Electrophysiology of nuclear membrane INSP3 receptor

描述（由申请人提供）：本提案的主要目标是表征涉及三磷酸肌醇受体（InsP 3R）Ca 2+释放通道的新型蛋白质相互作用。InsP 3R参与产生复杂的Ca 2+信号，调节许多生理过程。InsP 3R与细胞组分之间的相互作用可以提供将Ca 2+释放耦合到特定靶点或调节通道的Ca 2+释放特性的有效手段。最近，我们发现了一种新的机制，直接将InsP 3R与程序性细胞死亡联系起来。我们确认了BC！- XL，促凋亡和抗凋亡蛋白Bcl-2家族的促存活成员，作为与InsP 3R的相互作用物。Bel-XL结合使通道门控对存在于未刺激细胞中的极低InsP 3浓度敏感，从而减少内质网（ER）中的Ca 2+并增强Ca 2+信号传导。这种相互作用深刻地增强了细胞承受凋亡损伤的能力，从而确定了一种分子机制，该机制将ER，InsP 3R和Bcl-2蛋白与细胞存活联系在一起。由于细胞凋亡是由Bcl-2蛋白在正常的生理和病理生理，我们的识别Bcl-2蛋白的一个新的生理目标可能会提供新的机会，在人类疾病的干预涉及细胞凋亡，包括癌症，神经变性和心血管疾病。我们提出了三个目标来表征InsP 3R和Bcl-2蛋白之间相互作用的机制和功能。首先，我们将使用生物化学、细胞成像、天然ER膜中重组InsP 3R的单通道记录和生理测定的组合来定义Bcl-Xt通道调节的机制。其次，我们将确定的相互作用的结构基础，使用诱变，肽竞争和生物物理方法。我们还将通过NMR光谱直接检查InsP 3R/Bcl-2复合物的溶液结构。第三，我们将确定促生存Bcl-2蛋白与InsP 3R相互作用的生理相关性。首先，我们将确定这种相互作用赋予细胞凋亡抗性的机制，通过测试的假设，线粒体生物能量学是一个主要的传感器。第二，我们将确定是否破坏的相互作用影响细胞凋亡的阻力在体内。这些研究的结果应该提供重要的见解，调节细胞凋亡的分子机制，并在人类疾病的治疗干预的新目标的发展。

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.