Molecular determinants of Ca2+/calmodulin-dependent regulation of Cav2.1 channels
Molecular determinants of Ca2+/calmodulin-dependent regulation of Cav2.1 channels
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DOI：
10.1073/pnas.2237000100
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发表时间：
2003-12-23
影响因子：
11.1
通讯作者：
Catterall, WA
中科院分区：
文献类型：
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作者：
Lee, A;Zhou, H;Catterall, WA
Ca（v）2.1通道的Ca 2+依赖性易化和失活（CDF和CDI）调节突触前P/Q型Ca 2+电流，并促进活动依赖性突触可塑性。这种由Ca 2+进行的双重反馈调节涉及钙调蛋白（CaM）与α（1）亚基（α（1）2.1）的结合。Ca（v）2.1通道的Ca 2+依赖性调节的分子决定簇存在于CaM和α（1）2.1的C端结构域中的两个CaM结合位点，即CaM结合结构域（CBD）和IQ样结构域。在转染的tsA-201细胞中，CDF和CDI均通过CBD的缺失而降低。相比之下，IQ样结构域的前两个残基的丙氨酸取代（IM-AA）完全阻止CDF，但对CDI的影响不大，谷氨酸取代（IM-EE）大大加速电压依赖性失活，但不能阻止CDI。Ca 2+结合位点的CaM的突变分析表明，CaM的N-和C-末端叶所需的充分发展的促进，但只有N-末端叶是必不可少的CDI。在生化测定中，CaM 12和CaM 34不能结合CBD，而CaM 34而不是CaM 12，保留了与IQ样结构域的Ca 2+依赖性结合。这些发现支持了一个模型，其中Ca 2+结合到C-末端EF-手preassociated钙调素启动CDF通过与IQ样结构域的相互作用。进一步的Ca 2+结合到N-末端EF-手促进二级CaM与CBD的相互作用，这增强了促进作用并引起启动CDI的构象变化。这种多方面的机制允许响应于局部Ca 2+增加（CDF）的Ca 2+的正调节和在更全局的Ca 2+增加（CDI）期间的负调节。
Ca2+-dependent facilitation and inactivation (CDF and CDI) of Ca(v)2.1 channels modulate presynaptic P/Q-type Ca2+ currents and contribute to activity-dependent synaptic plasticity. This dual feedback regulation by Ca2+ involves calmodulin (CaM) binding to the alpha(1) subunit (alpha(1)2.1). The molecular determinants for Ca2+-dependent modulation of Ca(v)2.1 channels reside in CaM and in two CaM-binding sites in the C-terminal domain of alpha(1)2.1, the CaM-binding domain (CBD) and the IQ-like domain. In transfected tsA-201 cells, CDF and CDI were both reduced by deletion of CBD. In contrast, alanine substitution of the first two residues of the IQ-like domain (IM-AA) completely prevented CDF but had little effect on CDI, and glutamate substitutions (IM-EE) greatly accelerated voltage-dependent inactivation but did not prevent CDI. Mutational analyses of the Ca2+ binding sites of CaM showed that both the N- and C-terminal lobes of CaM were required for full development of facilitation, but only the N-terminal lobe was essential for CDI. In biochemical assays, CaM12 and CaM34 were unable to bind CBD, whereas CaM34 but not CaM12, retained Ca2+-dependent binding to the IQ-like domain. These findings support a model in which Ca2+ binding to the C-terminal EF-hands of preassociated CaM initiates CDF via interaction with the IQ-like domain. Further Ca2+ binding to the N-terminal EF-hands promotes secondary CaM interactions with CBD, which enhance facilitation and cause a conformational change that initiates CDI. This multifaceted mechanism allows positive regulation of Ca2+ in response to local Ca2+ increases (CDF) and negative regulation during more global Ca2+ increases (CDI).