Mechanisms, Structure, and Regulation of CFTR's NBDs

描述：CFTR（囊性纤维化跨膜传导调节因子）， 由CF中突变的基因编码的是ATP结合盒（ABC）蛋白 形成氯离子通道。通道孔的打开和关闭是 在CFTR的两个核苷酸结合处受ATP的结合和水解调节 结构域（NBD），其反过来由cAMP依赖性蛋白控制 激酶介导的磷酸化和特异性磷酸酶介导的 CFTR中集中的特定丝氨酸残基的去磷酸化 调节域。这项研究的目的是了解，在 分子细节，NBD的结构和功能机制， 它们之间的相互作用，以及它们被调节的机制。 了解控制CFTR打开和关闭的精确机制 Cl-通道可能允许CF患者的最终药物补救， 由于突变CFTR表达导致离子流不足的病变细胞 通道;这包括那些突变体，未能达到细胞表面， 足够的数量，那些具有减小的单沟道电导， 以及那些在足够长的时间内不开放的。两 该项目的具体目标保持不变。第一个解决了 问题：NBD看起来像什么，它们如何运作，以及 它们之间的相互作用机制？目前的假设是， NBD是相似的，因为它们都能够结合和水解ATP， 但它们在结构、功能和机制上有所不同--一个变得“活跃”， 只有在可水解核苷结合并可能水解后， 三磷酸，而核苷酸结合似乎足以激活 其他.第二个目标解决的问题：如何磷酸化（和在 哪个或哪些站点）允许通道打开？磷酸化是如何 额外的不稳定位点（或多个位点）促进通道开放的稳定 州？这是哪个网站（或网站）？野生型和突变型CFTR通道将 在卵母细胞和哺乳动物细胞中表达， 将使用电生理学，生物化学，分子生物学， 和生物物理方法（包括分子建模，希望， 最终是关键胞质结构域的晶体学）。开业率 和关闭在那些结构域中携带特定突变的单个CFTR通道 （通过利用它们的结构模型和晶体结构来选择） 相关分子）将被测量以探测它们的正常催化功能 以及他们的合作互动。

DESCRIPTION: CFTR (cystic fibrosis transmembrane conductance regulator), encoded by the gene mutated in CF, is an ATP binding cassette (ABC) protein that forms a Cl- ion channel. Opening and closing of the channel pore are regulated by binding and hydrolysis of ATP at CFTR's two nucleotide binding domains (NBDs) which, in turn, are controlled by cAMP-dependent protein kinase-mediated phosphorylation, and specific phosphatase-mediated dephosphorylation, of particular serine residues concentrated in CFTR's regulatory (R) domain. The goal of the proposed research is to understand, in molecular detail, the structure and mechanisms of function of the NBDs, the interactions between them, and the mechanisms by which they are regulated. Understanding the precise mechanisms that control opening and closing of CFTR Cl- channels might permit eventual pharmacological rescue in CF patients of diseased cells with inadequate ion flow due to expression of mutant CFTR channels; this includes those mutants that fail to reach the cell surface in sufficient numbers, those that have a diminished single-channel conductance, and those that are not open for a large enough fraction of the time. The two specific aims of the project remain unchanged. The first addresses the questions: What do the NBDs look like, how do they function, and what are the mechanisms of interactions between them? The working hypothesis is that the two NBDs are similar, in that they are both capable of binding and hydrolyzing ATP, but they differ in structure, function and mechanism - one becoming "active" only after binding, and likely hydrolysis, of a hydrolyzable nucleoside triphosphate, while nucleotide binding appears sufficient to activate the other. The second aim addresses the questions: How does phosphorylation (and at which site or sites) permit channel opening? How does phosphorylation of an additional labile site (or sites) promote stabilization of the channel open state? Which is that site (or sites)? Wild type and mutant CFTR channels will be expressed in oocytes and mammalian cells, and their structure and function will be analyzed using electrophysiological, biochemical, molecular biological, and biophysical methods (including molecular modeling and, hopefully, eventually crystallography of the key cytoplasmic domains). Rates of opening and closing of single CFTR channels bearing specific mutations in those domains (selected by exploiting models of their structure, and crystal structures of related molecules) will be measured to probe their normal catalytic functions and their cooperative interactions.