New paradigms of CFTR regulation

描述(由申请人提供)：此续订提案的主要目标是定义CFTR通道选通的基本原则，以及最常见的CF突变如何扰乱这一过程。与其他ABC转运蛋白一样，CFTR有两个核苷酸结合域(NBD)，它们在NBD二聚体的界面上将ATP结合在口袋中。CFTR也有一个独特的调控结构域(R结构域)，除非被蛋白激酶A(PKA)磷酸化，否则它会抑制通道的开放。悬而未决的问题包括ATP结合和通道开放之间的偶联程度，以及R结构域磷酸化如何刺激通道活性。在当前的资金支持期间，我们取得了几项重大发现，这些发现揭示了CFTR门控机制；值得注意的是：(I)连接NBDS与孔道的细胞质环中的某些点突变促进了结构性(ATP不依赖)的通道开放；(Ii)这些突变挽救了常见的CF突变通道(G551D)的缺陷门控；(Iii)CFTR门控可用变构机制很好地描述，其中ATP结合改变了先前存在的关闭和开放状态之间的平衡；(Iv)R结构域调节通道活性，而不依赖于ATP结合或NBD二聚作用。这些发现为我们项目的下一阶段奠定了基础，在这一阶段，我们将实现三个具体目标。目的1：测试一个模型，在该模型中，胞浆环起压缩弹簧的作用，强烈地抵抗未连接的CFTR通道的开放，并确定我们的CFTR发现是否可以推广到相关的ABC转运蛋白(酵母YOR1出口蛋白)。目的2：对变构门控模型进行测试预测，该模型解决了通道开放和核苷酸占有率之间的联系。目的3：明确R结构域的磷酸化与CFTR通道门控之间的联系。该项目将显著提高我们对CFTR通道门控的基本原理的理解，并可能导致治疗CFTR相关疾病的新方法。 公共卫生相关性：CFTR氯通道的突变导致囊性纤维化(CF)，这是美国最常见的遗传性疾病之一。该项目的主要目标是定义该通道如何正常运行，以及导致CF的突变如何扰乱其运行。由此得到的信息可能为如何纠正CF患者CFTR通道功能缺陷提供更好的线索。

DESCRIPTION (provided by applicant): The broad goal of this renewal proposal is to define the basic principles of CFTR channel gating and how the most common CF mutations disrupt this process. Like other ABC transporters, CFTR has two nucleotide binding domains (NBDs) that bind ATP in pockets at the interface of an NBD dimer. CFTR also has a unique regulatory domain (R domain) that inhibits channel opening unless phosphorylated by protein kinase A (PKA). Outstanding issues include the degree of coupling between ATP binding and channel opening and how R domain phosphorylation stimulates channel activity. During the current funding period we made several significant discoveries that shed light on the CFTR gating mechanism; notably; (i) certain point mutations in the cytosolic loops that connect the NBDs to the pore promote constitutive (ATP-independent) channel opening; (ii) these mutations rescue the defective gating of a common CF mutant channel (G551D); (iii) CFTR gating is well described by an allosteric mechanism in which ATP binding shifts the equilibrium between pre-existing closed and open states and (iv) the R domain regulates channel activity independent of either ATP binding or NBD dimerization. These findings set the stage for the next phase of our project in which we will pursue three specific aims. Aim 1: Test a model in which the cytosolic loops function as a compression spring that strongly resists unliganded CFTR channel opening, and determine whether our CFTR findings can be generalized to a related ABC transporter (yeast YOR1 exporter). Aim 2: Test predictions of an allosteric gating model that address the link between channel opening and nucleotide occupancy. Aim 3: Define the link between R domain phosphorylation and CFTR channel gating. This project should significantly improve our understanding of the basic principles of CFTR channel gating, and may lead to new approaches for treating CFTR-related diseases. PUBLIC HEALTH RELEVANCE: Mutations in the CFTR chloride channel cause cystic fibrosis (CF), one of the most common genetic diseases in the U.S. The main goals of this project are to define how this channel operates normally and how CF-causing mutations disrupt its operation. The resulting information may provide better clues as to how to correct defective CFTR channel function in CF patients.