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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通道家族包括人etherago-go相关基因（hERG，KCNH 2）钾通道和人etherago-go相关基因（hERG，KCNH 2）钾通道。一种在心脏中表达并负责动作电位复极化的通道， ether á go-go基因（EAG，KCNH 1）钾通道在神经元组织中表达，电兴奋性hERG在心脏健康中的作用已得到充分研究，hERG突变可导致长QT间期 2型综合征。相比之下，EAG的生理作用还相对缺乏研究，而人类EAG已超过在许多类型的癌症中表达，新发现的基因突变与致癫痫性坦普尔- Baraitser和Zimmerman-Leband综合征。此外，虽然EAG被钙传感器抑制，蛋白质CaM和S100 B的化学计量、钙占有率和协同性仍有待于揭示。虽然hERG和EAG通道具有高度的序列相似性、结构域拓扑结构和结构相似性，但它们具有高度发散的门控动力学和调节。我们假设每个KCNH通道都有不同的以及不同的门控动力学，其产生独特的通道动力学，以调节各个通道，生理作用和这些动力学改变生理相关的效应。在本提案中，我们测量和模拟结构解析的KCNH通道hERG和EAG的动力学。我们使用非- 典型氨基酸（ncAA）作为小的遗传编码的非扰动探针来研究通道动力学。我们研究了hERG的特征性缓慢失活，这部分归因于电压依赖性增强（VDP），并表现为失活的电压依赖性相比，到激活。在急性疾病期间可能发生的细胞外pH值降低导致VDP降低状态和加速hERG失活。我们将荧光ncAA 3-[（6-乙酰基-2-甲基-N-乙酰基）-N-甲基-N-乙酰基] 萘基）氨基]-L-丙氨酸（L-ANAP）在hERG和使用过渡金属Förster共振能量转移（tmFRET）以10- 20 μ m分辨率测量动态运动，以测量hERG VDP动力学并检查我们将使用从tmFRET获得的距离作为约束条件来可视化hERG中的VDP 罗塞塔模型然后，我们研究了高度保守的KCNH内在配体基序（IL）在 EAG动力学在EAG中，IL中的突变改变通道动力学以减慢激活并消除Cole-Moore通道。班我们将光交联ncAA 4-苯甲酰基-L-苯丙氨酸（BZF）在IL和使用紫外光来检查通道动力学上的EAG IL动力学的损失。最后，使用传统的FRET方法，目的是确定钙传感器蛋白调节EAG的保守性，并检查突变是否与TB/ZL综合征相关的改变EAG钙调节，因为尚不清楚钙依赖性通道抑制是否在疾病状态下消失。由于hERG在心脏兴奋性和心律失常中的作用，以及EAG在TB/ZL和癌症，确定这些通道的动态门控机制直接影响健康和疾病。