The Dynamic Nature of the CFTR Channel Pore: Coupling Gating to Permeation

描述（由申请人提供）：CFTR是ABC转运蛋白超家族的成员，但是已知具有离子通道活性的唯一成员。使用分子进化分析，我们已经确定了CFTR中的残基，这些残基似乎对从转运蛋白到通道的进化过渡至关重要。长期目标是了解CFTR通道的孔如何在打开和关闭状态之间改变其结构，ATP依赖性门控循环中的步骤如何控制孔门控，以及CFTR如何进化中断转运机制以获得离子通道功能的能力。该提案将测试以下假设：CFTR中氯离子通道活性通过将与胞质结构域处ATP结合和水解相关的膜结构域中的构象变化（如在真正的ABC转运蛋白中所发现的）转化为通过结构域间和结构域内相互作用形成稳定的开放状态而演变。上一个资助期的结果表明，在CFTR和相关ABC转运蛋白之间也被确定为不同的位点上，CFTR中的结构域内相互作用的破坏，在电导、选择性、药理学和多个传导状态之间的转换方面显著改变了通道行为。该更新申请提出使用精细的进化分析，结合基于卵母细胞中表达的CFTR通道的定量电生理学测定的结构/功能实验，以及CFTR同源性模型的模拟，来测试CFTR中特定残基在从转运蛋白到通道的切换中的重要性。目的#1是鉴定CFTR中通道活性进化的基础残基。在表现出与转运蛋白的进化分歧的位点，将确定突变对通道活性的影响。目的#2是确定细胞质结构域的ATP依赖性门控如何导致与通道功能相关的孔中的构象变化。这个目标将包括实验和分子模拟。在预测相互作用以稳定每个开放电导状态的位点，将确定与各种长度的双官能巯基修饰（SH）试剂交联的速率和状态依赖性，使我们能够构建和测试与开放和关闭相关的孔中的运动的方案。这些结果将用于验证所选CFTR同源模型的分子动力学模拟，以确定哪些结构最接近地反映真实的通道结构。目的#3是通过评估相互作用残基在CFTR和相关谷胱甘肽转运蛋白中的转运蛋白功能中的作用来验证相互作用残基的重要性。预计促进通道行为的相互作用将破坏转运蛋白行为，这可能影响CF的药物开发。这些研究将使我们能够将CFTR孔结构域中的分子运动与ATP依赖性门控循环中的步骤相关联，将使我们能够鉴定对CFTR中的离子通道功能至关重要的残基，并且可以鉴定药物可以对接以锁定开放的CFTR通道的位点，从而导致Cl-分泌增加。 公共卫生相关性：CFTR蛋白是三种破坏性疾病的关键因素：囊性纤维化（CF），分泌性腹泻和多囊肾病（PKD）。预计拟议的工作将提供有关通道开放状态如何稳定的信息，这将有助于合理设计药物，这些药物可以锁定CF细胞质膜中开放的CFTR通道，从而增加氯化物分泌并改善CF疾病。

DESCRIPTION (provided by applicant): CFTR is a member of the ABC Transporter superfamily, but is the only member known to bear ion channel activity. Using a molecular evolution analysis, we have identified residues in CFTR that appear to be critical to the evolutionary transition from transporter to channel. The long-term objective is to understand how the pore of the CFTR channel changes its structure between the open and closed states, how steps in the ATP-dependent gating cycle control pore gating, and how CFTR evolved the capability to interrupt a transporter mechanism in order to gain ion channel function. This proposal will test the hypothesis that chloride channel activity evolved in CFTR by converting the conformational changes in the membrane domain associated with binding and hydrolysis of ATP at the cytoplasmic domains, as are found in true ABC Transporters, into the formation of a stable open state, by means of inter- and intra-domain interactions. Results from the previous funding period show that disruption of intradomain interactions in CFTR, at sites that are also identified as divergent between CFTR and related ABC Transporters, dramatically alter channel behavior in terms of conductance, selectivity, pharmacology, and transitions between multiple conducting states. This renewal application proposes to use a refined evolutionary analysis, coupled to structure/function experiments based upon quantitative electrophysiological assays of CFTR channels expressed in oocytes, and simulations of CFTR homology models, to test the importance of specific residues in CFTR in the switch from transporter to channel. Aim #1 is to identify residues that underlie the evolution of channel activity in CFTR. At sites that exhibit evolutionary divergence from transporters, the impact of mutations on channel activity will be determined. Aim #2 is to determine how ATP-dependent gating at the cytoplasmic domains leads to conformational changes in the pore associated with channel function. This Aim will include both experiment and molecular simulation. At sites predicted to interact to stabilize each of the open conductance states, the rate and state-dependence of crosslinking with bifunctional sulfhydryl-modifying (SH) reagents of various lengths will be determined, allowing us to construct and test a scheme for the movements in the pore associated with both opening and closing. These results will be used to validate molecular dynamics simulations of selected CFTR homology models, to identify which structures most closely reflect the true channel structure. Aim #3 is to verify the importance of the interacting residues by assessing their role in transporter function, in both CFTR and a related glutathione transporter. It is expected that interactions that promote channel behavior will disrupt transporter behavior, which may impact drug development for CF. These studies will allow us to associate molecular motions in CFTR's pore domain with steps in the ATP- dependent gating cycle, will allow us to identify residues that are critical to ion channel function in CFTR, and may identify sites where drugs can be docked to lock open CFTR channels, leading to increased Cl- secretion. PUBLIC HEALTH RELEVANCE: The CFTR protein is a key element in three devastating diseases: cystic fibrosis (CF), secretory diarrhea, and polycystic kidney disease (PKD). The proposed work is expected to provide information on how the open state of the channel is stabilized, which will aid the rational design of drugs that can lock open CFTR channels in the plasma membranes of CF cells, leading to increased chloride secretion and amelioration of CF disease.