Caveolae-Dependent Regulation of Cardiac Ion Channels

心脏离子通道的小凹依赖性调节

• 批准号: 6999361
• 负责人: ERWIN F SHIBATA
• 金额: $ 36.01万
• 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/6999361
• 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。非磷酸化依赖的气体介导的峰值/Na的增加不是由于激活、失活、失活后的恢复或单通道振幅的变化。这些结果表明，细胞膜中功能Na+通道的数量有所增加。我们发现新的通道位于洞穴中（Yarbrough et al., 2002）。当小泡“颈”与质膜融合并打开，在细胞外空间和小泡内室之间建立电连续性时，小泡膜内的Na+通道开始发挥作用。该应用侧重于确定Na+和Ca2+通道在小泡中的共定位以及气体在小泡调节中的作用。具体而言，我们建议解决以下问题：