REGULATION OF CFTR CL CHANNELS BY THE NUCLEOTIDE BINDING DOMAINS

囊性纤维化跨膜电导调节因子(CFTR)是一种 质膜C1通道预测由五个结构域组成：两个 跨结构域的膜，这有助于形成 通道孔；一个R结构域，调节通道以响应 磷酸化；和两个核苷酸结合域(NBD)。该功能 一直令人费解，因为它们不是一个功能 在其他离子通道中发现；相反，它们在涉及的蛋白质中发现 在积极的运输中。此外，导致慢性萎缩性胃炎的许多突变是 在NBD里找到的。我们最近发现，需要镁ATP才能打开 Cftr C1通道。在我们的初步结果中，我们发现mgatp 与CFTR中的两个NbD交互，尽管两个NbD不是 在功能上相同。该项目的具体目标是：1) 了解MgATP如何控制CFTRC1-通道的打开和关闭。 为了充分了解MGATP和NBD如何调节CFTRC1-通道， 我们需要知道镁ATP是如何控制通道门控的。我们将使用 膜片钳技术研究细胞切取的膜片 重组野生型cftr和含定点的cftr的表达 突变。这一结果将在分子水平上提供一个理解 水平，如何与两个NBD相互作用，以调节CFTR。2)我们 将检查除镁三磷酸腺苷以外的化合物对调节的影响 Cftr.这些将包括ATP的非水解性类似物， 非核苷酸水解物和三磷酸腺苷水解物。 这些研究的结果应该会告诉我们NBD是如何与 用来维持渠道活跃性的镁ATP。3)我们将询问CF如何关联 NBD的突变改变了C1通道的调节。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突变是如何产生有缺陷的通道的。 从这些研究中获得的知识将使我们能够准确地-- 对这样的缺陷的坚持。

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.