Human ether-á-go-go-related gene (hERG) channels are key regulators of cardiac repolarization, neuronal excitability, and tumorigenesis. hERG channels contain N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBH) domains with many long-QT syndrome (LQTS)-causing mutations located at the interface between these domains. Despite the importance of PAS/CNBH domain interactions, little is known about their affinity. Here, we used the surface plasmon resonance (SPR) technique to investigate interactions between isolated PAS and CNBH domains and the effects of LQTS-causing mutations R20G, N33T, and E58D, located at the PAS/CNBH domain interface, on these interactions. We determined that the affinity of the PAS/CNBH domain interactions was ∼1.4 μM. R20G and E58D mutations had little effect on the domain interaction affinity, while N33T abolished the domain interactions. Interestingly, mutations in the intrinsic ligand, a conserved stretch of amino acids occupying the beta-roll cavity in the CNBH domain, had little effect on the affinity of PAS/CNBH domain interactions. Additionally, we determined that the isolated PAS domains formed oligomers with an interaction affinity of ∼1.6 μM. Coexpression of the isolated PAS domains with the full-length hERG channels or addition of the purified PAS protein inhibited hERG currents. These PAS/PAS interactions can have important implications for hERG function in normal and pathological conditions associated with increased surface density of channels or interaction with other PAS-domain-containing proteins. Taken together, our study provides the first account of the binding affinities for wild-type and mutant hERG PAS and CNBH domains and highlights the potential functional significance of PAS/PAS domain interactions.

中文翻译：

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研究 hERG 通道中 PAS 和 CNBH 结构域的相互作用以及长 QT 综合征引起突变与表面等离子共振的影响。

人类 ether-á-go-go 相关基因 (hERG) 通道是心脏复极、神经元兴奋性和肿瘤发生的关键调节因子。hERG 通道包含 N 末端 Per-Arnt-Sim (PAS) 和 C 末端环核苷酸结合同源性 (CNBH) 结构域，其中许多导致长 QT 综合征 (LQTS) 的突变位于这些结构域之间的界面。尽管 PAS/CNBH 域相互作用很重要，但对它们的亲和力知之甚少。在这里，我们使用表面等离子体共振 (SPR) 技术来研究孤立的 PAS 和 CNBH 结构域之间的相互作用，以及位于 PAS/CNBH 结构域界面的 LQTS 引起突变 R20G、N33T 和 E58D 对这些相互作用的影响。我们确定 PAS/CNBH 结构域相互作用的亲和力约为 1.4 μM。R20G 和 E58D 突变对域相互作用亲和力几乎没有影响，而 N33T 消除了域相互作用。有趣的是，内在配体（占据 CNBH 结构域中的 beta-roll 腔的一段保守氨基酸）的突变对 PAS/CNBH 结构域相互作用的亲和力几乎没有影响。此外，我们确定分离的 PAS 结构域形成了相互作用亲和力约为 1.6 μM 的寡聚体。分离的 PAS 结构域与全长 hERG 通道的共表达或添加纯化的 PAS 蛋白抑制 hERG 电流。这些 PAS/PAS 相互作用对正常和病理条件下的 hERG 功能具有重要意义，与通道表面密度增加或与其他含 PAS 结构域的蛋白质相互作用有关。综合起来，