Gα_o2Regulates Vesicular Glutamate Transporter Activity by Changing Its Chloride Dependence

Abstract

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 ␣ subunits of trimeric G-proteins, including G␣ o2 and G␣ q . Furthermore, glutamate uptake, mediated by vesicular glutamate transporters (VGLUTs), is decreased by the nonhydrolysable GTP-analog guanylylimidodiphosphate. Using mutant mice lacking various G␣ subunits, including G␣ o1 , G␣ o2 , G␣ q , and G␣ 11 , and a G␣ o2 -specific monoclonal antibody, we now show that VGLUTs are exclusively regulated by G␣ o2 . G-protein activation does not affect the electrochemical proton gradient serving as driving force for neurotransmitter uptake; rather, G␣ o2 exerts its action by specifically affecting the chloride dependence of VGLUTs. All VGLUTs show maximal activity at ϳ5 mM chloride. Activated G␣ o2 shifts this maximum to lower chloride concentrations. In contrast, glutamate uptake by vesicles isolated from G␣ o2 Ϫ/Ϫ mice have completely lost chloride activation. Thus, G␣ o2 acts on a putative regulatory chloride binding domain that appears to modulate transport activity of vesicular glutamate transporters.

Vesicular and Plasma Membrane Transporters for Neurotransmitters

“…A number of reports describe the regulation of vesicle filling by heterotrimeric G proteins, in particular Gao2 (Ahnert-Hilger et al 1998;Holtje et al 2003;Winter et al 2005). Although the molecular mechanism of modulation remains uncertain, it may involve a direct interaction of G protein with neurotransmitter transporter (Winter et al 2005;Brunk et al 2006).…”

“…Although the molecular mechanism of modulation remains uncertain, it may involve a direct interaction of G protein with neurotransmitter transporter (Winter et al 2005;Brunk et al 2006). …”

Synaptic and Vesicular Coexistence of VGLUT and VGAT in Selected Excitatory and Inhibitory Synapses

“…For neurotransmitter uptake experiments, immunoisolated subpopulations were taken from the lysis supernatant 1 (LS1) prepared from LS0 by a centrifugation step at 30,000 ϫ g for 20 min at 4°C. Some uptake experiments were performed with the usual synaptic vesicle fraction (LP2) prepared as given (Winter et al, 2005).…”

“…[ 3 H]Glutamate uptake was performed using SVs or immunoisolated SV subpopulations prepared from LS1 (immunoisolated by anti-Syp, -VGLUT1, -VGLUT2, or -VGAT antibodies or IgG for control) with the exception that potassium gluconate buffer (KGC buffer, containing 150 mM potassium gluconate) was used instead as given earlier (Winter et al, 2005). Uptake was started by adding 49.5 M K-glutamate and 0.5 M [ 3 H]glutamate to SVs.…”

“…To overcome this limitation, we analyzed the putative contribution of VGLUT activity to VGAT-mediated GABA uptake by using a SV preparation from whole brain and the specific VGLUT inhibitor trypan blue (Roseth et al, 1998). As previously shown, trypan blue selectively inhibits glutamate uptake comparable to an inhibition by dissipating the electrochemical gradient (Winter et al, 2005). In the presence of glutamate, trypan blue inhibited GABA uptake by 15%, indicating that VGLUT activity can improve GABA uptake.…”