Characterization of a H+-ATPase in rat brain synaptic vesicles. Coupling to L-glutamate transport.
Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.
Cidon
S
S
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021-6399.
Sihra
T S
TS
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000838:Anions; D001692:Biological Transport; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D008297:Male; D009550:Nigericin / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011522:Protons; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence; D013572:Synaptic Vesicles / Q000201:enzymology*; D014617:Vacuoles / Q000201:enzymology; D014634:Valinomycin / Q000494:pharmacology
D000838:Anions; D002712:Chlorides; D005971:Glutamates; D011522:Protons; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D009550:Nigericin