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

L型Ca^<2+>通道是Ca^<2+>信号调节的主要步骤。本课题旨在通过以下方法阐明L型Ca ^2+通道α_亚基（Ca_v1.2）门控调控的分子机制<1C>：1）L型Ca^2+通道α_亚基（Ca_v1.2）相关信号分子复合物的阐明<1C>：利用酵母双杂交筛选法从小鼠胚胎（E11.5）cDNA文库中筛选Ca_v1.2羧基端相关蛋白。我们发现Ca_v1.2的C端与磷脂酰胆碱转移蛋白样蛋白（PCTP-L/STARD 10）相互作用。北方印迹和西方印迹分析表明，PCTP-L在肝脏，肾脏，睾丸和胚胎心脏中表达，在成年啮齿动物心脏中，心房，而不是在心室。PCTP-L与Ca_v1.2在大鼠心房内共定位并共沉淀。当PCTP-L与Ca_v1.2共表达时，在BHK 6细胞中，PCTP-L促进了Ca_v1.2的表达。 ...更多信息 通过与Ca_v1.2的特异性相互作用改变失活机制来抑制Ca_v1.2。通过RNAi敲低PCTP-L可导致新生大鼠心房肌细胞动作电位平台期缩短和自发性Ca^2+瞬变频率增加。这些结果在一定程度上归因于PCTP-L减轻了L型Ca^&lt;2+&gt;通道的易化调节作用，从而下调了L型Ca^&lt;2+&gt;通道的活性。2）Ca_v1.2的Ca^&lt;2+&gt;选择性孔区周围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Ca_v1.2的孔形成IIIS 5-S6连接区的氨基酸拓扑，包括<1112><1115>对DHP结合至关重要的Phe^和Ser^。以MTSET阻断Ca^2+通道电流来评价亲水途径的半胱氨酸取代氨基酸的可及性。我们发现，<1112>在去极化条件下，Phe^暴露于亲水环境，而Ser^<1115>似乎被埋在疏水环境中，从而表明Phe^周围的电压依赖性构象变化<1112>可能是导致DHP与Ca^2+通道结合亲和力的电压依赖性变化的原因，这与我们先前的Ca_v1.2的DHP结合口袋的同源模型非常一致。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