New paradigms of CFTR regulation

描述（由申请人提供）：本更新提案的总体目标是定义CFTR通道门控的基本原理以及最常见的CF突变如何破坏这一过程。与其他ABC转运蛋白一样，CFTR具有两个核苷酸结合结构域（NBD），可在NBD二聚体界面的口袋中结合ATP。CFTR也有一个独特的调节结构域（R结构域），除非被蛋白激酶a （PKA）磷酸化，否则它会抑制通道打开。悬而未决的问题包括ATP结合和通道打开之间的耦合程度以及R结构域磷酸化如何刺激通道活性。在目前的资助期内，我们取得了几项重大发现，阐明了CFTR控制机制；值得注意的是;(i)连接nbd和孔的细胞质环中的某些点突变促进了构成性（不依赖于atp）通道的开放；（ii）这些突变挽救了常见CF突变通道（G551D）的缺陷门控；（iii） CFTR门控可以通过一种变构机制很好地描述，其中ATP结合改变了先前存在的关闭状态和开放状态之间的平衡；（iv） R结构域调节通道活性，不依赖于ATP结合或NBD二聚化。这些发现为我们项目的下一阶段奠定了基础，我们将追求三个具体目标。目的1：测试细胞质环作为压缩弹簧的模型，该模型强烈抵抗非配体CFTR通道打开，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：测试预测的变构门控模型，解决通道打开和核苷酸占用之间的联系。目标3：定义R结构域磷酸化与CFTR通道门控之间的联系。该项目将显著提高我们对CFTR通道门控基本原理的认识，并可能为CFTR相关疾病的治疗提供新的途径。

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.