Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p).

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Chan CY, Prudom C, Raines SM, Charkhzarrin S, Melman SD, De Haro LP, Allen C, Lee SA, Sklar LA, Parra KJ

Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p).

J Biol Chem. 2012 Mar 23;287(13):10236-50. doi: 10.1074/jbc.M111.321133. Epub 2012 Jan 3.

PubMed ID

22215674 [ View in PubMed

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Abstract

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 muM) and thonzonium bromide (EC(50) = 69 muM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 muM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Thonzonium	V-type proton ATPase subunit C 1	Protein	Humans	Yes	Inhibitor	Details