Winter Sandra, Brunk Irene, Walther Diego J, Höltje Markus, Jiang Meisheng, Peter Jens-Uwe, Takamori Shigeo, Jahn Reinhard, Birnbaumer Lutz, Ahnert-Hilger Gudrun
AG Funktionelle Zellbiologie, Centrum für Anatomie, Charité Universitätsmedizin Berlin, D-10115 Berlin, Germany.
J Neurosci. 2005 May 4;25(18):4672-80. doi: 10.1523/JNEUROSCI.0549-05.2005.
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.
经典神经递质，包括单胺类、乙酰胆碱、谷氨酸、γ-氨基丁酸（GABA）和甘氨酸，通过特定转运体被装载到突触小泡中。囊泡单胺转运体受到三聚体G蛋白α亚基的负调控，包括Gα(o2)和Gα(q)。此外，由囊泡谷氨酸转运体（VGLUTs）介导的谷氨酸摄取，会被不可水解的GTP类似物鸟苷酰亚胺二磷酸降低。利用缺乏各种Gα亚基的突变小鼠，包括Gα(o1)、Gα(o2)、Gα(q)和Gα11，以及一种Gα(o2)特异性单克隆抗体，我们现在表明VGLUTs仅受Gα(o2)调控。G蛋白激活并不影响作为神经递质摄取驱动力的电化学质子梯度；相反，Gα(o2)通过特异性影响VGLUTs对氯离子的依赖性发挥作用。所有VGLUTs在约5 mM氯离子浓度下显示最大活性。激活的Gα(o2)将这个最大值转移到更低的氯离子浓度。相比之下，从Gα(o2 -/-)小鼠分离的囊泡对谷氨酸的摄取完全丧失了氯离子激活作用。因此，Gα(o2)作用于一个假定的调节性氯离子结合结构域，该结构域似乎可调节囊泡谷氨酸转运体的转运活性。
