Atkinson Peter J, Young Kenneth W, Ennion Steven J, Kew James N C, Nahorski Stefan R, Challiss R A John
Department of Cell Physiology and Pharmacology, University of Leicester, Maurice Shock Medical Sciences Building, University Road, Leicester, LE1 9HN, UK.
Mol Pharmacol. 2006 Jan;69(1):174-84. doi: 10.1124/mol.105.014258. Epub 2005 Oct 18.
The metabotropic glutamate (mGlu) receptors mGlu1 and mGlu5 mediate distinct inositol 1,4,5-trisphosphate (IP(3)) and Ca(2+) signaling patterns, governed in part by differential mechanisms of feedback regulation after activation. Single cell imaging has shown that mGlu1 receptors initiate sustained elevations in IP(3) and Ca(2+), which are sensitive to agonist concentration. In contrast, mGlu5 receptors are subject to cyclical PKC-dependent uncoupling and consequently mediate coincident IP(3) and Ca(2+) oscillations that are largely independent of agonist concentration. In this study, we investigated the contribution of G(q/11)alpha protein expression levels in shaping mGlu1/5 receptor-mediated IP(3) and Ca(2+) signals, using RNA interference (RNAi). RNAi-mediated knockdown of G(q/11)alpha almost abolished the single-cell increase in IP(3) caused by mGlu1 and mGlu5 receptor activation. For the mGlu1 receptor, this unmasked baseline Ca(2+) oscillations that persisted even at maximal agonist concentrations. mGlu5 receptor-activated Ca(2+) oscillations were still observed but were only initiated at high agonist concentrations. Recombinant overexpression of G(q)alpha enhanced IP(3) signals after mGlu1 and mGlu5 receptor activation. It is noteworthy that although mGlu5 receptor-mediated IP(3) and Ca(2+) oscillations in control cells were largely insensitive to agonist concentration, increasing G(q)alpha expression converted these oscillatory signatures to sustained plateau responses in a high proportion of cells. In addition to modulating temporal Ca(2+) signals, up- or down-regulation of G(q/11)alpha expression alters the threshold for the concentration of glutamate at which a measurable Ca(2+) signal could be detected. These experiments indicate that altering G(q/11)alpha expression levels differentially affects spatiotemporal aspects of IP(3) and Ca(2+) signaling mediated by the mGlu1 and mGlu5 receptors.
代谢型谷氨酸(mGlu)受体mGlu1和mGlu5介导不同的肌醇1,4,5 - 三磷酸(IP(3))和Ca(2+)信号模式,这部分受激活后反馈调节的不同机制所支配。单细胞成像显示,mGlu1受体引发IP(3)和Ca(2+)的持续升高,这对激动剂浓度敏感。相比之下,mGlu5受体受到周期性蛋白激酶C(PKC)依赖性解偶联的影响,因此介导基本独立于激动剂浓度的IP(3)和Ca(2+)振荡。在本研究中,我们使用RNA干扰(RNAi)研究了G(q/11)α蛋白表达水平在塑造mGlu1/5受体介导的IP(3)和Ca(2+)信号中的作用。RNAi介导的G(q/11)α敲低几乎消除了由mGlu1和mGlu5受体激活引起的单细胞IP(3)增加。对于mGlu1受体,这揭示了即使在最大激动剂浓度下仍持续存在的基线Ca(2+)振荡。仍观察到mGlu5受体激活的Ca(2+)振荡,但仅在高激动剂浓度下才启动。G(q)α的重组过表达增强了mGlu1和mGlu5受体激活后的IP(3)信号。值得注意的是,尽管对照细胞中mGlu5受体介导的IP(3)和Ca(2+)振荡对激动剂浓度基本不敏感,但增加G(q)α表达在很大比例的细胞中将这些振荡特征转变为持续的平台反应。除了调节Ca(2+)信号的时间外,G(q/11)α表达的上调或下调还改变了可检测到可测量Ca(2+)信号的谷氨酸浓度阈值。这些实验表明,改变G(q/11)α表达水平会不同程度地影响mGlu1和mGlu5受体介导的IP(3)和Ca(2+)信号的时空方面。