Allosteric Regulation of KCNH Channels

KCNH 通道的变构调节

• 批准号: 10461772
• 负责人: Gail A Robertson
• 金额: $ 42.84万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10461772
• 关键词: Affect; Affinity; Allosteric Regulation; Antibodies; Arrhythmia; Behavior; Binding; Biochemical; Biochemistry; Biological Assay; Biological Markers; Biophysics; Brain; C-terminal; Calcium; Calmodulin; Clinical; Collaborations; Computer Models; Cryoelectron Microscopy; Crystallization; Cyclic Nucleotides; Data; Development; Disease; Epilepsy; Ethers; Evaluation; Event; Family; Hematopoietic Neoplasms; Immobilization; Individual; Ion Channel; Laboratories; Learning; Ligands; Malignant Neoplasms; Maps; Memory; Modeling; Molecular Machines; Monitor; Motion; Movement; Mutagenesis; N-terminal; Nervous system structure; Peer Review; Physiological; Predisposition; Presynaptic Terminals; Process; Property; Protein Isoforms; Proteins; Publications; Publishing; Reagent; Reporting; Roentgen Rays; Role; Schizophrenia; Side; Structure; System; Technology; Testing; Therapeutic; Tissues; Transforming Protein ERG; Translations; Universities; Up-Regulation; Wisconsin; Work; base; cancer type; clinically relevant; crosslink; experimental study; insight; novel; novel strategies; sensor; single molecule; stoichiometry; structural biology; sudden cardiac death; therapeutic target; tool; translational potential; tumor; unnatural amino acids; voltage; waveguide

KCNH channels such as EAG and hERG serve important physiological roles in the nervous system and are targets for disease such as epilepsy and cardiac arrhythmia. They are emerging biomarkers for malignancy and proliferation in a wide range of blood cancers and tumors. Unique to this family of channels are highly conserved intracellular domains that have evolved over the millennia to serve unique physiological roles. In the previous project period, the PI and Co-I resolved details about the role of the C-terminal cyclic nucleotide- binding homology domain (CNBhD) in gating, and provided first insights into dynamic behavior of the Per-Arnt- Sim (PAS) domain. We generated new reagents in the form of single-chain fragment (scFv) antibodies, which we showed exerted therapeutic potential with a beneficial ceiling effect that could confer protection against arrhythmia. Here we have developed a new model for dynamic modulation of gating via the interactions of the PAS domain, CNBhD and the C-linker based on recent cryo-EM structures of closed and open channels. We will test hypotheses emerging from the EAG1 cryo-EM structure with calmodulin (CaM) to elucidate the mechanism by which CaM inhibits EAG1 channel function. We will test a new hypothesis for how the PAS-cap modulates channel gating scFv antibodies as tools to monitor state-dependent changes in accessibility and by immobilizing the domain by crosslinking substituted unnatural amino acids. We will count the number of PAS domains in heteromeric hERG channels comprising PAS-containing (1a) and PAS-less (1b) subunits in both heterologous systems and native tissues. To answer this long-standing question of hERG stoichiometry, we will use isoform-specific scFvs in combination with a novel single-molecule technology that can detect individual binding events of antibodies with modest affinities to untagged channel subunits and is equally applicable to native tissues. The broad range of biochemical, biophysical and functional approaches reflect highly complementary strengths of the participating laboratories. Given the importance of the KCNH family of channels to so many physiological and disease processes, the advances expected from the work here, made possible by recent cutting-edge conceptual and technical developments, will have broad implications.

EAG和HERG等KCNH渠道在神经系统中起重要的生理作用，并且是 疾病的靶标，例如癫痫和心律不齐。他们正在新兴的恶性生物标志物 以及在各种血液癌和肿瘤中的增殖。这个频道家族独有的很高 在千年中演变为独特的生理作用的保守细胞内结构域。在 上一个项目期，PI和CO-I解决了有关C末端循环核苷酸的作用的细节 在门控中结合同源域（CNBHD），并提供了对人类的动态行为的首先见解 SIM（PAS）域。我们以单链片段（SCFV）抗体的形式生成了新试剂，该试剂的形式 我们显示出具有有益上限效果的治疗潜力 心律不齐。在这里，我们开发了一种新的模型，用于通过相互作用的相互作用进行动态调制。 PAS结构域，CNBHD和C-LINKER基于封闭和开放通道的最新冷冻EM结构。我们 将测试从EAG1 Cryo-EM结构中出现的假设，以阐明 CAM抑制EAG1通道功能的机制。我们将测试一个新的假设，以了解PAS-CAP 调节通道门控SCFV抗体作为监视访问性和通过状态依赖性变化的工具 通过交联取代的非天然氨基酸来固定结构域。我们将计算PA的数量 杂物HERG通道中的域，包括含PAS的（1A）和PAS-INS-be-pa-neber（1B）亚基 异源系统和天然组织。为了回答这个长期存在的HERG化学计量学问题，我们 将与可以检测到的新型单分子技术结合使用同工型特异性SCFV 具有适度亲和力的抗体的单个结合事件，对未标记的通道亚基，同样是 适用于天然组织。广泛的生化，生物物理和功能方法反映了 参与实验室的高度互补优势。考虑到KCNH家族的重要性 在这里的工作预期的进步，到达了许多生理和疾病过程的渠道 通过最近的最先进的概念和技术发展，可能会有广泛的影响。

项目成果

Screening for Non-Pore-Binding Modulators of EAG K+ Channels.

• DOI: 10.1177/1087057116636592
• 发表时间: 2016-08
• 期刊: Journal of biomolecular screening
• 作者: Fernandes AS;Morais-Cabral JH;Harley CA
• 通讯作者: Harley CA

Administration of Non-Torsadogenic human Ether-à-go-go-Related Gene Inhibitors Is Associated with Better Survival for High hERG-Expressing Glioblastoma Patients.

• DOI: 10.1158/1078-0432.ccr-15-3169
• 发表时间: 2017-01-01
• 期刊: Clinical cancer research : an official journal of the American Association for Cancer Research
• 作者: Pointer KB;Clark PA;Eliceiri KW;Salamat MS;Robertson GA;Kuo JS
• 通讯作者: Kuo JS

Structural properties of PAS domains from the KCNH potassium channels.

• DOI: 10.1371/journal.pone.0059265
• 发表时间: 2013
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Adaixo R;Harley CA;Castro-Rodrigues AF;Morais-Cabral JH
• 通讯作者: Morais-Cabral JH

Hysteretic hERG channel gating current recorded at physiological temperature.

• DOI: 10.1038/s41598-022-10003-7
• 发表时间: 2022-04-08
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Jones DK

Molecular Insights into the Mechanism of Calmodulin Inhibition of the EAG1 Potassium Channel.

• DOI: 10.1016/j.str.2016.07.020
• 发表时间: 2016-10-04
• 期刊: STRUCTURE
• 影响因子: 5.7
• 作者: Marques-Carvalho, Maria Joao;Oppermann, Johannes;Munoz, Eva;Fernandes, Andreia S.;Gabant, Guillaume;Cadene, Martine;Heinemann, Stefan H.;Schoenherr, Roland;Morais-Cabral, Joao Henrique
• 通讯作者: Morais-Cabral, Joao Henrique