Takahashi Shoji X, Miriyala Jayalakshmi, Tay Lai Hock, Yue David T, Colecraft Henry M
Calcium Signals Laboratory, Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
J Gen Physiol. 2005 Oct;126(4):365-77. doi: 10.1085/jgp.200509354.
Auxiliary Ca(2+) channel beta subunits (Ca(V)beta) regulate cellular Ca(2+) signaling by trafficking pore-forming alpha(1) 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 Ca(V)beta SH3-GK module regulates multiple Ca(2+) channel functions are not well understood. Here, using a split-domain approach, we investigated the role of the interrelationship between Ca(V)beta 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 Ca(V)beta 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 Ca(V)beta trafficking and gating properties: alpha(1C) 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 alpha(1C) (Ca(V)1.2) subunit. The results reveal that Ca(V)beta SH3 and GK domains function codependently to tune Ca(2+) channel trafficking and gating properties, and suggest new paradigms for physiological and therapeutic regulation of Ca(2+) channel activity.
辅助性钙通道β亚基（Ca(V)β）通过将形成孔道的α(1)亚基转运至细胞膜并使通道门控正常化来调节细胞内钙信号传导。这些效应是通过一个具有特征性的src同源3/鸟苷酸激酶（SH3-GK）结构模块介导的，该设计特征与支架蛋白膜相关鸟苷酸激酶（MAGUK）家族相同。然而，Ca(V)β SH3-GK模块调节多种钙通道功能的机制尚不清楚。在此，我们采用结构域拆分方法，研究了Ca(V)β SH3和GK结构域之间的相互关系在定义通道特性中的作用。这些研究基于先前鉴定的一对结构域拆分对，该对表现出反式SH3-GK相互作用，并能完全重建Ca(V)β对通道转运、激活门控和开放概率增加（P(o)）的影响。在此，通过改变用于分离SH3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SH3-GK相互作用，我们鉴定出一对特定的结构域拆分对，其反式SH3-GK相互作用的构型发生了细微改变。值得注意的是，这一对拆分对区分了Ca(V)β的转运和门控特性：α(1C)靶向细胞膜的功能得以完全重建，而激活门控的改变和P(o)功能的增加则选择性丧失。一个更极端的情况是，反式SH3-GK相互作用被选择性消除，产生了一对结构域拆分对，其既不能重建转运功能也不能重建门控调节功能，尽管两个部分都能独立与α(1C)（Ca(V)1.2）亚基上各自的结合位点结合。结果表明，Ca(V)β SH3和GK结构域相互依赖发挥作用，以调节钙通道的转运和门控特性，并为钙通道活性的生理和治疗调节提出了新的范例。
