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，但是已知的唯一具有离子通道活性的成员。通过分子进化分析，我们已经确定了CFTR中似乎对从转运体到通道的进化转变至关重要的残基。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如何进化出中断转运体机制以获得离子通道功能的能力。本研究将验证以下假设：CFTR中的氯离子通道活性是通过将与胞质结构域ATP结合和水解相关的膜结构域的构象变化（如在真正的ABC转运体中发现的）通过结构域间和结构域内的相互作用转化为稳定的开放状态而进化的。先前资助期的结果表明，CFTR中域内相互作用的破坏，也被认为是CFTR和相关ABC转运蛋白之间的分歧位点，在电导率、选择性、药理学和多种导电状态之间的转换方面显著改变了通道行为。本更新申请建议使用改进的进化分析，结合基于卵母细胞中表达的CFTR通道的定量电生理分析的结构/功能实验，以及CFTR同源模型的模拟，来测试CFTR中特定残基在从转运体到通道转换中的重要性。目的1是确定CFTR中通道活性演变的基础残基。在表现出与转运体进化差异的位点，突变对通道活性的影响将被确定。目的2是确定细胞质域的atp依赖性门控如何导致与通道功能相关的孔的构象变化。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打开电导状态的位点上，将确定与不同长度的双功能巯基修饰（SH）试剂交联的速率和状态依赖性，从而使我们能够构建和测试与打开和关闭相关的孔中的运动方案。这些结果将用于验证所选CFTR同源模型的分子动力学模拟，以确定哪些结构最能反映真实的通道结构。目的#3是通过评估相互作用残基在转运蛋白功能中的作用来验证其重要性，包括CFTR和相关的谷胱甘肽转运蛋白。预计促进通道行为的相互作用将破坏转运体行为，这可能会影响CF的药物开发。这些研究将使我们能够将CFTR孔域的分子运动与ATP依赖性门控循环的步骤联系起来，将使我们能够识别对CFTR离子通道功能至关重要的残基，并可能确定药物可以停靠以锁定打开的CFTR通道的位置，从而导致Cl-分泌增加。

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.