Gαo2 regulates vesicular glutamate transporter activity by changing its chloride dependence

Classical neurotransmitters, including monoamines, acetylcholine, glutamate, GABA, and glycine, are loaded into synaptic vesicles by means of specific transporters. Vesicular monoamine transporters are under negative regulation by alpha subunits of trimeric G-proteins, including G alpha(o2) and G alpha(q). Furthermore, glutamate uptake, mediated by vesicular glutamate transporters (VGLUTs), is decreased by the nonhydrolysable GTP-analog guanylylimidodiphosphate. Using mutant mice lacking various G alpha subunits, including G alpha(o1), G alpha(o2), G alpha(q), and G alpha(11), and a G alpha(o2)-specific monoclonal antibody, we now show that VGLUTs are exclusively regulated by G alpha(o2). G-protein activation does not affect the electrochemical proton gradient serving as driving force for neurotransmitter uptake; rather, G alpha(o2) exerts its action by specifically affecting the chloride dependence of VGLUTs. All VGLUTs show maximal activity at similar to 5 mM chloride. Activated G alpha(o2) shifts this maximum to lower chloride concentrations. In contrast, glutamate uptake by vesicles isolated from G alpha(-/-)(o2) mice have completely lost chloride activation. Thus, G alpha(o2) acts on a putative regulatory chloride binding domain that appears to modulate transport activity of vesicular glutamate transporters.