Electrophysiology of nuclear membrane INSP3 receptor

描述（由申请人提供）：本提案的总体目标是表征涉及肌醇三磷酸受体（InsP3R） Ca2+释放通道的新型蛋白质相互作用。InsP3R参与产生复杂的Ca2+信号，调节许多生理过程。InsP3R与细胞组分之间的相互作用可以提供将Ca2+释放偶联到特定靶点的有效手段，或调节通道的Ca2+释放特性。最近，我们发现了一种将InsP3R与程序性细胞死亡直接联系起来的新机制。我们确定了BC！- xl， Bcl-2家族促生存蛋白和抗凋亡蛋白的成员，作为与InsP3R的相互作用。Bel-XL结合使通道门控对存在于未刺激细胞中的极低的InsP3浓度敏感，减少内质网（ER）中的Ca2+并增强Ca2+信号传导。这种相互作用极大地增强了细胞抵抗凋亡损伤的能力，从而在一种新的范式中确定了将ER、InsP3R和Bcl-2蛋白与细胞存活联系起来的分子机制。由于细胞凋亡在正常生理和病理生理中是由Bcl-2蛋白调控的，我们对Bcl-2蛋白的新生理靶点的鉴定可能为干预包括癌症、神经退行性疾病和心血管疾病在内的与细胞凋亡有关的人类疾病提供新的机会。我们提出了三个目标来表征InsP3R和Bcl-2蛋白之间相互作用的机制和功能。首先，我们将结合生物化学、细胞成像、天然内质网膜中重组InsP3R的单通道记录和生理分析来确定Bcl-Xt调节通道的机制。其次，我们将利用诱变、肽竞争和生物物理方法确定相互作用的结构基础。我们还将通过核磁共振光谱直接研究InsP3R/Bcl-2配合物的溶液结构。第三，我们将确定促生存Bcl-2蛋白与InsP3R相互作用的生理相关性。首先，我们将通过测试线粒体生物能量学是主要传感器的假设，确定这种相互作用赋予细胞凋亡抗性的机制。其次，我们将确定相互作用的破坏是否会影响体内的细胞凋亡抗性。这些研究的结果应该为调控细胞凋亡的分子机制提供重要的见解，并为人类疾病治疗干预的新靶点的发展提供重要的见解。

DESCRIPTION (provided by applicant): The broad goal of this proposal is to characterize novel protein interactions involving the inositol trisphosphate receptor (InsP3R) Ca2+ release channel. The InsP3R participates in generation of complex Ca2+ signals that regulate many physiological processes. Interactions between the InsP3R and cellular components could provide effective means to couple Ca2+ release to specific targets, or to regulate Ca2+ release properties of the channel. Recently, we identified a novel mechanism that directly links the InsP3R to programmed cell death. We identified BC!-XL, a pro-survival member of the Bcl-2 family of pro- and anti-apoptotic proteins, as an interactor with the InsP3R. Bel-XL binding sensitizes channel gating to extremely low InsP3 concentrations that exist in unstimulated cells, reducing Ca2+ in the endoplasmic reticulum (ER) and enhancing Ca2+ signaling. The interaction profoundly enhances the ability of cells to withstand apoptotic insults, thereby identifying a molecular mechanism that links the ER, InsP3R and Bcl-2 proteins to cell survival in a novel paradigm. Because apoptosis is orchestrated by Bcl-2 proteins in normal physiology and pathophysiology, our identification of a novel physiological target of Bcl-2 proteins may provide new opportunities for interventions in human diseases involving apoptosis, including cancer, neurodegeneration and cardiovascular disease. We propose three aims to characterize the mechanisms and function of the interaction between the InsP3R and Bcl-2 proteins. First, we will define the mechanisms of Bcl-Xt regulation of the channel using a combination of biochemistry, cell imaging, single channel recording of recombinant InsP3R in native ER membranes, and physiological assays. Second, we will determine the structural bases for the interaction, using mutagenesis, peptide competition and biophysical approaches. We will also examine directly the solution structure of the InsP3R/Bcl-2 complex by NMR spectroscopy. Third, we will determine the physiological relevance of the interaction of pro-survival Bcl-2 proteins with the InsP3R. First, we will identify the mechanisms whereby this interaction confers apoptosis resistance, by testing the hypothesis that mitochondrial bioenergetics is a primary sensor. Second, we will determine if disruption of the interaction affects apoptosis resistance in vivo. The results of these studies should provide important insights into the molecular mechanisms that regulate apoptosis, and into the development of novel targets for therapeutic interventions in human diseases.