Program in Neuroscience and 2 Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
J Gen Physiol. 2011 Mar;137(3):315-25. doi: 10.1085/jgp.201010582.
Human ether-á-go-go-related gene (hERG) potassium channels have voltage-dependent closing (deactivation) kinetics that are unusually slow. A Per-Arnt-Sim (PAS) domain in the cytoplasmic N-terminal region of hERG regulates slow deactivation by making a direct interaction with another part of the hERG channel. The mechanism for slow deactivation is unclear, however, because the other regions of the channel that participate in regulation of deactivation are not known. To identify other functional determinants of slow deactivation, we generated hERG channels with deletions of the cytoplasmic C-terminal regions. We report that hERG channels with deletions of the cyclic nucleotide-binding domain (CNBD) had accelerated deactivation kinetics that were similar to those seen in hERG channels lacking the PAS domain. Channels with dual deletions of the PAS domain and the CNBD did not show further acceleration in deactivation, indicating that the PAS domain and the CNBD regulate deactivation by a convergent mechanism. A recombinant PAS domain that we previously showed could directly regulate PAS domain-deleted channels did not regulate channels with dual deletions of the PAS domain and CNBD, suggesting that the PAS domain did not interact with CNBD-deleted channels. Biochemical protein interaction assays showed that glutathione S-transferase (GST)-PAS (but not GST) bound to a CNBD-containing fusion protein. Coexpression of PAS domain-deleted subunits (with intact C-terminal regions) and CNBD-deleted subunits (with intact N-terminal regions) resulted in channels with partially restored slow deactivation kinetics, suggesting regulatory intersubunit interactions between PAS domains and CNBDs. Together, these data suggest that the mechanism for regulation of slow deactivation in hERG channels is an interaction between the N-terminal PAS domain and the C-terminal CNBD.
人 ether-á-go-go 相关基因 (hERG) 钾通道具有电压依赖性关闭（失活）动力学，其失活动力学非常缓慢。hERG 细胞质 N 端区域中的 Per-Arnt-Sim (PAS) 结构域通过与 hERG 通道的另一部分直接相互作用来调节缓慢失活。然而，缓慢失活的机制尚不清楚，因为参与失活调节的通道的其他区域尚不清楚。为了确定缓慢失活的其他功能决定因素，我们生成了具有细胞质 C 端区域缺失的 hERG 通道。我们报告说，具有环核苷酸结合结构域 (CNBD) 缺失的 hERG 通道具有加速的失活动力学，类似于缺乏 PAS 结构域的 hERG 通道。具有 PAS 结构域和 CNBD 双重缺失的通道在失活中没有进一步加速，表明 PAS 结构域和 CNBD 通过收敛机制调节失活。我们之前表明可以直接调节 PAS 结构域缺失通道的重组 PAS 结构域不能调节具有 PAS 结构域和 CNBD 双重缺失的通道，表明 PAS 结构域不与 CNBD 缺失的通道相互作用。生化蛋白质相互作用测定表明，谷胱甘肽 S-转移酶 (GST)-PAS（而不是 GST）与含有 CNBD 的融合蛋白结合。表达 PAS 结构域缺失亚基（具有完整的 C 端区域）和 CNBD 缺失亚基（具有完整的 N 端区域）导致部分恢复缓慢失活动力学的通道，表明 PAS 结构域和 CNBD 之间存在调节性亚基相互作用。这些数据表明，hERG 通道中缓慢失活调节的机制是 N 端 PAS 结构域与 C 端 CNBD 之间的相互作用。
