The Molecular Basis of KCNH Channel Regulation by the EAG Domain

Abstract

The KCNH voltage dependent potassium channels are key regulators of cellular excitability, involved in cardiac long QT syndrome type 2 (LQTS2), epilepsy, schizophrenia and cancer. The intracellular domains of KCNH channels are structurally distinct from other voltage-gated channels. The amino-terminal region contains an eag domain, which includes a Per-Arnt-Sim (PAS) module and a PAS-cap region, while the carboxy-terminal region encompasses a cyclic nucleotide-binding homology domain (CNBHD), connected to the pore domain through a C-linker domain. These specialized intracellular domains are the site of many disease-causing mutations and bestow unique gating and regulation on KCNH channels. It has been suggested that the eag domain may interact with either the S4-S5 linker or the CNBHD in human ERG (hERG) and EAG channels. We have used fluorescence approaches to determine that the eag domain and the CNBHD, from the mEAG1 channel, form a complex in solution, with an apparent affinity of 13.2 ± 2.3 μM. Moreover, an equimolar mixture of purified eag domain and CNBHD produced co-crystals of the complex, belonging to the P65 space group, which diffracted to 2.0 A resolution. The structure of the eag domain-CNBHD complex was solved using molecular replacement with mEAG1 CNBHD as a model. Harboring many LQTS2 and cancer-associated mutations, the eag domain-CNBHD interface involves three important regions: (i) the intrinsic ligand motif, a unique structural feature of the CNBHD; (ii) the post-CNBHD region, known to mediate EAG channels regulation by a variety of cellular signaling events; and finally, (iii) the PAS-cap region, which constitutes the first 25 amino acids of the eag domain, and forms a highly conserved amphipathic helix (αCAP). The structure of the EAG domain-CNBHD complex of mEAG1 provides a detailed physiological and pathophysiological description of the intracellular domain of the KCNH channels.