Caveolae-Dependent Regulation of Cardiac Ion Channels

心脏离子通道的小凹依赖性调节

• 批准号: 6835682
• 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/6835682
• 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（由申请方提供）：除了对离子通道的磷酸化依赖性作用外，G蛋白对心脏Na+通道的调节具有额外的“直接”作用。非磷酸化依赖性Gas介导的峰/Na增加不是由于激活、失活、从失活恢复的变化或单通道振幅的变化。这些结果表明，功能性Na+通道的数量增加的膜。我们表明，新的通道是在小窝（Yarbrough等人，2002年）。当小窝“颈”与质膜融合并打开以在细胞外空间和小窝内隔室之间建立电连续性时，小窝膜内的Na+通道变得有功能。本申请的重点是确定共同定位的Na+和Ca 2+通道的小窝和气体的调节小窝的作用。具体而言，我们建议处理以下问题： 1)Na+和Ca 2+通道被分为相同还是不同的小窝？Yarbrough等人，（2002）表明，在小窝膜中发现了Na+通道。此外，我们的初步数据表明，抗a1 C抗体识别的蛋白质信号中的小窝丰富的部分大鼠心室肌细胞。我们假设Na+和Ca 2+通道被分类到相同的小窝。我们将使用免疫沉淀，Western印迹分析，免疫荧光，免疫电镜技术，并使用tip-dip方法直接膜片钳记录来研究这一假设。 2)Gas的N-末端在调节小窝中的功能作用是什么？Lu等人，（1999）表明，Gas可以以不依赖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融合的研究，N-和C-末端，所有的细胞内段循环，和域间循环的大鼠心脏Na+通道，Galphas不直接与通道也不Gby。然而，Galphas的短N-末端肽（a.a. 27-42）可以模拟增加Na+电流的作用。我们推测Galphas的N-末端在小窝开放的调节中起着重要的作用。使用Na+电流作为我们的测定，我们将使用Galphas/Glyphat和Galphas/Galphai嵌合体和短N-末端Galphas寡聚物来检查Gas的N-末端的功能效应。我们还将探测Galphas与之相互作用以调节小窝开放的底物。3)小窝是动态的Ω形结构，其膜融合和分裂机制几乎是未知的。这个特定的目标将测试参与膜相关蛋白质的小窝对接和/或融合事件。我们还将探测气体与之相互作用的基质，以调节小窝颈部的打开和关闭。内皮细胞中的小窝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。我们将测试参与毒素敏感性小突触泡蛋白（VAMP，囊泡相关膜蛋白）在心脏心室小窝对接和/或融合使用抗体识别成员的VAMP家族和神经毒素，破伤风毒素，蛋白水解裂解VAMP蛋白。我们将分析Na+电流的变化。将使用VAMP和小窝蛋白-3抗体的免疫组织化学方法测试VAMP在小窝结构上的定位。VAMP与Cav-3的共定位对于将Ω形膜结构鉴定为小窝而不是另一种囊泡将是重要的。