Visualizing the divergent conformational dynamics of KCNH channels

可视化 KCNH 通道的不同构象动力学

基本信息

批准号：
10682486
负责人：
Sara J. Codding
金额：
$ 12.5万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-11 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10682486
关键词：
Acceleration Action Potentials Acute Disease Alanine Amino Acids Arrhythmia Binding Sites Biophysics Brain Calcium Calcium Channel Calmodulin Cancer Biology Cardiac Cardiac Myocytes Cardiac health Cells Characteristics Chemosensitization Cryoelectron Microscopy DNA Sequence Alteration Data Dependence Development Disease Electrodes Electrophysiology (science)Epilepsy Ethers Exhibits Exposure to Family Fluorescence Fluorescence Resonance Energy Transfer Fluorometry Functional disorder Genes Goals Health Heart Human Individual Inherited Kinetics Lead Ligands Link Malignant Neoplasms Measurable Measures Membrane Membrane Potentials Metal Binding Site Modeling Molecular Conformation Motion Movement Mutagenesis Mutation Nature Neurons Phenylalanine Physiologic pulse Physiological Physiology Potassium Potassium Channel Proteins Recovery Regulation Reporting Research Resolution Role Seizures Site Structure Sudden Death Syndrome Testing Tissues Transition Elements Type 2 Long QT syndrome Ultraviolet Rays Visualization Voltage-Gated Potassium Channel cancer type comparative experimental study extracellular gain of function heart rhythm insight overexpression patch clamp response sensor stoichiometry sudden cardiac death therapeutic target therapeutically effective voltage voltage clamp

项目摘要

Project Summary The KCNH channel family includes both the Human ether á go-go related gene (hERG, KCNH2) potassium channel that is expressed in the heart and responsible for repolarizing the action potential and, the mammalian ether á go-go gene (EAG, KCNH1) potassium channel is expressed in neuronal tissue and contributes to electrical excitability. The role of hERG in cardiac health is well studied and mutations in hERG cause Long QT type 2 syndrome. Comparatively, the physiological role of EAG is relatively unstudied, yet human EAG is over expressed in many types of cancer and newly identified genetic mutations are linked to epileptogenic Temple- Baraitser and Zimmerman-Leband syndromes. Additionally, although EAG is inhibited by calcium sensor proteins CaM and S100B, the stoichiometry, calcium occupancy and cooperativity remain to be uncovered. While hERG and EAG channels share high sequence similarity, domain topology, and structural similarity they have highly divergent gating kinetics and regulation. We hypothesize that each KCNH channel has divergent and distinct gating dynamics that give rise to unique channel kinetics to tune individual channels for their precise physiological roles and these dynamics are altered by physiologically relevant effectors. In this proposal we measure and model the dynamics of the structurally solved KCNH channels hERG and EAG. We use non- canonical amino acids (ncAA) as small genetically encoded non-perturbing probes to study channel dynamics. We examine the characteristic slow deactivation of hERG that has been partially attributed to voltage dependent potentiation (VDP) and manifests as a hyperpolarizing shift in the voltage dependence of deactivation compared to activation. VDP is reduced in response to lowered extracellular pH which can occur during acute disease states and accelerates hERG deactivation. We incorporate the fluorescent ncAA 3-[(6-acetyl-2- naphthalenyl)amino]-L-alanine (L-ANAP) in hERG and use transition metal Förster resonance energy transfer (tmFRET) to measure dynamic motions at 10-20Å resolution to measure hERG VDP dynamics and examine how it is altered by pH. We will use distances obtained from tmFRET as constraints to visualize VDP in hERG with Rosetta modeling. We then examine the role of the highly conserved KCNH intrinsic ligand motif (IL) in EAG kinetics. In EAG, mutations in the IL alter channel kinetics to slow activation and abolish the Cole-Moore shift. We incorporate the photo-crosslinkable ncAA 4-benzoyl-L-phenylalanine (BZF) at the IL and use ultraviolet light to examine the loss of EAG IL dynamics on channel kinetics. Finally, with a traditional FRET approach we aim to determine the conserved nature of calcium sensor protein regulation of EAG and examine if mutations linked to TB/ZL syndromes alter EAG calcium regulation as it is unclear if calcium dependent channel inhibition is lost in disease states. Due to the roles of hERG in cardiac excitability and arrhythmia, and EAG in TB/ZL and cancer, determining the dynamic gating mechanisms of these channels directly impacts health and disease.

项目摘要 KCNH频道家族包括人类醚ÁGo-Go相关基因（HERG，KCNH2）钾在心脏中表达的渠道，负责重新振动动作电位，哺乳动物以太余基因（EAG，KCNH1）钾通道在神经元组织中表达，并有助于电气令人兴奋。 HERG在心脏健康中的作用很好地研究了，而在HERG中的突变导致QT很长类型2综合征。相比之下，EAG的身体作用相对未被研究，但人EAG已经结束在许多类型的癌症和新鉴定的遗传突变中表达与癫痫发生寺庙有关 Baraitser和Zimmerman-Leband综合征。另外，尽管EAG被钙传感器抑制蛋白质CAM和S100B，化学计量，钙占用率和协调仍有待发现。 HERG和EAG通道具有很高的序列相似性，域拓扑和结构相似性具有高度不同的门控动力学和调节。我们假设每个KCNH通道都有不同以及独特的门控动力学，引起独特的频道动力学，以调整单个渠道的精确渠道生理角色和这些动力学因物理相关的影响而改变。在这个建议中，我们测量和建模结构解决的KCNH通道的动力学HERG和EAG。我们使用非 - 规范氨基酸（NCAA）作为小遗传编码的非扰动问题，以研究通道动力学。我们检查了HERG的特征缓慢停用，该远离电压依赖性的部分归因于电压增强（VDP）并表现为停用电压依赖性的过度变化激活。 VDP响应于急性疾病期间可能发生的细胞外pH值降低国家并加速HERG停用。我们结合了荧光NCAA 3 - [（6-乙酰基-2-- HERG中的萘基）氨基] -l-丙氨酸（L-ANAP），并使用过渡金属Förster共振能量转移（TMFRET）以10-20Å分辨率测量动态运动以测量HERG VDP动力学并检查如何通过pH改变它。我们将使用从tmfret获得的距离作为约束来可视化herg中的VDP 与Rosetta建模。然后，我们检查高度组成的KCNH固有配体基序（IL）在 EAG动力学。在EAG中，IL中的突变改变了通道动力学以减慢激活和废除Cole-Moore 我们在IL上融合了可互连接的NCAA NCAA 4-苯甲酰l-苯丙氨酸（BZF），并使用紫外线光以检查通道动力学上EAG IL动力学的损失。最后，采用传统的肉体方法目的是确定EAG钙传感器蛋白调节的保守性质和检查突变与TB/ZL综合征有关改变EAG钙调节的链接，因为尚不清楚钙依赖性通道抑制是否存在在疾病状态下丢失。由于HERG在心脏兴奋性和心律不齐中的作用，以及在TB/ZL中的EAG和EAG 癌症，确定这些通道的动态门控机制直接影响健康和疾病。