Determining the Role of the hERG Intracellular Loop Regions in Regulating Channel Gating

Abstract

Human ether-a-go-go-related gene (hERG) potassium channels exhibit unique kinetic properties, including unusually slow deactivation kinetics, which help to specialize them for their role in the heart. Previously, we demonstrated that an N-terminal Per-Arnt-Sim (PAS) domain is essential in regulating channel deactivation kinetics, and that this regulation occurs through a direct interaction with a cyclic nucleotide binding domain (CNBD) in the C-terminal tail. Recent evidence suggests the intracellular loop between the S4 and S5 transmembrane domains (S4-S5 linker) may also be of importance in regulating both activation and deactivation gating and that modulation of gating by the N-terminal PAS domain may act via the S4-S5 linker; however, the mechanism for this remains unclear. Here, we sought to investigate the role of the S4-S5 linker, together with the PAS domain, in regulating channel gating by performing site-directed mutagenesis within the S4-S5 linker region. We found that mutations in the S4-S5 linker produced channels with altered gating properties, including accelerated deactivation kinetics and shifts in the steady-state activation curves, supporting the notion that this region may play an important role in gating. For example, hERG E544R mutant channels exhibited faster deactivation kinetics and a rightward shift in the steady-state activation curve to more positive potentials. On the other hand, hERG Y545A mutant channels also exhibited accelerated deactivation kinetics, but instead showed a leftward shift in the steady-state activation curve to more negative potentials. Together, these experiments work towards elucidating the role of the S4-S5 linker region in regulating hERG channel gating.