Leyris J-P, Gondeau C, Charnet A, Delattre C, Rousset M, Cens T, Charnet P
CRBM, CNRS UMR 5237, Université de Montpellier 1, 34293 Montpellier cedex, France.
FASEB J. 2009 Aug;23(8):2627-38. doi: 10.1096/fj.08-122135. Epub 2009 Mar 30.
RGK (Rad-Gem-Rem) GTPases have been described as potent negative regulators of the Ca(2+) influx via high-threshold voltage-activated Ca(2+) channels. Recent work, mostly performed on Ca(V)1.2 Ca(2+) channels, has highlighted the crucial role played by the channel auxiliary Ca(V)beta subunits and identified several GTPase and beta-subunit protein domains involved in this regulation. We now extend these conclusions by producing the first complete characterization of the effects of Gem, Rem, and Rem2 on the neuronal Ca(V)2.1 Ca(2+) channels expressed with Ca(V)beta(1) or Ca(V)beta(2) subunits. Current inhibition is limited to a decrease in amplitude with no modification in the voltage dependence or kinetics of the current. We demonstrate that this inhibition can occur for Ca(V)beta constructs with impaired capacity to induce current potentiation, but that it is lost for Ca(V)beta constructs deleted for their beta-interaction domain. The RGK C-terminal last approximately 80 amino acids are sufficient to allow potent current inhibition and in vivo beta-subunit/Gem interaction. Interestingly, although Gem and Gem carboxy-terminus induce a completely different pattern of beta-subunit cellular localization, they both potently inhibit Ca(V)2.1 channels. These data therefore set the status of neuronal Ca(V)2.1 Ca(2+) channel inhibition by RGK GTPases, emphasizing the role of short amino acid sequences of both proteins in beta-subunit binding and channel inhibition and revealing a new mechanism for channel inhibition.
RGK（Rad-Gem-Rem）GTP酶被描述为通过高阈值电压激活的Ca(2+)通道对Ca(2+)内流具有强大负调控作用的因子。最近的研究工作大多是在Ca(V)1.2 Ca(2+)通道上进行的，突出了通道辅助性Ca(V)β亚基所起的关键作用，并确定了参与这种调控的几个GTP酶和β亚基蛋白结构域。我们现在通过首次全面表征Gem、Rem和Rem2对与Ca(V)β(1)或Ca(V)β(2)亚基共同表达的神经元Ca(V)2.1 Ca(2+)通道的影响来扩展这些结论。电流抑制仅限于幅度降低，而电流的电压依赖性或动力学没有改变。我们证明，对于诱导电流增强能力受损的Ca(V)β构建体，这种抑制可能会发生，但对于其β相互作用结构域缺失的Ca(V)β构建体，这种抑制作用会丧失。RGK C末端最后大约80个氨基酸足以实现强大的电流抑制和体内β亚基/Gem相互作用。有趣的是，尽管Gem和Gem羧基末端诱导出完全不同的β亚基细胞定位模式，但它们都能有效抑制Ca(V)2.1通道。因此，这些数据确定了RGK GTP酶对神经元Ca(V)2.1 Ca(2+)通道的抑制状态，强调了两种蛋白质的短氨基酸序列在β亚基结合和通道抑制中的作用，并揭示了一种新的通道抑制机制。
