Structural and Functional Studies of Potassium Channels by Solid-State NMR

描述（由申请人提供）：一组大的、普遍存在的和同源的离子通道在真核生物中执行关键功能，包括神经信号传导、心律调节和GPCR信号转导途径的调节剂。特别地，K+通道是所有人类细胞特有的，其中它们参与通过使离子穿过细胞膜来控制电化学势。电压门控K+（Kv）通道产生肌肉、神经和内分泌系统功能所必需的电脉冲，并且是细胞增殖、凋亡和癌细胞增殖等行为的基础。这些系统已经证明是非常具有挑战性的研究传统的结构方法。该研究的目的是应用和扩展固态NMR方法，以获得电压门控K+通道的结构和功能信息。这项工作将开始与通道KcsA从变铅青链霉菌。易于制备，样品稳定性，与其他K+通道的同源性，以及现有的研究，包括SSNMR，使KcsA成为探测离子选择，结合传输和释放所涉及的基本生物物理学的理想系统。计划KcsA的实验包括化学位移分配和张量测量的pH值，K+浓度的范围内，并在季铵离子的存在下。这些数据将被用来开发高度模糊的SSNMR数据的结构测定方法。在该方案中，在本提案中被称为“自上而下”SSNMR，结构模型将针对未分配的NMR数据进行筛选以确保一致性。NMR约束的分子动力学（MD），然后将与特定位点和模糊信息的组合进行。在此之后，更精细的原核系统如KvAP和哺乳动物系统如Kv1.2或可能的Kv10.1将被靶向。对通道（如Kv1.2）的结构研究说明并提示了电压传感器、孔门控和其他亚基调节的功能机制。这些系统的开放状态已经稳定在一个更真实的功能形式。如果得到资助，这个项目将为我提供结构和机械生物学方面的重要培训。这项拟议工作对公共卫生的影响是通过我的长期目标，即将SSNMR应用于关键的癌症和心脏病相关目标。该计划最终将利用SSNMR的力量来研究大型膜蛋白，这是人类疾病中相当重要的参与者。

DESCRIPTION (provided by applicant): A large, ubiquitous, and homologous group of ion channels perform crucial functions in eukaryotes including neural signaling, cardiac rhythm regulation, and modulators of GPCR signal transduction pathways. In particular, K+ channels are endemic to all human cells where they are involved in controlling the electrochemical potential by passing ions through the cell membrane. Voltage-gated K+ (Kv) channels generate electric impulses essential to the function of muscles, nerves, and the endocrine system and are fundamental to such behaviors as cell proliferation, apoptosis, and cancer cell proliferation. These systems have proven exceedingly challenging to study by traditional structure methods. The goal of the proposed research is to apply and extend solid-state NMR methodology to obtain structural and functional information on voltage-gated K+ channels. This work will begin with the channel KcsA from Streptomyces lividans. The ease of preparation, sample stability, homology with other K+ channels, and extant studies, including SSNMR, makes KcsA an ideal system for probing the basic biophysics involved in ion selection, binding transmission, and release. Experiments planned for KcsA include chemical shift assignments and tensor measurements over a range of pH, K+ concentration, and in the presence of quaternary ammonium ions. This data will be leveraged to develop structure determination methods for highly ambiguous SSNMR data. In this scheme, referred to in this proposal as "top-down" SSNMR, structural models will be screened against unassigned NMR data for consistency. NMR constrained molecular dynamics (MD) will then be performed with a combination of site-specific and ambiguous information. Following this, more elaborate prokaryotic systems such as KvAP and mammalian systems such as Kv1.2 or possibly Kv10.1 will be targeted. Structural studies of channels such as Kv1.2 illustrate and suggest a mechanism for the function of voltage sensors, pore gating, and regulation by additional subunits. The open states of these systems have been stabilized in a more authentically functional form. If funded, this project would provide me with crucial training in structural and mechanistic biology. The impact of this proposed work on public health is through my long-term goal of applying SSNMR to crucial cancer and heart disease related targets. This program would ultimately harness the power of SSNMR to study large membrane proteins, a sizeable and critically important players in human disease.