Visualizing the divergent conformational dynamics of KCNH channels

可视化 KCNH 通道的不同构象动力学

• 批准号: 10525010
• 负责人: Sara J. Codding
• 金额: $ 9.81万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 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/10525010
• 关键词: 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; Long QT Syndrome; Malignant Neoplasms; Measurable; Measures; Membrane; Membrane Potentials; Metal Binding Site; Modeling; Molecular Conformation; Motion; Movement; Mutagenesis; Mutation; Nature; Neurons; Phenylalanine; Physiological; Physiology; Plant Roots; Potassium; Potassium Channel; Proteins; Recovery; Regulation; Reporting; Research; Resolution; Role; Seizures; Site; Structure; Sudden Death; Syndrome; Testing; Tissues; Transition Elements; Ultraviolet Rays; Voltage-Gated Potassium Channel; cancer type; comparative; crosslink; experimental study; extracellular; gain of function; heart rhythm; insight; patch clamp; response; sensor; stoichiometry; sudden cardiac death; therapeutic target; therapeutically effective; voltage; voltage clamp

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.

项目概要 KCNH 通道家族包括人类 ether á go-go 相关基因 (hERG、KCNH2) 钾 在心脏中表达的通道，负责使动作电位复极化，并且，哺乳动物 etherá go-go 基因 (EAG、KCNH1) 钾通道在神经元组织中表达，有助于 hERG 在心脏健康中的作用已得到充分研究，hERG 突变会导致 QT 间期延长。 相比之下，EAG 的生理作用相对较少被研究，但人类 EAG 已经结束。 在许多类型的癌症中表达，新发现的基因突变与癫痫源性寺庙有关 此外，尽管 EAG 受到钙传感器的抑制，但 Baraitser 和 Zimmerman-Leband 综合征。 CaM 和 S100B 蛋白的化学计量、钙占有率和协同性仍有待揭示。 虽然 hERG 和 EAG 通道具有高度的序列相似性、域拓扑和结构相似性，但它们 我们率先提出每个 KCNH 通道都具有不同的门控动力学和调节。 和独特的门控动力学，产生独特的通道动力学，以调整各个通道的精确度 生理作用和这些动态被生理相关效应器改变。 我们使用非结构求解的 KCNH 通道 hERG 和 EAG 进行测量和建模。 规范氨基酸（ncAA）作为小型基因编码的非扰动探针来研究通道动力学。 我们研究了 hERG 缓慢失活的特征，部分原因是电压依赖性 增强（VDP）并表现为失活电压依赖性的超极化转变 VDP 会因急性疾病期间发生的细胞外 pH 值降低而降低。 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) 并使用过渡金属福斯特共振能量转移 (tmFRET) 以 10-20Å 分辨率测量动态运动，以测量 hERG VDP 动态并检查 我们将使用从 tmFRET 获得的距离作为约束来可视化 hERG 中的 VDP。 然后，我们通过 Rosetta 模型研究了高度保守的 KCNH 内在配体基序 (IL) 在 EAG 动力学。在 EAG 中，IL 的突变会改变通道动力学以减缓激活并消除 Cole-Moore。 我们在 IL 中加入了可光交联的 ncAA 4-苯甲酰基-L-苯丙氨酸 (BZF)，并使用紫外线。 最后，我们使用传统的 FRET 方法来检查 EAG IL 动力学对通道动力学的损失。 旨在确定 EAG 钙传感器蛋白调节的保守性并检查是否发生突变 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 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。