Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes

Petr Obrdlik (1) , Kerstin Diekert , Natalie Watzke , Christine Keipert , Ulrich Pehl , Catrin Brosch , Nicole Boehm , Inga Bick , Maarten Ruitenberg , Walter Volknandt , Bela Kelety

1 IonGate Biosciences GmbH

Vesicular V-type H+-ATPase (V-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed solid supported membrane (SSM)-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 bafilomycin A1 (BafA1) and by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In plasma membranes, the currents were inhibited by the Na+/K+-ATPase inhibitor digitoxigenin. 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 K0.5 of 51 ± 7 µM and were inhibited by ADP in a negatively cooperative manner with IC50 of 1.2 ± 0.6 µM. 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, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the chloride conductance but not the V-ATPase. The presented data 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.

Mots clés: Life Sciences

Petr Obrdlik, Kerstin Diekert, Natalie Watzke, Christine Keipert, Ulrich Pehl, et al.. Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes. Biochemical Journal, 2010, 427 (1), pp.151-159. ⟨10.1042/BJ20091380⟩. ⟨hal-00479251⟩