ANALYSIS OF A KINASE DOMAIN WITHIN A NOVEL ION CHANNEL

新型离子通道内激酶结构域的分析

• 批准号: 6434732
• 负责人: MONICA Jane NADLER
• 金额: $ 11.95万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-15 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6434732
• 关键词: SDS polyacrylamide gel electrophoresis; calcium channel; cell line; densitometry; enzyme activity; immunoprecipitation; intracellular; mathematical model; membrane channels; phosphorylation; protein kinase; protein structure function; site directed mutagenesis; voltage /patch clamp

Little is known of calcium channels underlying flux in lymphocytes and other non-excitable cells. I have recently cloned a novel ion channel, designated LTRPC7, that is expressed in many types of non-excitable cells including all immune system tissues. This channel is a member of the LTRPC TRP sub-family and is unique in that it contains a catalytic kinase domain homology to eEF-2K at its C-terminus. Preliminary data indicate that this protein is bi-functional: it is a cation channel that is permeable to calcium and magnesium, and it has kinase activity. We have observed that the ion permeability of LTRPC7 is sensitive to and suppressed by Mg-ATP. These data suggest that the kinase activity and/or changes in conformation of the kinase domain upon phosphorylation could be critical to the gating and/or modulation of this channel. Experiments are proposed to fully characterize the enzymatic potential of this domain (Aim 1). In addition, experiments are proposed to explore the regions of LTRPC7 that mediate and influence the Mg- ATP suppression effect (Aim 2). Finally, preliminary functional characterization of LTRPC7 demonstrates that over-expression of it in HEK293 cells is toxic and/or induces growth arrest. In addition, production of a double allele LTRPC7 knockout cell line in the DT-40 B-cell system results in lethality. These results suggest that LTRPC7 function is linked to basic cellular processes required for cell survival. Experiments to address the unique structural features of LTRPC7 that could account for these functional effects and assessment of its ability to affect intracellular calcium are also proposed (Aim 3).

对于淋巴细胞和其他不可兴奋细胞中流动的钙通道知之甚少。我最近克隆了一种新型离子通道，命名为 LTRPC7，它在多种类型的非兴奋性细胞（包括所有免疫系统组织）中表达。该通道是 LTRPC TRP 亚家族的成员，其独特之处在于其 C 末端包含与 eEF-2K 同源的催化激酶结构域。初步数据表明，这种蛋白质具有双功能：它是一种可渗透钙和镁的阳离子通道，并且具有激酶活性。我们观察到 LTRPC7 的离子渗透性对 Mg-ATP 敏感并受 Mg-ATP 抑制。这些数据表明磷酸化时激酶活性和/或激酶结构域构象的变化可能对该通道的门控和/或调节至关重要。建议通过实验来充分表征该结构域的酶促潜力（目标 1）。此外，还建议进行实验来探索 LTRPC7 介导和影响 Mg-ATP 抑制作用的区域（目标 2）。最后，LTRPC7 的初步功能表征表明，其在 HEK293 细胞中的过度表达具有毒性和/或诱导生长停滞。此外，在 DT-40 B 细胞系统中产生双等位基因 LTRPC7 敲除细胞系会导致致死性。这些结果表明 LTRPC7 功能与细胞生存所需的基本细胞过程相关。还提出了解决 LTRPC7 独特结构特征的实验，这些特征可以解释这些功能效应并评估其影响细胞内钙的能力（目标 3）。