Role of Kir6.1 subunits of K+ channels in heart

描述(申请者的逐字描述)：ATP敏感(KATP) 通道将能量代谢和应激与膜兴奋性联系起来，并 在心里高度表达。它们在心脏和冠状动脉血管系统中的作用 已经通过使用药物制剂进行了广泛的研究，这些药物可以打开或 阻断KATP通道。KATP通道亚基的分子克隆揭示 KATP通道由形成孔的Kir6亚基组成，与 调节性SUR亚基。有两个Kir6成员(Kir6.1和Kir6.2)-都是 都是在内心高度表达的。心脏KATP通道被认为包括 Kir6.2/SUR2复合体。然而，Kir6.1的作用尚不清楚。总目标 本申请的目的是研究Kir6.1亚基在细胞内的功能 心血管系统。因为蛋白质的局部化可以指示 它的功能，我们将考察区域、细胞和亚细胞 Kir6.1亚基在心肌细胞中的分布模式、传导 系统，在冠状动脉血管系统和内皮细胞中使用 免疫组织化学技术(我们已经开发出优秀的抗体 此目的)。我们从生化和电生理学的角度证明了Kir6.1和 Kir6.2亚基可以在异源表达系统中共组装，这表明 这也可能发生在内心。我们将研究这种联合组装是否 发生在天然Kir6蛋白上。我们还将调查功能 使用膜片钳技术的异构体Kir6通道的特性。我们 将产生靶向干扰Kir6.1基因的小鼠。LoxP站点将 被引入Kir6.1基因座，这些小鼠将与其他小鼠杂交 Cre在心脏中过度表达。我们将直接利用这些老鼠来检测 Kir6.1亚基在心脏和血管功能中的作用 孤立的，兰登多夫灌流的心脏技术。冠脉血流量和 -在缺血期间，储备将与心脏功能一起测量， 再灌流和缺血预适应。这些研究将提供一个 用来理解KATP通道复杂性的框架 心血管系统，特别是Kir6.1亚基的作用，并将 提供对动物体内KATP通道功能的分子洞察力 正常和病理生理条件。

DESCRIPTION (the applicant's description verbatim): ATP-sensitive (KATP) channels couple energy metabolism and stress to membrane excitability and are highly expressed in heart. Their function in the heart and coronary vasculature has been studied extensively by the use of pharmacological agents that open or block KATP channels. The molecular cloning of subunits of KATP channel revealed that KATP channels consist of pore-forming Kir6 subunits in association with regulatory SUR subunits. There are two Kir6 members (Kir6.1 and Kir6.2) - both are highly expressed in heart. Cardiac KATP channels are believed to consist of Kir6.2/SUR2 complexes. However, the role of Kir6.1 is unknown. The overall goal of this application is to examine the function of Kir6.1 subunits in the cardiovascular system. Since the localization of a protein can be indicative of its function, we will examine the regional, cellular and subcellular distribution patterns of Kir6.1 subunits in cardiac myocytes, the conduction system, in the coronary vasculature and in endothelial cells using immunohistochemistry techniques (we have developed excellent antibodies for this purpose). We show biochemically and electrophysiologically that Kir6.1 and Kir6.2 subunits can co-assemble in heterologous expression systems, suggesting that this may also occur in heart. We will examine whether such co-assembly takes place for native Kir6 proteins. We will also investigate the functional characteristics of heteromeric Kir6 channels using patch clamp techniques. We will generate mice with targeted disruption of the Kir6.1 gene. LoxP sites will be introduced in the Kir6.1 locus and these mice will be crossed with others overexpressing Cre in the heart. We will utilize these mice directly to examine the role of Kir6.1 subunits in the function of heart and vasculature using isolated, Langendorff-perfused heart techniques. Coronary blood flow and -reserve will be measured along with heart function during ischemia, reperfusion and ischemic preconditioning. These studies will provide a framework in which to understand the complexity of KATP channels in the cardiovascular system, in particular the role of Kir6.1 subunits, and will provide molecular insights into the function of KATP channels in animals during normal and pathophysiological conditions.