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

描述(申请人提供)：在真核生物中，大量的、普遍存在的和同源的离子通道发挥着重要的功能，包括神经信号、心律调节和gpr信号转导通路的调节器。特别是，钾通道是所有人类细胞特有的，它们通过通过细胞膜传递离子来控制电化学势。电压门控K(Kv)通道产生对肌肉、神经和内分泌系统的功能至关重要的电脉冲，是细胞增殖、凋亡和癌细胞增殖等行为的基础。事实证明，用传统的结构方法研究这些系统具有极大的挑战性。这项研究的目标是应用和扩展固态核磁共振方法来获得电压门控K通道的结构和功能信息。这项工作将从变铅青链霉菌的通道KCSA开始。KCSA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SS核磁共振，使KCSA成为探测涉及离子选择、结合传递和释放的基本生物物理的理想系统。KCSA计划进行的实验包括在pH、K浓度范围内的化学位移分配和张量测量，以及在季铵离子存在的情况下。这些数据将被用来开发高度模糊的SS核磁共振数据的结构确定方法。在这个方案中，这个方案被称为“自上而下”的SS核磁共振，结构模型将根据未分配的核磁共振数据进行筛选，以确保一致性。核磁共振约束分子动力学(MD)将结合特定部位和不明确的信息进行。在此之后，更复杂的原核系统，如KvAP和哺乳动物系统，如Kv1.2或可能的Kv10.1，将成为目标。对通道的结构研究，如Kv1.2，说明并建议了电压传感器的功能、孔门和由额外亚基调节的机制。这些系统的开放状态已经以更真实的功能形式稳定下来。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健康的影响是通过我的长期目标--将SS核磁共振应用于关键的癌症和心脏病相关目标--来实现的。该计划最终将利用SS核磁共振的力量来研究大型膜蛋白，这是人类疾病中相当大和至关重要的参与者。

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.