Allosteric Regulation of KCNH Channels

KCNH通道如EAG和hERG在神经系统中起重要的生理作用， 癫痫和心律失常等疾病的靶点。它们是恶性肿瘤的新兴生物标志物 以及在广泛的血液癌症和肿瘤中的增殖。这一系列渠道的独特之处在于， 保守的细胞内结构域已经进化了数千年，以发挥独特的生理作用。在 在上一个项目期间，PI和Co-I解决了关于C末端环核苷酸作用的细节- 结合同源结构域（CNBhD）的门控，并提供了第一个洞察到的Per-Arnt的动态行为， Sim（PAS）结构域。我们以单链片段（scFv）抗体的形式产生了新的试剂， 我们显示出了发挥治疗潜力的有益的天花板效应， 心律不齐在这里，我们开发了一个新的模型，通过门控的相互作用， PAS结构域，CNBhD和C-接头基于最近的封闭和开放通道的冷冻电镜结构。我们 将用钙调蛋白（CaM）检验EAG 1冷冻EM结构中出现的假设，以阐明 CaM抑制EAG 1通道功能的机制。我们将测试一个新的假设， 调节通道门控scFv抗体作为监测可及性的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所述结构域。我们将统计PAS的数量 异聚体hERG通道中的结构域，在两个结构域中都包含含PAS（1a）和无PAS（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 将使用同种型特异性scFv与一种新的单分子技术相结合， 与未标记通道亚基具有适度亲和力的抗体的单独结合事件，并且同样 适用于天然组织。生物化学、生物物理和功能方法的广泛范围反映了 参与实验室的优势高度互补。鉴于KCNH家族的重要性， 通道这么多的生理和疾病过程，从这里的工作预期的进展， 通过最近的尖端概念和技术发展，将产生广泛的影响。

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.