A CaVbeta SH3/guanylate kinase domain interaction regulates multiple properties of voltage-gated Ca2+ channels.
A CaVbeta SH3/guanylate kinase domain interaction regulates multiple properties of voltage-gated Ca2+ channels.
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DOI：
10.1085/jgp.200509354
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发表时间：
2005-10
期刊：
影响因子：
--
通讯作者：
Colecraft HM
中科院分区：
文献类型：
--
作者：
Takahashi SX;Miriyala J;Tay LH;Yue DT;Colecraft HM
辅助钙通道β亚基（CaVβ）通过将成孔α1亚基运输至细胞膜并使通道门控正常化来调节细胞内钙信号。这些作用通过特征性src同源性3/鸟苷酸激酶（SH 3-GK）结构模块介导，该结构模块是与支架蛋白的膜相关鸟苷酸激酶（MAGUK）家族共有的设计特征。然而，CaVβ SH 3-GK模块调节多种Ca 2+通道功能的机制尚不清楚。在这里，使用分裂域方法，我们研究了CaVβ SH 3和GK结构域之间的相互关系在定义通道特性中的作用。这些研究建立在先前鉴定的显示反式SH 3-GK相互作用的分裂结构域对的基础上，并完全重建了CaVβ对通道运输、激活门控和增加开放概率（Po）的作用。在这里，通过改变用于分离SH 3和GK结构域的精确位置，并通过荧光共振能量转移（FRET）监测随后的SH 3-GK相互作用，我们确定了一个特定的分裂结构域对，其显示了反式SH 3-GK相互作用的微妙改变的构型。值得注意的是，这对区分CaVβ运输和门控特性：靶向膜的α1C完全重建，而激活门控和增加的PO功能的变化选择性丢失。在一个更极端的情况下，反式SH 3-GK相互作用被选择性地消除，产生了一个分裂结构域对，它既不能重建运输功能，也不能重建门控调节功能，尽管这两个部分都可以独立地与α1C（CaV 1.2）亚基上各自的结合位点结合。结果表明，CaVβ SH 3和GK结构域在调节Ca 2+通道的转运和门控特性方面具有协同作用，为Ca 2+通道活性的生理和治疗调控提供了新的范例。
Auxiliary Ca2+ channel β subunits (CaVβ) regulate cellular Ca2+ signaling by trafficking pore-forming α1 subunits to the membrane and normalizing channel gating. These effects are mediated through a characteristic src homology 3/guanylate kinase (SH3–GK) structural module, a design feature shared in common with the membrane-associated guanylate kinase (MAGUK) family of scaffold proteins. However, the mechanisms by which the CaVβ SH3–GK module regulates multiple Ca2+ channel functions are not well understood. Here, using a split-domain approach, we investigated the role of the interrelationship between CaVβ SH3 and GK domains in defining channel properties. The studies build upon a previously identified split-domain pair that displays a trans SH3–GK interaction, and fully reconstitutes CaVβ effects on channel trafficking, activation gating, and increased open probability (P o). Here, by varying the precise locations used to separate SH3 and GK domains and monitoring subsequent SH3–GK interactions by fluorescence resonance energy transfer (FRET), we identified a particular split-domain pair that displayed a subtly altered configuration of the trans SH3–GK interaction. Remarkably, this pair discriminated between CaVβ trafficking and gating properties: α1C targeting to the membrane was fully reconstituted, whereas shifts in activation gating and increased P o functions were selectively lost. A more extreme case, in which the trans SH3–GK interaction was selectively ablated, yielded a split-domain pair that could reconstitute neither the trafficking nor gating-modulation functions, even though both moieties could independently engage their respective binding sites on the α1C (CaV1.2) subunit. The results reveal that CaVβ SH3 and GK domains function codependently to tune Ca2+ channel trafficking and gating properties, and suggest new paradigms for physiological and therapeutic regulation of Ca2+ channel activity.
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