REGULATION OF CFTR CL CHANNELS BY THE NUCLEOTIDE BINDING DOMAINS

囊性纤维化跨膜传导调节因子（CFTR）是一种 质膜C1通道预测由五个结构域组成：两个 跨膜结构域，有助于形成 通道孔; R结构域，其响应于 磷酸化;和两个核苷酸结合结构域（NBD）。 功能 CFTR中的NBD一直令人困惑，因为它们是一个功能， 在其他离子通道中发现;相反，它们在涉及的蛋白质中发现 在积极的运输。 此外，许多导致CF的突变是 在NBD中找到。 我们最近发现MgATP需要打开 CFTR C1频道 在我们的初步结果中我们发现mgatp 与CFTR中的两个NBD相互作用，尽管两个NBD不 功能等同。 该项目的具体目标是：1） 了解MgATP如何控制CFTR C1通道的打开和关闭。 为了充分理解mgatp和NBD如何调节CFTR C1通道， 我们需要知道MgATP如何控制通道门控 我们将使用 膜片钳技术研究从细胞上切下的膜片 表达重组野生型CFTR和含有位点定向的 突变。 结果将提供一个理解，在分子 水平，MgATP如何与两个NBD相互作用以调节CFTR。2)我们 将检查MgATP以外的化合物对调节的影响， 的CFTR。 这些将包括ATP的不可水解类似物， 非核苷酸可水解化合物和ATP水解产物。 这些研究的结果应该告诉我们NBD如何与 MgATP以维持通道活性。 3)我们会问CF相关的 NBD中的突变改变C1通道调节。 如果我们要 了解CF的发病机制并开发创新的新疗法， 我们需要了解CFTR的突变如何产生有缺陷的通道。 从这些研究中获得的知识将允许一个精确的下- 这种缺陷的存在。

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane C1- channel predicted to consist of five domains: two membrane spanning domains, that contribute to the formation of the channel pore; an R domain, that regulates the channel in response to phosphorylation; and two nucleotide binding domains (NBDs). The function of the NBDs in CFTR has been puzzling because they are a feature not found in other ion channels; instead, they are found in proteins involved in active transport. Moreover, many of the mutations that cause CF are found within the NBDs. We recently found that MgATP is required to open the CFTR C1 channel. In our preliminary results we found that mgatp interacts with both NBDs in CFTR, although the two NBDs are not functionally equivalent. The specific aims of this project are: 1) To learn how MgATP controls the opening and closing of the CFTR C1- channel. To fully understand how mgatp and the NBDs regulate the CFTR C1- channel, we need to know how MgATP controls channel gating. We will use the patch-clamp technique to study excised membrane patches from cells expressing recombinant wild-type CFTR and CFTR containing site-directed mutations. The results will provide an understanding, at the molecular level, of how MgATP interacts with the two NBDs to regulate CFTR. 2) We will examine the effect of compounds other than MgATP on the regulation of CFTR. These will include nonhydrolyzable analogs of ATP, nonnucleotide hydrolyzable compounds, and the products of ATP hydrolysis. The results of these studies should teach us how the NBDs interact with MgATP to maintain channel activity. 3) We will ask how CF-associated mutations in the NBDs alter C1- channel regulation. If we are to understand the pathogenesis of CF and develop innovative new therapies, we need to understand how mutations in CFTR produce defective channels. The knowledge obtained from these studies will allow a precise under- standing of such defects.