Obrdlik P.,IonGate Biosciences |
Diekert K.,IonGate Biosciences |
Watzke N.,IonGate Biosciences |
Keipert C.,IonGate Biosciences |
And 9 more authors.
Biochemical Journal | Year: 2010
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′-diisothiocyanostilbene-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 μM and were inhibited by ADP in a negatively co-operative mannerwith an 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, 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. © The Authors. Source
Balannik V.,Northwestern University |
Obrdlik P.,IonGate Biosciences |
Inayat S.,Northwestern University |
Steensen C.,IonGate Biosciences |
And 6 more authors.
Pflugers Archiv European Journal of Physiology | Year: 2010
Influenza A virus encodes an integral membrane protein, A/M2, that forms a pH-gated proton channel that is essential for viral replication. The A/M2 channel is a target for the anti-influenza drug amantadine, although the effectiveness of this drug has been diminished by the appearance of naturally occurring point mutations in the channel pore. Thus, there is a great need to discover novel anti-influenza therapeutics, and, since the A/M2 channel is a proven target, approaches are needed to screen for new classes of inhibitors for the A/M2 channel. Prior in-depth studies of the activity and drug sensitivity of A/M2 channels have employed labor-intensive electrophysiology techniques. In this study, we tested the validity of electrophysiological measurements with solid-supported membranes (SSM) as a less labor-intensive alternative technique for the investigation of A/M2 ion channel properties and for drug screening. By comparing the SSM-based measurements of the activity and drug sensitivity of A/M2 wild-type and mutant channels with measurements made with conventional electrophysiology methods, we show that SSM-based electrophysiology is an efficient and reliable tool for functional studies of the A/M2 channel protein and for screening compounds for inhibitory activity against the channel. Source