N-Terminal Region Regulation of hERG Potassium Channel Gating: Contributions of the Eag/Pas Domain and the Proximal N-Terminus

Abstract

The Human Ether-á-go-go Related Gene (hERG) encodes a voltage-activated K+ channel. hERG contributes to the repolarization of the ventricular action potential as part of the IKr current and has also been shown to modulate neuronal firing frequency. hERG gating is characterized by rapid inactivation upon depolarization and rapid recovery from inactivation and slow closing (deactivation) upon repolarization. These factors combine to create a resurgent hERG current, where the current amplitude is paradoxically larger with repolarization than with depolarization. Previous data have shown that the N-terminal region of hERG regulates gating kinetics, however the molecular mechanisms of this regulation are not fully understood. Deletion of the hERG N-terminus (amino acids 2-354) has been shown to speed channel deactivation and recovery from inactivation compared to that of wild-type hERG channels. Relative outward current amplitude is also increased during the depolarization phase, leading to reduced current rectification. A genetically encoded eag domain fragment (amino acids 1-135) was shown to restore slow deactivation to N-truncated channels by forming a stable interaction with the C-terminal cyclic nucleotide binding domain (CNBD). Our present study sought to further investigate the contributions of the eag domain and proximal N-terminal region (amino acids 136-354) to hERG gating kinetics. We coexpressed genetically encoded N-terminal region fragments with hERG channels bearing a deletion of the full N-terminus (D2-354) or the eag domain (D2-135) in Xenopus oocytes and measured current with two-electrode voltage-clamp recordings. Here we report that eag domain-containing fragments (N1-135, N1-354) were demonstrated to reduce relative outward current, slow deactivation, and slow recovery from inactivation, resulting in channels with properties similar to those measured in wild-type hERG. The proximal N-terminal region was shown to be involved in steady-state activation.