Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels.
Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels.
复制标题
对电压依赖性钙通道的 RGK GTPase 抑制进行工程选择性。
DOI：
10.1073/pnas.1811024115
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发表时间：
2018
影响因子：
11.1
通讯作者：
Colecraft,HenryM
中科院分区：
文献类型：
--
作者：
Puckerin,AkilA;Chang,DonaldD;Shuja,Zunaira;Choudhury,Papiya;Scholz,Joachim;Colecraft,HenryM
电压依赖性 Ca2+(CaV) 通道的基因编码抑制剂 (GECCIs) 是有用的研究工具和潜在的治疗方法。 Rad/Rem/Rem2/Gem (RGK) 蛋白是 Ras 样 G 蛋白，可有效抑制高压激活 (HVA) Ca2+（CaV1/CaV2 家族）通道，但其非选择性限制了其潜在应用。我们假设 RGK 抑制的非选择性源自其与辅助 CaVβ 亚基的结合。为了研究潜在的 CaVβ 独立抑制成分，我们将每个 RGK 与在 HEK293 细胞中用野生型 (WT) β2a 或不结合 RGK 的突变版本 (β2a,TM) 重建的 CaV1 (CaV1.2/CaV1.3) 或 CaV2 (CaV2.1/CaV2.2) 通道单独共表达。所有四种 RGK 均强烈抑制用 WT β2a 重建的 CaV1/CaV2 通道。相比之下，当用β2a,TM重建通道时，Rem仅抑制CaV1.2，Rad选择性抑制CaV1.2和CaV2.2，而Gem和Rem2无效。我们生成了无法结合 WT CaVβ 的突变体 RGK（Rem[R200A/L227A] 和 Rad[R208A/L235A]），这通过荧光共振能量转移得到证实。 Rem[R200A/L227A]选择性阻断重组CaV1.2，而Rad[R208A/L235A]抑制CaV1.2/CaV2.2，但不抑制CaV1.3/CaV2.1。 Rem[R200A/L227A]和Rad[R208A/L235A]均抑制心室心肌细胞中的内源性CaV1.2通道，并分别选择性阻断背根神经节体感神经元中25%和62%的HVA电流，这与它们对CaV1.2和CaV1.2/CaV2.2通道的独特选择性相对应。因此，我们利用对特定 CaV1/CaV2 通道的潜在 β 结合独立的 Rem 和 Rad 抑制来开发选择性 GECCI，其特性是目前小分子 CaV 通道阻滞剂无法比拟的。
Genetically encoded inhibitors for voltage-dependent Ca2+(CaV) channels (GECCIs) are useful research tools and potential therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like G proteins that potently inhibit high voltage-activated (HVA) Ca2+(CaV1/CaV2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary CaVβ-subunits. To investigate latent CaVβ-independent components of inhibition, we coexpressed each RGK individually with CaV1 (CaV1.2/CaV1.3) or CaV2 (CaV2.1/CaV2.2) channels reconstituted in HEK293 cells with either wild-type (WT) β2aor a mutant version (β2a,TM) that does not bind RGKs. All four RGKs strongly inhibited CaV1/CaV2 channels reconstituted with WT β2a. By contrast, when channels were reconstituted with β2a,TM, Rem inhibited only CaV1.2, Rad selectively inhibited CaV1.2 and CaV2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT CaVβ, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted CaV1.2 while Rad[R208A/L235A] inhibited CaV1.2/CaV2.2 but not CaV1.3/CaV2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous CaV1.2 channels in ventricular cardiomyocytes and selectively blocked 25 and 62%, respectively, of HVA currents in somatosensory neurons of the dorsal root ganglion, corresponding to their distinctive selectivity for CaV1.2 and CaV1.2/CaV2.2 channels. Thus, we have exploited latent β-binding–independent Rem and Rad inhibition of specific CaV1/CaV2 channels to develop selective GECCIs with properties unmatched by current small-molecule CaVchannel blockers.