1985

Bibliographical note

Funding Information:
The authors wish to thank Ms. Jocelyn Matsumoto-Pon for her technical assistance. The electron microscopic studies were performed by Mr. Gary Hradek through the courtesy of the Liver Core Center Electron Microscopy Facihty under the direction of Dr. Albert L. Jones. The authors also which to thank Dr. Antony Mc-Donagh (AM 26307) for generously making available the spectrofluorimeter used in these studies, the Liver Center Spectroscopy Core Facility (Dr. Almira Correia) and Ms. Diana Fedorchak and Mr. Michael Karasik for their help m preparation of the manuscript. This work was supported in part by NIH Grants AM 26270, AM 26307, AM 06743, and grants from the American Liver Foundation, the American Gastroenterological Association and the Walter C. Pew Fund for Gastrointestinal Research.


  • (Brain vesicle)
  • ATP dependence
  • Acidification
  • Clathrin
  • Proton transport


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