Similar molecular determinants on Rem mediate two distinct modes of inhibition of CaV1.2 channels.
Similar molecular determinants on Rem mediate two distinct modes of inhibition of CaV1.2 channels.
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Rem 上类似的分子决定簇介导 CaV1.2 通道的两种不同的抑制模式。
DOI：
10.1080/19336950.2016.1180489
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Colecraft,HenryM
中科院分区：
文献类型：
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作者：
Puckerin,AkilA;Chang,DonaldD;Subramanyam,Prakash;Colecraft,HenryM
Rad/Rem/Rem 2/Gem（RGK）蛋白是Ras样GTP酶，其有效地抑制所有高电压门控钙（CaV 1/CaV 2）通道，并且因此处于良好位置以调节多种生理过程。了解RGK蛋白如何抑制CaV通道对于了解其（病理）生理作用非常重要，并且可以促进其作为遗传编码CaV通道阻断剂的开发和使用。我们先前报道了Rem可以通过两种独立的方式阻断表面CaV1.2通道，这些方式与通道复合物的不同组分结合：（1）通过结合辅助β亚基（β结合依赖性抑制，或BBD）;（2）通过结合成孔α 1C亚基N端（α 1C结合依赖性抑制，或ABD）。相比之下，Gem只使用BBD机制来阻止CaV1.2。BBD CaV1.2抑制所需的Rem分子决定簇是远端C-末端和鸟嘌呤核苷酸结合G-结构域，它们分别与质膜和CaVβ相互作用。然而，ABD CaV1.2抑制的Rem决定因素是未知的。在这里，结合荧光共振能量转移，电生理学，系统截断，和Rem/Gem嵌合体，我们发现，相同的Rem远端C-末端和G-结构域也介导ABD CaV1.2抑制，但与不同的相互作用伙伴。Rem远端C-末端与α 1 CN-末端相互作用以锚G-结构域，其可能与尚未鉴定的位点相互作用。与以前的一些研究相反，Rem和Gem的C-末端都不足以抑制CaV 1/CaV 2通道。结果表明，Rem上相似的分子决定簇被重新利用以启动CaV1.2抑制的2种独立机制。
Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like GTPases that potently inhibit all high-voltage-gated calcium (CaV1/CaV2) channels and are, thus, well-positioned to tune diverse physiological processes. Understanding how RGK proteins inhibit CaVchannels is important for perspectives on their (patho)physiological roles and could advance their development and use as genetically-encoded CaVchannel blockers. We previously reported that Rem can block surface CaV1.2 channels in 2 independent ways that engage distinct components of the channel complex: (1) by binding auxiliary β subunits (β-binding-dependent inhibition, or BBD); and (2) by binding the pore-forming α1Csubunit N-terminus (α1C-binding-dependent inhibition, or ABD). By contrast, Gem uses only the BBD mechanism to block CaV1.2. Rem molecular determinants required for BBD CaV1.2 inhibition are the distal C-terminus and the guanine nucleotide binding G-domain which interact with the plasma membrane and CaVβ, respectively. However, Rem determinants for ABD CaV1.2 inhibition are unknown. Here, combining fluorescence resonance energy transfer, electrophysiology, systematic truncations, and Rem/Gem chimeras we found that the same Rem distal C-terminus and G-domain also mediate ABD CaV1.2 inhibition, but with different interaction partners. Rem distal C-terminus interacts with α1CN-terminus to anchor the G-domain which likely interacts with an as-yet-unidentified site. In contrast to some previous studies, neither the C-terminus of Rem nor Gem was sufficient to inhibit CaV1/CaV2 channels. The results reveal that similar molecular determinants on Rem are repurposed to initiate 2 independent mechanisms of CaV1.2 inhibition.