Caveolae-Dependent Regulation of Cardiac Ion Channels

描述(由申请人提供)：除了依赖于离子通道的磷酸化作用外，G蛋白对心脏钠通道的调节还有一个额外的“直接”效应。非磷酸化依赖的Gas介导的Peak/Na升高不是由于激活、失活、失活恢复或单通道幅度的改变。这些结果表明，膜上功能性钠通道的数量增加。我们证明了新的通道是在洞穴中(Yarbough等人，2002年)。当小窝“颈”与质膜融合并开放以建立细胞外间隙和小窝内室之间的电连续性时，小窝内的NA通道开始起作用。这项应用着重于确定Na和Ca~(2+)通道在小窝中的共定位以及GAS在小窝调节中的作用。具体地说，我们建议解决以下问题： 1)钠通道和钙通道是否分属于相同或不同的小窝？Yarbough等人(2002)表明，在小凹细胞膜中发现了钠通道。此外，我们的初步数据显示，抗A1C抗体识别大鼠心室肌细胞中富含小凹的部分的蛋白质信号。我们假设Na和Ca~(2+)通道属于同一小窝。我们将使用免疫沉淀、Western印迹分析、免疫荧光、免疫电子显微镜技术和使用尖端浸渍方法的直接膜片钳记录来研究这一假说。 2)GAS的N-末端在小窝的调节中起什么作用？Lu等人(1999)证明了气体能以一种不依赖阵营的方式增加钠电流的大小。我们的数据还表明，在酵母双杂交筛选和GST融合研究中，大鼠心脏钠通道的N-末端和C-末端、所有细胞内片段环和域间环，Galphas和Gby都不直接与该通道相互作用。然而，Galphas(A.A.)的一个短N端肽。27-42)可以模拟钠电流增大的影响。我们推测Galphas的N端在腔泡开放的调节中起着重要作用。以钠电流为基础，我们将使用Galphas/Galphat和Galphas/Galphai嵌合体和短N-末端Galphas齐聚物来研究Gas的N-端的功能效应。我们还将探索Galpas与之相互作用的底物，以调节小窝的打开。3)小窝是一种动态的omega状结构，其膜融合和分裂机制几乎未知。这一特定的目的将测试膜相关蛋白在腔泡对接和/或融合事件中的参与。我们还将探索气体与之相互作用的底物，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的介导囊泡形成、停靠和/或融合的关键蛋白。我们将使用识别VAMP家族成员的抗体和破伤风毒素(它能分解VAMP蛋白的神经毒素)来测试毒素敏感突触短杆菌(VAMP，囊泡相关膜蛋白)在心室腔空泡对接和/或融合中的作用。我们将分析钠电流的变化。VAMP在腔泡结构上的定位将用VAMP和Caveolin-3抗体的免疫组织化学方法进行测试。VAMP与Cav-3的共定位对于鉴定omega形膜结构是小窝而不是另一种囊泡具有重要意义。

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.