Molecular mechanism underlying the gating regulation of Ca^<2+> permeable channel via Ca^<2+> complex

L型钙通道是钙信号调节的第一步。本课题旨在通过以下途径阐明L钙通道α_&lt；2+&gt；通道α_&lt；1C&gt；亚单位(Ca_v1.2)门控调控的分子机制：1)阐明与L钙通道亚单位(Ca_v1.2)相关的信号分子复合体：采用酵母双杂交筛选方法，从小鼠胚胎(E11.5)的cDNA文库中筛选出与Ca_v1.2羧基末端相关的蛋白质。我们发现CaV1.2的C末端与磷脂酰胆碱转运蛋白样蛋白(PCTP-L/STARD10)相互作用。Northern印迹和Western印迹分析表明，PCTP-L在肝脏、肾脏、睾丸和胚胎心脏中均有表达，在成年啮齿动物心脏中有表达，在心房中表达，但在心室中不表达。PCTP-L与Ca_v1.2在大鼠心房内共定位并免疫共沉淀。当PCTP-L与Ca_v1.2在BHK6细胞中共表达时，PCTP-L促进了…通过与Ca_v1.2的特异性相互作用，改变失活机制，进一步激活Ca_v1.2。RNA干扰抑制心肌细胞PCTP-L后，动作电位平台期缩短，自发性钙瞬变频率增加。这些结果部分归因于PCTP对钙通道的易化调节作用，导致L型钙通道活性下调。2)钙通道选择性孔区氨基酸结构拓扑的功能分析：我们用取代半胱氨酸可及性方法分析了钙通道成孔IIIS5-S6连接区氨基酸的拓扑结构。MTSET将亲水途径中的半胱氨酸取代氨基酸的可及性作为钙通道电流的阻滞物。我们发现，Phe^&lt；1112&gt；在去极化条件下暴露在亲水环境中，而Ser^&lt；1115&gt；似乎被埋在疏水环境中，从而表明Phe^&lt；1112&gt；周围的电压依赖构象变化可能是DHP与Ca^&lt；2+&gt；通道结合亲和力随电压变化的原因，这与我们之前对Ca_v1.2的DHP结合口袋的同源模型很好地一致。较少

L-type Ca^<2+> channel serves as a primary step in the regulation of Ca^<2+> signaling. In this project, we aimed at clarifying the molecular mechanism underlying the gating regulation of L-type Ca^<2+> channel α_<1C> subunit (Ca_v1.2) through the following approaches :1)Clarification of signaling molecular complex associated with L-type Ca^<2+> channel α_<1C> subunit (Ca_v1.2) :We screened proteins associated with the carboxyl terminus of Ca_v1.2 in the cDNA library of mouse embryo (E11.5) with yeast two-hybrid screening method. We found that the C-terminus of Ca_v1.2 interacts with phosphatidylcholine transfer protein-like protein (PCTP-L/STARD10). Northern blot and Western blot analysis revealed that PCTP-L was expressed in liver, kidney, and testis as well as in embryonic heart and, in adult rodent hearts, in atria but not in ventricle. PCTP-L colocalized and was coimmunoprecipitated with Ca_v1.2 in rat atria. When PCTP-L was coexpressed with Ca_v1.2, in BHK6 cells, PCTP-L facilita … More ted Ca_v1.2 via the alteration of the inactivation mechanism through the specific interaction with Ca_v1.2. The knockdown of PCTP-L in neonatal atrial myocytes by RNAi resulted in the shortening of the plateau phase of action potentials and an increase in the frequency of spontaneous Ca^<2+> transients. These results are attributed, in a part, to the down-regulation of the activity of the L-type Ca^<2+> channel as a result of the relief of the facilitatory modulation by PCTP-L.2)Functional analysis of structural topology of amino acids around Ca^<2+> selective pore region of Ca_v1.2 :We analyzed the topology of amino acids in the pore forming IIIS5-S6 linker region of Ca_v1.2, including Phe^<1112> and Ser^<1115> that are critical for DHP binding, with substituted cysteine accessibility method. The accessibility of cysteine-substituted amino acids from hydrophilic pathway was assed as the block of Ca^<2+> channel current by MTSET. We found that Phe^<1112> is exposed to hydrophilic environment at depolarized condition, while Ser^<1115> appears to be buried in hydrophobic environment, thus suggesting that the voltage-dependent conformational change around Phe^<1112> may be responsible for the voltage-dependent change of the binding affinity of DHP to the Ca^<2+> channel, which is in good agreement with our previous homology model of the DHP binding pocket of Ca_v1.2. Less