CONFORMATIONAL DYNAMICS IN ION CHANNEL SELECTIVITY AND GATING

 描述(申请人提供)：离子通道控制细胞内静息膜电位和动作电位。因此，离子通道的不正常生理功能导致多种疾病状态，包括心脏和神经功能障碍、癫痫、哮喘、听力损失等。适当的离子通道功能需要适当的离子选择性和通道门控来控制跨生物膜的离子通量。然而，我们对离子通道的这些基本性质的了解仍然不完整，因为它们是由结构和动力学的复杂相互作用产生的。我们将使用溶液核磁共振来实验测试蛋白质动力学在各向同性双胞体中溶解的蜡状芽孢杆菌的NAK通道的离子选择性和门控中的作用。NaK是一个理想的模型系统，因为微小的突变可以改变它的选择性；它的结构类似于真核细胞的钾 通道孔域，特别是生物医学上重要的环核苷酸门控和HERG钾通道；它高度稳定，易于进行结构和生物物理表征。核磁共振提供了一种独特的方法，可以同时定量探测离子通道的结构和动力学，并具有特定位置的分辨率。核磁共振测量不同时间尺度上的动力学并检测瞬时和低填充状态的能力使我们能够通过实验评估蛋白质动力学在从离子选择性到通道门控等过程中的作用。在这个提案中，我们将使用NaK的核磁共振研究来测试三个目标：选择性是否来自离子结合对骨架动力学的协同效应？N端M0螺旋是如何调节NAK门控的？什么结构和动态特征允许钾通道中选择性过滤器和内门之间的变构通信？

 DESCRIPTION (provided by applicant): Ion channels control both the resting membrane potential and the action potential in cells. As a result, improper physiological function of ion channels leads to a wide variety of disease states, including cardiac and neurological dysfunction, epilepsy, asthma, hearing loss, and others. Proper ion channel function requires appropriate ion selectivity and channel gating to control ion flux across biological membranes. However, our understanding of these fundamental properties of ion channels is still incomplete because they arise from a complex interplay of structure and dynamics. We will use solution NMR to experimentally test the role of protein dynamics in ion selectivity and gating of the NaK channel from Bacillus cereus solubilized in isotropic bicelles. NaK is an ideal model system because small mutations can shift its selectivity; its structure is similar to eukaryotic potassium channel pore domains, particularly biomedically important cyclic nucleotide gated and HERG potassium channels; and it is highly stable and amenable to structural and biophysical characterization. NMR provides a unique method to simultaneously and quantitatively probe ion channel structure and dynamics with site-specific resolution. The ability of NMR to measure dynamics on diverse timescales and detect transient and lowly populated states allows us to experimentally evaluate the role of protein dynamics on processes ranging from ion selectivity to channel gating. In this proposal we will use NMR studies of NaK to test three aims: Does selectivity arise from cooperative effects of ion binding on backbone dynamics? How does the N-terminal M0 helix regulate NaK gating? What structural and dynamic features allow allosteric communication between the selectivity filter and inner gate in potassium channels?