High-resolution crystallographic and functional studies of K+ channel function.

K 通道功能的高分辨率晶体学和功能研究。

基本信息

批准号：
10197146
负责人：
Luis Gonzalo Cuello
金额：
$ 30.6万
依托单位：
TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10197146
关键词：
Affect Applications Grants Arrhythmia Autoimmune Diabetes Autoimmune Diseases Binding Biochemical Cells Coupling Crystallization Crystallography Diabetes Mellitus Differential Scanning Calorimetry Disease Drug Targeting Electrophysiology (science)Engineering Epilepsy Functional disorder Gland Glycine Heart Diseases Human Human Pathology Immunoglobulin Fragments Ion Channel Ions Kinetics Methodology Molecular Conformation Mutagenesis Mutate Mutation Myocardium Nervous system structure Pathologic Pharmaceutical Preparations Pharmacologic Substance Point Mutation Positioning Attribute Potassium Channel Production Property Protocols documentation Reaction Resolution Role Skeletal Muscle Smooth Muscle Structure Syndrome System Therapeutic Therapeutic Agents Time X-Ray Crystallography computer studies experience improved mutant nervous system disorder next generation

项目摘要

K+ channels are key regulators of cell excitability in the nervous system, skeletal, smooth and cardiac muscle and secretory glands. Therefore, it is not surprising that dysfunction of K+ channels are the underlie cause of uncountable human pathologies, such as: neurological disorders, cardiac diseases and diabetes. For this reason, it is extremely important to understand at the atomic level the properties of K+ channels that determine cell excitability. Understanding ion selectivity, permeation and gating at atomic detail will allow us to identify highly-specific therapeutic agents that can recognize with precision a specific channel's kinetic state that need to be regulated to correct a given channelopathy. It follows that for two decades, functional, structural and computational studies, performed on the KcsA-closed structure, have improved our understanding of how the structure defines the function of K+ channels. Recently, we have made two important scientific contributions: the first atomic-resolution description of KcsA's minimal kinetic cycle and the quantification of the energetics associated with each kinetic cycle reaction. However, important unanswered questions remain, mostly due to our inability to conduct simultaneous structural and functional studies in: 1 ) the open-state of the channel 2) mutants of the highly conserved glycine residues in the selectivity filter, which are known to affect inactivation gating, ion selectivity and/or ion binding in the closed and open states of the channel, 3) tandem-tetramers to dissect cooperativity of ion channel function, and 4) mutants that dissect the non-conductive open states of KcsA by precisely uncoupling activation-gate opening from the onset of ion permeation/inactivation at the selectivity filter. Consequently, we propose the following Specific Aims: 1) To characterize the structure-function correlations between the selectivity filter, ion occupancy and conduction properties of KcsA “trapped” with its activation gate open 2) To determine the structure-function correlations of KcsA subunit cooperativity using tandem hetero-tetramers 3) To understand the role of KcsA's allosteric coupling on the onset of ion permeation, C-type inactivation and ion selectivity and 4) To understand the structural and functional roles of the glycine residues within the K+ channel selectivity filter. The novelty of our experimental approaches, together with our vast experience working with ion channels, fully qualifies us to perform the proposed project. Finally, the completion of this project will bring us closer to a complete atomistic understanding of ion-channel function, allowing us to identify ion-channels kinetic intermediates more suitable as pharmaceutical targets for the next generation of more specific and safer therapeutic drugs.

K+ 通道是神经系统、骨骼肌、平滑肌和心肌细胞兴奋性的关键调节因子因此，K+通道功能障碍是其根本原因也就不足为奇了。无数的人类疾病，例如：神经系统疾病、心脏病和糖尿病。在原子水平上了解决定细胞的 K+ 通道的特性极其重要了解原子细节的离子选择性、渗透和门控将使我们能够识别高度特异性的治疗剂，可以精确识别特定的需要调节通道的动力学状态以纠正给定的通道病变。二十年来，对 KcsA 封闭结构进行的功能、结构和计算研究已经提高了我们对结构如何定义 K+ 通道功能的理解。两项重要的科学贡献：KcsA 最小动力循环的第一个原子分辨率描述然而，与每个动力循环反应相关的能量的量化很重要。悬而未决的问题仍然存在，主要是由于我们无法同时进行结构和功能研究领域：1) 通道的开放状态 2) 高度保守的甘氨酸残基的突变体的选择性过滤器，已知会影响关闭和开放状态下的失活门控、离子选择性和/或离子结合通道状态，3) 串联四聚体，用于剖析离子通道功能的合作，以及 4) 突变体通过从一开始就精确解耦激活门打开来剖析 KcsA 的非导电打开状态为了检查选择性过滤器处的离子渗透/失活，我们提出以下具体目标：1) 表征选择性过滤器、离子占据和传导之间的结构-功能相关性 KcsA 的属性“被困”且其激活门打开 2) 确定结构-功能相关性使用串联异四聚体的 KcsA 亚基协同性 3) 了解 KcsA 变构的作用耦合对离子渗透开始、C 型失活和离子选择性的影响，以及 4) 了解 K+ 通道选择性过滤器中甘氨酸残基的结构和功能作用实验方法，加上我们在离子通道方面的丰富经验，使我们完全有资格最后，该项目的完成将使我们更接近完整的原子化。了解离子通道功能，使我们能够识别更合适的离子通道动力学中间体作为下一代更特异、更安全的治疗药物的药物靶点。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A cost-effective protocol for the over-expression and purification of fully-functional and more stable Erwinia chrysanthemi ligand-gated ion channel.

一种经济高效的方案，用于过表达和纯化功能齐全且更稳定的菊欧文氏菌配体门控离子通道。

DOI：
10.1016/j.pep.2017.03.006
发表时间：
2017
期刊：
Protein expression and purification
影响因子：
1.6
作者：
Elberson,BenjaminW;Whisenant,TyE;Cortes,DMarien;Cuello,LuisG
通讯作者：
Cuello,LuisG

The nonconducting W434F mutant adopts upon membrane depolarization an inactivated-like state that differs from wild-type Shaker-IR potassium channels.

DOI：
10.1126/sciadv.abn1731
发表时间：
2022-09-16
期刊：
Science advances
影响因子：
13.6
作者：
通讯作者：

An improved method for the cost-effective expression and purification of large quantities of KcsA.

一种用于经济有效地表达和纯化大量 KcsA 的改进方法。

DOI：
10.1016/j.pep.2016.07.002
发表时间：
2016
期刊：
Protein expression and purification
影响因子：
1.6
作者：
Tilegenova,Cholpon;Vemulapally,Spandana;Cortes,DorisM;Cuello,LuisG
通讯作者：
Cuello,LuisG

CW-EPR Spectroscopy and Site-Directed Spin Labeling to Study the Structural Dynamics of Ion Channels.

CW-EPR 光谱和定点自旋标记研究离子通道的结构动力学。

DOI：
10.1007/978-1-4939-7362-0_21
发表时间：
2018
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
0
作者：
Tilegenova,Cholpon;Elberson,BenjaminW;MarienCortes,D;Cuello,LuisG
通讯作者：
Cuello,LuisG

Activation of the mechanosensitive ion channel MscL by mechanical stimulation of supported Droplet-Hydrogel bilayers.

通过机械刺激支持的液滴-水凝胶双层来激活机械敏感离子通道 MscL。

DOI：
10.1038/srep45180
发表时间：
2017
期刊：
Scientific reports
影响因子：
4.6
作者：
Rosholm,KadlaR;Baker,MatthewAB;Ridone,Pietro;Nakayama,Yoshitaka;Rohde,PaulR;Cuello,LuisG;Lee,LawrenceK;Martinac,Boris
通讯作者：
Martinac,Boris