title: Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes.
abstract: Vesicular V-ATPase (V-type H+-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at the synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed SSM (solid supported membrane)-based electrophysiological analyses of synaptic vesicles and plasma membranes prepared from rat brains by sucrose-gradient fractionation. Acidification experiments revealed V-ATPase activity in fractions containing the vesicles but not in the plasma membrane fractions. For the SSM-based electrical measurements, the ATPases were activated by ATP concentration jumps. In vesicles, ATP-induced currents were inhibited by the V-ATPase-specific inhibitor BafA1 (bafilomycin A1) and by DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate). In plasma membranes, the currents were inhibited by the Na+/K+-ATPase inhibitor digitoxigenin. The distribution of the V-ATPase- and Na+/K+-ATPase-specific currents correlated with the distribution of vesicles and plasma membranes in the sucrose gradient. V-ATPase-specific currents depended on ATP with a K0.5 of 51+/-7 microM and were inhibited by ADP in a negatively co-operative manner with an IC50 of 1.2+/-0.6 microM. Activation of V-ATPase had stimulating effects on the chloride conductance in the vesicles. Low micromolar concentrations of DIDS fully inhibited the V-ATPase activity, whereas the chloride conductance was only partially affected. In contrast, NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] inhibited the chloride conductance but not the V-ATPase. The results presented describe electrical characteristics of synaptic V-ATPase and Na+/K+-ATPase in their native surroundings, and demonstrate the feasibility of the method for electrophysiological studies of transport proteins in native intracellular compartments and plasma membranes.
authors.affiliation: IonGate Biosciences, Industriepark Hoechst, 65926 Frankfurt am Main, Germany. petrobrdlik@web.de
authors.forename: Petr
authors.initials: P
authors.lastname: Obrdlik
authors.forename: Kerstin
authors.initials: K
authors.lastname: Diekert
authors.forename: Natalie
authors.initials: N
authors.lastname: Watzke
authors.forename: Christine
authors.initials: C
authors.lastname: Keipert
authors.forename: Ulrich
authors.initials: U
authors.lastname: Pehl
authors.forename: Catrin
authors.initials: C
authors.lastname: Brosch
authors.forename: Nicole
authors.initials: N
authors.lastname: Boehm
authors.forename: Inga
authors.initials: I
authors.lastname: Bick
authors.forename: Maarten
authors.initials: M
authors.lastname: Ruitenberg
authors.forename: Walter
authors.initials: W
authors.lastname: Volknandt
authors.forename: Bela
authors.initials: B
authors.lastname: Kelety
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology*; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D011188:Potassium / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013570:Synaptic Membranes / Q000201:enzymology*; D013572:Synaptic Vesicles / Q000201:enzymology*; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D018942:Macrolides; C040929:bafilomycin A1; D000255:Adenosine Triphosphate; D012964:Sodium; D025262:Vacuolar Proton-Translocating ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium