Abstract

Human KCNH2 (hKCNH2, Ether-a-go-go (EAG)-Related Gene, hERG) are best known for their role in cardiac action potentials repolarization and have key roles in various pathologies. As other KCNH family members, hKCNH2 contains a unique intracellular complex crucial for channel function, consisting of an N-terminal eag domain and a C-terminal cyclic nucleotide-binding homology domain (CNBHD). Previous studies demonstrated that the CNBHD is occupied by an intrinsic ligand motif (ILM), in a self-liganded conformation, providing a structural mechanism for the lack of KCNH channels regulation by cyclic nucleotides. While significant advancements in structural and functional characterizations of the CNBHD of KCNH channels have been made, a high-resolution structure of the hKCNH2 intracellular complex was missing. Here, we report the 1.5 [A] resolution structure of the hKCNH2 channel CNBHD. The structure reveals the canonical fold shared by other KCNH family members, where the spatial organization of the ILM is preserved within the {beta}-roll region. Moreover, measurements of small-angle X-ray scattering profile in solution, as well as comparison with a recent nuclear magnetic resonance (NMR) analysis of hKCNH2, revealed high agreement with the structure, indicating an overall low flexibility in solution. Importantly, we identified a novel salt-bridge (E807-R863), which was not previously resolved in the NMR and cryogenic electron microscopy (cryo-EM) structures. Strikingly, electrophysiological analysis of charge reversal mutations revealed its crucial role for hKCNH2 function. Moreover, comparison with other KCNH members revealed the structural conservation of this salt-bridge, consistent with its functional significance. Together with the available structure of the mouse KCNH1 intracellular complex, and previous electrophysiological and spectroscopic studies of KCNH family members, we propose that this salt-bridge serves as a strategically positioned linchpin to support both the spatial organization of the ILM and the maintenance of the intracellular complex interface.\n\nSummaryHuman KCNH2 are key channels governing cardiac repolarization. Here, a 1.5 [A] resolution structure of their cyclic nucleotide-binding homology domain is presented. Structural analysis and electrophysiological validation reveal a novel salt-bridge, playing an important role in hKCNH2 functional regulation.