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

通过固态核磁共振研究钾通道的结构和功能

• 批准号: 8055358
• 负责人: Benjamin James Wylie
• 金额: $ 5.3万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8055358
• 关键词: Affect; Ammonium; Apoptosis; Behavior; Binding; Biology; Biophysics; Cell Proliferation; Cell membrane; Cells; Chemicals; Complement; Complex; Data; Data Analyses; Data Set; Endocrine system; Eukaryota; Family; Four-dimensional; Funding; Future; G Protein-Coupled Receptor Signaling; Goals; Health; Heart Diseases; Homology Modeling; Human; Ion Channel; Ions; Length; Light; Literature; Malignant Neoplasms; Measurement; Measures; Membrane; Membrane Proteins; Methodology; Methods; Molecular; Muscle function; Nerve; Potassium Channel; Preparation; Proteins; Public Health; Regulation; Reporting; Research; Research Training; Resolution; Roentgen Rays; Sampling; Scheme; Side; Signal Transduction; Signal Transduction Pathway; Site; Spectrum Analysis; Streptomyces lividans; Structural Models; Structure; System; Techniques; Testing; Training; Variant; Vertebral column; Voltage-Gated Potassium Channel; Work; base; cancer cell; experience; heart rhythm; human disease; insight; molecular dynamics; novel; programs; relating to nervous system; research study; restraint; sensor; solid state nuclear magnetic resonance; three dimensional structure; transmission process; voltage

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.

描述（由申请人提供）：一大群普遍存在且同源的离子通道在真核生物中发挥着关键功能，包括神经信号传导、心律调节和 GPCR 信号转导途径的调节剂。特别是，K+通道是所有人类细胞所特有的，它们通过使离子穿过细胞膜来控制电化学势。电压门控 K+ (Kv) 通道产生对肌肉、神经和内分泌系统功能至关重要的电脉冲，并且是细胞增殖、凋亡和癌细胞增殖等行为的基础。事实证明，通过传统结构方法研究这些系统极具挑战性。本研究的目标是应用和扩展固态 NMR 方法来获取电压门控 K+ 通道的结构和功能信息。这项工作将从浅青紫链霉菌的 KcsA 通道开始。易于制备、样品稳定性、与其他 K+ 通道的同源性以及现有研究（包括 SSNMR）使 KcsA 成为探测涉及离子选择、结合传输和释放的基本生物物理学的理想系统。 KcsA 计划进行的实验包括在一定范围的 pH、K+ 浓度以及季铵离子存在下的化学位移分配和张量测量。该数据将用于开发高度模糊的 SSNMR 数据的结构确定方法。在该方案中，在本提案中称为“自上而下”SSNMR，结构模型将根据未指定的 NMR 数据进行筛选，以确保一致性。然后将结合特定位点和模糊信息来执行 NMR 约束分子动力学 (MD)。在此之后，更复杂的原核系统（例如 KvAP）和哺乳动物系统（例如 Kv1.2 或可能的 Kv10.1）将成为目标。 Kv1.2 等通道的结构研究阐明并提出了电压传感器、孔门控和附加亚基调节功能的机制。这些系统的开放状态已经以更真实的功能形式稳定下来。如果获得资助，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实现的，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 的力量来研究大膜蛋白，大膜蛋白在人类疾病中发挥着相当大且至关重要的作用。