Galphao2 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 Galpha(o2) and Galpha(q). Furthermore, glutamate uptake, mediated by vesicular glutamate transporters (VGLUTs), is decreased by the nonhydrolysable GTP-analog guanylylimidodiphosphate. Using mutant mice lacking various Galpha subunits, including Galpha(o1), Galpha(o2), Galpha(q), and Galpha11, and a Galpha(o2)-specific monoclonal antibody, we now show that VGLUTs are exclusively regulated by Galpha(o2). G-protein activation does not affect the electrochemical proton gradient serving as driving force for neurotransmitter uptake; rather, Galpha(o2) exerts its action by specifically affecting the chloride dependence of VGLUTs. All VGLUTs show maximal activity at approximately 5 mm chloride. Activated Galpha(o2) shifts this maximum to lower chloride concentrations. In contrast, glutamate uptake by vesicles isolated from Galpha(o2-/-) mice have completely lost chloride activation. Thus, Galpha(o2) acts on a putative regulatory chloride binding domain that appears to modulate transport activity of vesicular glutamate transporters.
Winter
Sandra
S
AG Funktionelle Zellbiologie, Centrum für Anatomie, Charité Universitätsmedizin Berlin, D-10115 Berlin, Germany.
Brunk
Irene
I
Walther
Diego J
DJ
Höltje
Markus
M
Jiang
Meisheng
M
Peter
Jens-Uwe
JU
Takamori
Shigeo
S
Jahn
Reinhard
R
Birnbaumer
Lutz
L
Ahnert-Hilger
Gudrun
G
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015153:Blotting, Western / Q000379:methods; D002712:Chlorides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000172:deficiency / Q000276:immunology / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D011189:Potassium Chloride / Q000494:pharmacology; D050683:R-SNARE Proteins / Q000378:metabolism; D051381:Rats; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000276:immunology; D014316:Tritium / Q000493:pharmacokinetics; D050496:Vesicular Glutamate Transport Proteins / Q000145:classification / Q000378:metabolism*
D000906:Antibodies; D002712:Chlorides; D050683:R-SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050496:Vesicular Glutamate Transport Proteins; D014316:Tritium; D006165:Guanylyl Imidodiphosphate; D018698:Glutamic Acid; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go