Calmodulin的N环和C环与心肌CaV1.2钙通道的多个结合位点交互作用介导其Ca2+依赖性失活的机制研究

The activity of the CaV1.2 channel is limited by the increased Ca2+, so called Ca2+-dependent inactivation (CDI), which is thought as a physiological safety mechanism against a harmful Ca2+ overload in the cell, notably during large Ca2+ currents through the channels. It is believed that CDI is mediated by calmodulin (CaM) through its binding to the channel. The CaM binding sites on CaV1.2 channels include an isoleucine-glutamine motif (referred as IQ motif), a preIQ motif and a CaM-binding motif in the N-terminal tail (NT). Structurally a CaM has two lobes, each of which contains two EF-hand conformational Ca2+-binding sites. The two Ca2+-binding sites of the C-terminal lobe (C-lobe) have higher affinity for Ca2+ than those of the N-terminal lobe (N-lobe). We previously studied the regulatory mechanism of the basal activity of L-type Ca2+ channel by cytoplasmic factors, and we found that CaM may recover the channel activity after its run-down in cell-free patches at resting Ca2+ concentration ([Ca2+]) level. From the view of this point, we studied the CDI process with a wide range of [Ca2+] starting from the resting level of [Ca2+], and we obtained some interesting results. The results showed that at resting [Ca2+] level all the three CaM binding sites of CaV1.2 channel can bind the N-lobe of CaM. With the increase of [Ca2+], the affinity of the bindings between the two lobes and the three CaM binding sites change, and at 2 mM Ca2+, both N-lobe and C-lobe can bind to the three CaM binding sites. Based on these findings, we proposed a new model in which multiple CaM molecules might link the three main CaM binding sites on CaV1.2 channel with their N-lobes and C-lobes, which are NT-CaM-preIQ, NT-CaM-IQ and preIQ-CaM-IQ. These cross-linking relationships are formed step by step to strengthen CDI, and the present project intends to clarify the model we proposed. By using molecular biological, biochemical and electrophysiological techniques, we will construct CaM binding site mutated channel, N-lobe and C-lobe of CaM, and Ca2+ binding site mutated CaM. By comparing the changes of CDI process, we will try to elucidate the dynamic processes of the formation of the cross-linking interactions and their Ca2+-dependences. Finally, we will make CaM binding site mutated knock-in mice to observe the changes of CDI and its relationship with ischemia-reperfusion injury degree. The aim of the project is to verify the hypothesis that CaM can form multiple cross-linking conformations among the CaM binding sites of CaV1.2 channel, and then to understand the molecular mechanism of CDI and its pathophysiological significance. This project is also of significance for searching new targets of bio-inspired Ca2+ channel antagonists.

心肌CaV1.2钙通道的钙依赖性失活（CDI）是限制较大钙内流诱发钙超载的生理保护机制。Calmodulin（钙调蛋白，CaM）两端的N-lobe和C-lobe上有钙结合位点，CaM与钙通道结合介导了CDI。申请人基于钙通道基本活性维持机制研究的背景，从静息钙水平开始研究高钙诱导的CDI。结果发现静息钙水平下CaV1.2三个主要CaM结合位点上均结合CaM的N-lobe；钙升高后N-、C-lobe与CaM结合位点的结合呈不同的钙依赖性增加，由此提出了由CaM的N-lobe与静息通道结合起始的、多个CaM分子参与的伴有多重交联结构形成的CDI新机制模型。本研究拟制备CaM结合位点突变钙通道和钙结合位点突变CaM，观察CDI发生与通道CaM结合状态变化间的关系，验证所提机制模型；并制备通道CaM结合位点突变小鼠，观察CDI在预防心肌缺血再灌注损伤中的作用，为生物钙通道拮抗剂新药研发提供新靶点。

心肌 CaV1.2 钙通道的钙依赖性失活（CDI）是心肌细胞限制较大钙内流诱发钙超载的生理保护机制。Calmodulin（钙调蛋白，CaM）两端的 N-lobe 和 C-lobe 上各有两个钙结合位点，CaM通过与钙通道结合介导了 CDI。本项目的通过观察CaM与钙通道的相互作用，探讨了CDI的发生机制。研究制备了CaM结合位点突变钙通道和钙结合位点突变CaM，观察了CDI发生与通道CaM结合状态变化间的关系；观察了诱发长QT综合征的CaM突变体与钙通道结合的变化；采用大鼠心肌肥厚模型，观察CDI在心肌肥厚发生中的作用；同时基于CaM对钙通道调节作用，设计了以钙通道为靶点的新型多肽药物Athycaltide-1，并观察了其抗心肌肥厚的作用。结果表明，单独的N-lobe和C-lobe可以触发浓度和钙依赖性易化作用，但不能触发浓度和钙依赖失活作用，提示CaM可能以一个lobe结合到通道上就足以维持通道的基本活性，但失活作用可能需要N-lobe和C-lobe的同时参与。与野生型CaM相比，长QT突变体CaME141G与的结合量明显减少，提示该种突变可能通过减弱CDI导致QT间期延长。Athycaltide-1具有抗心肌肥厚作用，拟进行进一步的研发。课题探讨了遗传性长QT综合征的发病机制，设计并观察了用于治疗心肌肥厚的新型多肽药物，有重要的理论和实际意义。研究发表论文37篇，SCI论文15篇，中文论文22篇，学术会议交流：13次，申请发明专利2项。组织国际会议1项，开展大学生创新计划项目研究11项，继续教育项目1项。