Melliti K, Meza U, Fisher R, Adams B
Department of Physiology and Biophysics, University of Iowa, College of Medicine, Iowa City, Iowa 52242-1109, USA.
J Gen Physiol. 1999 Jan;113(1):97-110. doi: 10.1085/jgp.113.1.97.
Regulators of G protein signaling (RGS) proteins bind to the alpha subunits of certain heterotrimeric G proteins and greatly enhance their rate of GTP hydrolysis, thereby determining the time course of interactions among Galpha, Gbetagamma, and their effectors. Voltage-gated N-type Ca channels mediate neurosecretion, and these Ca channels are powerfully inhibited by G proteins. To determine whether RGS proteins could influence Ca channel function, we recorded the activity of N-type Ca channels coexpressed in human embryonic kidney (HEK293) cells with G protein-coupled muscarinic (m2) receptors and various RGS proteins. Coexpression of full-length RGS3T, RGS3, or RGS8 significantly attenuated the magnitude of receptor-mediated Ca channel inhibition. In control cells expressing alpha1B, alpha2, and beta3 Ca channel subunits and m2 receptors, carbachol (1 microM) inhibited whole-cell currents by approximately 80% compared with only approximately 55% inhibition in cells also expressing exogenous RGS protein. A similar effect was produced by expression of the conserved core domain of RGS8. The attenuation of Ca current inhibition resulted primarily from a shift in the steady state dose-response relationship to higher agonist concentrations, with the EC50 for carbachol inhibition being approximately 18 nM in control cells vs. approximately 150 nM in RGS-expressing cells. The kinetics of Ca channel inhibition were also modified by RGS. Thus, in cells expressing RGS3T, the decay of prepulse facilitation was slower, and recovery of Ca channels from inhibition after agonist removal was faster than in control cells. The effects of RGS proteins on Ca channel modulation can be explained by their ability to act as GTPase-accelerating proteins for some Galpha subunits. These results suggest that RGS proteins may play important roles in shaping the magnitude and kinetics of physiological events, such as neurosecretion, that involve G protein-modulated Ca channels.
G蛋白信号调节（RGS）蛋白与某些异源三聚体G蛋白的α亚基结合，并极大地提高其GTP水解速率，从而决定Gα、Gβγ及其效应器之间相互作用的时间进程。电压门控N型钙通道介导神经分泌，这些钙通道受到G蛋白的强烈抑制。为了确定RGS蛋白是否会影响钙通道功能，我们记录了在人胚肾（HEK293）细胞中与G蛋白偶联的毒蕈碱（m2）受体及各种RGS蛋白共表达的N型钙通道的活性。全长RGS3T、RGS3或RGS8的共表达显著减弱了受体介导的钙通道抑制的幅度。在表达α1B、α2和β3钙通道亚基及m2受体的对照细胞中，与仅表达外源性RGS蛋白的细胞中约55%的抑制相比，卡巴胆碱（1μM）抑制全细胞电流约80%。RGS8保守核心结构域的表达也产生了类似的效果。钙电流抑制的减弱主要源于稳态剂量反应关系向更高激动剂浓度的转变，卡巴胆碱抑制的EC50在对照细胞中约为18 nM，而在表达RGS的细胞中约为150 nM。RGS也改变了钙通道抑制的动力学。因此，在表达RGS3T的细胞中，预脉冲易化的衰减较慢，并且激动剂去除后钙通道从抑制状态的恢复比对照细胞更快。RGS蛋白对钙通道调节的作用可以通过它们作为某些Gα亚基的GTP酶加速蛋白的能力来解释。这些结果表明，RGS蛋白可能在塑造涉及G蛋白调节钙通道的生理事件（如神经分泌）的幅度和动力学方面发挥重要作用。
