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.

DIDS (4,4-Diisothiocyanatostilbenedisulphonic Acid) Induces Apoptotic Cell Death in a Hippocampal Neuronal Cell Line and Is Not Neuroprotective against Ischemic Stress

“…Alternatively, inhibition of ATPases, protein synthesis and translocation, DNA repair, Ca 2+ homeostasis, or proper neurotransmitter function/responses at both the pre- and post-synaptic cell may all contributed to the induction of apoptosis and spread of cell death by DIDS in our study [13], [14], [15], [16], [17], [18], [20], [54]. For example, in our experiments, DIDS activates the JNK3 immune pathway and mitochondrial cytochrome C release, and activates multiple caspase-dependent pathways, including caspases 3 and 6 (since DIDS induces lamin A cleavage, which is a caspase-6 dependent process that leads to nuclear deregulation and cell death [55], [56], [57], cellular events which are also induced by DIDS in our study), indicating that multiple secondary mechanisms are likely activated by DIDS that contribute to the overall deleterious phenotype.…”

“…DIDS prevents protein translocation across the ER membrane, uncouples mitochondrial respiration, and directly binds to the DNA binding site of RAD51, an essential eukaryotic DNA recombinase, competitively inhibiting DNA-RAD51 binding activity and double stranded DNA repair [13], [14], [15]. Furthermore, DIDS affects the regulation of ionic and neurotransmitter homeostasis: DIDS reduces tonic astrocyte glutamate release and synaptic ATP uptake in brain, inhibits post-synaptic γ-amino butyric acid (GABA) A receptors, inhibits renal Na + /K + -ATPase and vesicular H + -ATPase activity, and reduces mitochondrial V m and Ca 2+ buffering in bacterial cells [15], [16], [17], [18], [19], [20]. DIDS also inhibits cellular proliferation in some cell types [15], [21], induces apoptosis in tumors and stem cells [21], [22], and perhaps more importantly, increases cell death in some disease models of pathology.…”

Olfactory bulb proteome dynamics during the progression of sporadic Alzheimer's disease: identification of common and distinct olfactory targets across Alzheimer-related co-pathologies

“…In intermediate stages, we have detected an imbalance in the protein composition of V-type proton ATPase (subnetwork B). This multi-subunit protein drives the loading of neurotransmitters into synaptic vesicles [ 55 ], indicating that the cycling of neurotransmitters at the olfactory synapse is not totally efficient. Moreover, several Collagen VI chains (subnetwork C) and its potential interactor AKAP12 are de-regulated in intermediate stages, suggesting that modulation of its protein levels may be part of the protective mechanisms against β-amyloid in olfactory neurons of AD patients [ 31 , 33 ].…”

Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters

“…A number of different electrogenic proteins (ion pumps, transporters and channels) have been tested using solid-supported membrane (SSM) electrophysiology [66][67][68][69]. More recently, this technique has been applied to intracellular transporters to overcome the inaccessibilty of endomembranes [70,71].…”