Caveolae-Dependent Regulation of Cardiac Ion Channels

心脏离子通道的小凹依赖性调节

• 批准号: 6712202
• 负责人: ERWIN F SHIBATA
• 金额: $ 36.88万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-15 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6712202
• 关键词: G protein; antiantibody; calcium channel; caveolas; immunocytochemistry; immunoelectron microscopy; immunofluorescence technique; immunoprecipitation; membrane fusion; membrane proteins; protein isoforms; protein localization; protein protein interaction; protein structure function; sodium channel; tetanus toxin; voltage /patch clamp; western blottings

DESCRIPTION (provided by applicant): In addition to the phosphorylation-dependent effects on ion channels, G-proteins have an additional "direct" effect on the regulation of cardiac Na+ channels. The non-phosphorylation-dependent Gas-mediated increase of peak/Na is not due to a change in activation, inactivation, recovery from inactivation or a change in single channel amplitude. These results suggest that the number of functional Na+ channels have increased in the membrane. We showed that the new channels are in caveolae (Yarbrough et al., 2002). Na+ channels within the caveolae membrane become functional when the caveolae "neck" fuses with the plasma membrane and opens to establish electrical continuity between the extracellular space and the intracaveolae compartment. This application focuses on determining the co-localization of Na+ and Ca2+ channels in caveolae and the role of Gas in the regulation of caveolae. Specifically, we propose to address the following questions: 1) Are Na+ and Ca 2+ channels sorted to the same or different caveolae? Yarbrough et al., (2002) showed that Na+ channels are found in caveolae membranes. In addition, our preliminary data show that the anti-a1C antibody recognizes a protein signal in the caveolae-rich fraction of rat ventricular myocytes. We hypothesize that Na+ and Ca2+ channels are sorted to the same caveolae. We will investigate this hypothesis using immunoprecipitation, Western blot analysis, immuno-fluorescence, immuno-electron microscopy techniques, and direct patch clamp recordings using the tip-dip method. 2) What is the functional role of the N-terminus of Gas in the regulation of caveolae? Lu et al., (1999) showed that Gas could enhance the size of Na+ current in a cAMP-independent fashion. Our data also show that in yeast two-hybrid screens and GST fusion studies, the N- and C-terminals, all of the intracellular segment loops, and interdomain loops of the rat cardiac Na+ channel, Galphas does not interact directly with the channel nor does Gby. However, a short N-terminal peptide of Galphas (a.a. 27-42) can mimic the effects of increasing the Na+ current. We hypothesize that the N-terminal of Galphas plays an important role in the regulation of caveolar opening. Using the Na+ current as our assay, we will examine the functional effects of the N-terminal of Gas using Galphas/Galphat and Galphas/Galphai chimeras and short N-terminal Galphas oligomers. We will also probe for the substrate that Galphas is interacting with to regulate the opening of caveolae. 3) Caveolae are dynamic omega-shaped structures whose membrane fusion and fission mechanisms are virtually unknown. This specific aim will test the involvement of membrane-associated proteins in caveolar docking and/or fusion events. We will also probe for the substrate that Gas is interacting with to regulate the opening and closing of caveolae necks. Caveolae in endothelial cells have been shown to contain key proteins known to mediate vesicle formation, docking, and/or fusion. We will test for the involvement of toxin-sensitive synaptobrevin (VAMP, Vesicle-Associated Membrane Protein) in cardiac ventricular caveolar docking and/or fusion using antibodies recognizing members of the VAMP family and by the neurotoxin, tetanus toxin, which proteolytically cleave VAMP proteins. We will assay changes in the Na+ current. The localization of VAMP on caveolar structures will be tested using immunohistochemical approaches with antibodies to VAMP and Caveolin-3. Co-localization of VAMP with Cav-3 will be important to identify the omega -shaped membrane structure as a caveolae rather than another kind of vesicle.

描述（由申请人提供）：除了对离子通道的磷酸化依赖性作用之外，G蛋白对心脏Na+通道的调节还具有额外的“直接”作用。非磷酸化依赖性气体介导的峰/Na 的增加不是由于激活、失活、从失活恢复或单通道振幅的变化引起的。这些结果表明膜中功能性 Na+ 通道的数量有所增加。我们表明新通道位于小凹中（Yarbrough et al., 2002）。当小窝“颈部”与质膜融合并打开以在细胞外空间和小窝内室之间建立电连续性时，小窝膜内的 Na+ 通道开始发挥作用。本应用的重点是确定小凹中 Na+ 和 Ca2+ 通道的共定位以及气体在小凹调节中的作用。具体来说，我们建议解决以下问题： 1) Na+ 和Ca 2+ 通道是否被分类到相同或不同的小凹？ Yarbrough 等人 (2002) 表明，在小凹膜中发现了 Na+ 通道。此外，我们的初步数据表明，抗 a1C 抗体可识别大鼠心室肌细胞富含小凹部分的蛋白质信号。我们假设 Na+ 和 Ca2+ 通道被分类到相同的小凹。我们将使用免疫沉淀、蛋白质印迹分析、免疫荧光、免疫电子显微镜技术以及使用尖端浸入法的直接膜片钳记录来研究这一假设。 2）Gas N 末端在小凹调节中的功能作用是什么？ Lu 等人 (1999) 表明，Gas 可以以不依赖于 cAMP 的方式增强 Na+ 电流的大小。我们的数据还表明，在酵母双杂交筛选和 GST 融合研究中，大鼠心脏 Na+ 通道的 N 端和 C 端、所有细胞内节段环和域间环，Galphas 不直接与通道相互作用，Gby 也不与通道相互作用。然而，Galphas 的短 N 端肽（a.a. 27-42）可以模拟增加 Na+ 电流的效果。我们假设 Galphas 的 N 末端在小凹开口的调节中发挥重要作用。使用 Na+ 电流作为我们的测定，我们将使用 Galphas/Galphat 和 Galphas/Galphai 嵌合体以及短 N 端 Galphas 寡聚物来检查 Gas N 端的功能效应。我们还将探究 Galphas 与之相互作用以调节小凹开口的底物。 3）小凹是动态的欧米茄形结构，其膜融合和裂变机制实际上是未知的。这一具体目标将测试膜相关蛋白在小凹对接和/或融合事件中的参与。我们还将探测与气体相互作用的基质，以调节小窝颈部的打开和关闭。内皮细胞中的小凹已被证明含有已知介导囊泡形成、对接和/或融合的关键蛋白质。我们将使用识别 VAMP 家族成员的抗体以及通过蛋白水解裂解 VAMP 蛋白的神经毒素、破伤风毒素来测试毒素敏感的突触短蛋白（VAMP，囊泡相关膜蛋白）在心室小窝对接和/或融合中的参与情况。我们将测定 Na+ 电流的变化。 VAMP 在小凹结构上的定位将使用 VAMP 和 Caveolin-3 抗体的免疫组织化学方法进行测试。 VAMP 与 Cav-3 的共定位对于将 omega 形膜结构识别为小凹而不是另一种囊泡非常重要。