Characterization of the solubilized and reconstituted ATP-dependent vesicular glutamate uptake system

We have previously provided evidence for ATP-dependent glutamate uptake into synaptic vesicles, and, based upon the unique properties of the vesicular uptake system, we have proposed that the vesicular glutamate translocator plays a crucial role in selecting glutamate for neurotransmission. In this study, we have solubilized the vesicular glutamate uptake system, proposed to consist of at least a glutamate translocator and a proton pump Mg-ATPase, from rat brain synaptic vesicles, and reconstituted the functional ATP-dependent glutamate uptake system into liposomes. The glutamate uptake in the reconstituted system is dependent upon ATP, markedly potentiated by low millimolar concentrations of chloride and inhibited by agents known to dissipate electrochemical proton gradients. Moreover, it exhibited low affinity for glutamate (Km = 2 mM), yet high specificity for glutamate; thus, it did not recognize aspartate and other agents known to interact with glutamate receptors. These properties are indistinguishable from those observed in intact synaptic vesicles. The solubilized functional components of the glutamate uptake system, alone or as a complex, have been estimated to have a Stokes radius in the range of 69 to 84 A. The reconstitution experiments described here provide a functional assay for the solubilized vesicular glutamate uptake system and represent an initial step towards the purification of the glutamate translocator.