Allosteric Regulation of KCNH Channels

KCNH通道，如EAG和HERG，在神经系统中发挥重要的生理作用，并 癫痫和心律失常等疾病的靶标。它们是恶性肿瘤的新兴生物标记物 以及在多种血癌和肿瘤中的增殖。这一系列渠道的独特之处在于 保守的细胞内结构域，经过数千年的进化，发挥着独特的生理作用。在 在之前的项目期间，PI和Co-I解决了关于C末端环核苷酸的作用的细节- 在门控中结合同源结构域(CNBhD)，并首次提供了对Per-Arnt-Arnt-R动态行为的见解。 SIM(PAS)域。我们以单链片段(ScFv)抗体的形式产生了新的试剂， 我们表现出了发挥治疗潜力的有益的天花板效应，可以提供保护 心律不齐。在这里，我们开发了一个新的模型，通过相互作用来实现浇注的动态调节 PAS结构域、CNBhD和基于最近闭合和开放通道的冷冻-EM结构的C-连接子。我们 将用钙调蛋白(CaM)测试从EAG1冷冻-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，即PAS-CAP如何 调节通道门控scFv抗体作为工具来监控可访问性的状态依赖变化，并通过 通过交联化取代的非天然氨基酸固定结构域。我们将清点传球的数量 含PAS(1a)和不含PAS(1b)亚基的异构体HERG通道中的结构域 异种系统和天然组织。为了回答赫尔格化学计量学这个长期存在的问题，我们 将使用异构体特异性单链抗体与一种新的单分子技术相结合，这种技术可以检测到 与未标记的通道亚基具有适度亲和力的抗体的个别结合事件 适用于天然组织。广泛的生化、生物物理和功能方法反映了 参与实验室的优势互补性很强。鉴于KCNH家族的重要性 许多生理和疾病过程的通道，从这里的工作中预期的进展，取得了 最近的前沿概念和技术发展所带来的可能性，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.