Kinoshita-Kawada Mariko, Oberdick John, Xi Zhu Michael
Department of Neuroscience and the Center for Molecular Neurobiology, The Ohio State University, 168 Rightmire Hall, 1060 Carmack Road, Columbus, OH 43210, USA.
Brain Res Mol Brain Res. 2004 Dec 6;132(1):73-86. doi: 10.1016/j.molbrainres.2004.09.007.
L7/Pcp-2 is a GoLoco domain protein encoded by a Purkinje cell dendritic mRNA. Although biochemical interactions of GoLoco proteins with Galpha(o) and Galpha(i) are well documented, little is known about effector function modulation resulting from these interactions. The P-type Ca2+ channels might be physiological effectors of L7 because (1) they are the major voltage-dependent Ca2+ channels (VDCC) that modulate Purkinje cell output and (2) they are regulated by G(i/o) proteins. As a first step towards validating this hypothesis and to further understand the possible physiological effect of L7 protein and its two isoforms, we have coexpressed Ca(v)2.1 channels and kappa-opioid receptors (KORs) with varying amounts of L7A or L7B in Xenopus oocytes and measured ionic currents by two-electrode voltage clamping. Without receptor activation L7 did not alter the Ca2+ channel activity. With tonic and weak activation of the receptors, however, the Ca2+ channels were inhibited by 40-50%. This inhibition was enhanced by low, but dampened by high, expression levels of L7A and L7B and differences were observed between the two isoforms. The enhancing effect of L7 was occluded by overexpression of Gbetagamma, whereas the disinhibition was antagonized by overexpression of Galpha(o). We propose that L7 differentially affects the Galpha and Gbetagamma arms of receptor-induced G(i/o) signaling in a concentration-dependent manner, through which it increases the dynamic range of regulation of P/Q-type Ca2+ channels by G(i/o) protein-coupled receptors. This provides a framework for designing further experiments to determine how dendritic local fluctuations in L7 protein levels might influence signal processing in Purkinje cells.
L7/Pcp-2是一种由浦肯野细胞树突状mRNA编码的含GoLoco结构域的蛋白质。尽管GoLoco蛋白与Gα(o)和Gα(i)之间的生化相互作用已有充分记录，但对于这些相互作用所导致的效应器功能调节却知之甚少。P型Ca2+通道可能是L7的生理效应器，因为：（1）它们是调节浦肯野细胞输出的主要电压依赖性Ca2+通道（VDCC）；（2）它们受G(i/o)蛋白调节。作为验证这一假设以及进一步了解L7蛋白及其两种异构体可能的生理效应的第一步，我们在非洲爪蟾卵母细胞中共同表达了不同量的L7A或L7B与Ca(v)2.1通道和κ-阿片受体（KOR），并通过双电极电压钳测量离子电流。在未激活受体的情况下，L7不会改变Ca2+通道活性。然而，当受体受到持续性和微弱激活时，Ca2+通道被抑制了40 - 50%。L7A和L7B的低表达水平增强了这种抑制作用，但高表达水平则使其减弱，并且在两种异构体之间观察到了差异。L7的增强作用被Gβγ的过表达所阻断，而解除抑制作用则被Gα(o)的过表达所拮抗。我们提出，L7以浓度依赖的方式差异性地影响受体诱导的G(i/o)信号传导的Gα和Gβγ分支，通过这种方式它增加了G(i/o)蛋白偶联受体对P/Q型Ca2+通道调节的动态范围。这为设计进一步的实验提供了一个框架，以确定L7蛋白水平在树突中的局部波动如何影响浦肯野细胞中的信号处理。
