Mechanistic Insight into Human ether-à-go-go-related Gene (hERG) K+ Channel Deactivation Gating from the Solution Structure of the EAG Domain

Abstract

Human ether-à-go-go-related gene (hERG) K+ channels have a critical role in cardiac repolarization. hERG channels close (deactivate) very slowly, and this is vital for regulating the time course and amplitude of repolarizing current during the cardiac action potential. Accelerated deactivation is one mechanism by which inherited mutations cause long QT syndrome and potentially lethal arrhythmias. hERG deactivation is highly dependent upon an intact EAG domain (the first 135 amino acids of the N terminus). Importantly, deletion of residues 2–26 accelerates deactivation to a similar extent as removing the entire EAG domain. These and other experiments suggest the first 26 residues (NT1–26) contain structural elements required to slow deactivation by stabilizing the open conformation of the pore. Residues 26–135 form a Per-Arnt-Sim domain, but a structure for NT1–26 has not been forthcoming, and little is known about its site of interaction on the channel. In this study, we present an NMR structure for the entire EAG domain, which reveals that NT1–26 is structurally independent from the Per-Arnt-Sim domain and contains a stable amphipathic helix with one face being positively charged. Mutagenesis and electrophysiological studies indicate that neutralizing basic residues and breaking the amphipathic helix dramatically accelerate deactivation. Furthermore, scanning mutagenesis and molecular modeling studies of the cyclic nucleotide binding domain suggest that negatively charged patches on its cytoplasmic surface form an interface with the NT1–26 domain. We propose a model in which NT1–26 obstructs gating motions of the cyclic nucleotide binding domain to allosterically stabilize the open conformation of the pore.

Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels

“…The N-terminal N-Cap domains of Kv11.1 channels have been proposed to bind to various regions of the channel protein including the S4S5 linker (36,37) or the cNBH domain (38). Recently, Haitin et al (18) proposed that two charged residues, Arg 7 and Arg 8 , in the N-Cap domain of Kv10.1 channels form a functional interaction with E627R of the cNBH domain.…”

Demonstration of Physical Proximity between the N Terminus and the S4-S5 Linker of the Human ether-à-go-go-related Gene (hERG) Potassium Channel

“…However, it is difficult to translate our data to real distances because the probability of collision between the cysteine pairs forming disulfide bonds is greatly increased by fluctuations of otherwise flexible structures, even though they are located at a certain distance (40,42,(45)(46)(47). There is evidence that the N-terminal-most segments of the hERG N terminus and the S4-S5 linker constitute highly flexible and/or unstructured regions (28,33,36,39). The protein regions studied here are expected to be dynamically modulated during channel function, so it could be expected that collisions between the cysteine pairs may also be influenced by the molecular rearrangements associated with gating.…”

“…Therefore, it sheds significant new insight into the molecular basis of the protein-protein interactions between the N-terminal distal regions and the S4-S5 linker involved in the control of gating properties and hormonal modulation of hERG (1,2,24,(27)(28)(29)(30)(31)(32)(33)36).…”

Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels

“…Subsequent NMR studies from a number of laboratories have shown that the N-Cap contains an amphipathic helix (N-Cap helix, residues 13-23) and a flexible * This work was supported by Grant-in-aid G11S 5829 from the Health Foun-N-terminal tail (N-tail, residues 1-12) (13)(14)(15). More recently, using a construct in which the flexible N-terminal tail was deleted, Morais-Cabral and co-workers (26) showed that the N-Cap helix can fold back and interact with a surface-exposed hydrophobic patch on the remainder of the PAS domain.…”