The hERG potassium channel intrinsic ligand regulates N- and C-terminal interactions and channel closure

Abstract

Human ether-à-go-go–related gene (hERG, KCNH2) voltage-activated potassium channels are critical for cardiac excitability. hERG channels have characteristic slow closing (deactivation), which is auto-regulated by a direct interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). hERG channels are not activated by the binding of extrinsic cyclic nucleotide ligands, but rather bind an “intrinsic ligand” that is composed of residues 860–862 within the CNBHD and mimics a cyclic nucleotide. The intrinsic ligand is located at the PAS–CNBHD interface, but its mechanism of action in hERG is not well understood. Here we use whole-cell patch-clamp electrophysiology and FRET spectroscopy to examine how the intrinsic ligand regulates gating. To carry out this work, we coexpress PAS (a PAS domain fused to cyan fluorescent protein) in trans with hERG “core” channels (channels with a deletion of the PAS domain fused to citrine fluorescent protein). The PAS domain in trans with hERG core channels has slow (regulated) deactivation, like that of WT hERG channels, as well as robust FRET, which indicates there is a direct functional and structural interaction of the PAS domain with the channel core. In contrast, PAS in trans with hERG F860A core channels has intermediate deactivation and intermediate FRET, indicating perturbation of the PAS domain interaction with the CNBHD. Furthermore, PAS in trans with hERG L862A core channels, or PAS in trans with hERG F860G,L862G core channels, has fast (nonregulated) deactivation and no measurable FRET, indicating abolition of the PAS and CNBHD interaction. These results indicate that the intrinsic ligand is necessary for the functional and structural interaction between the PAS domain and the CNBHD, which regulates the characteristic slow deactivation gating in hERG channels.

Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge

“…Unlike the cyclic-nucleotide gated (CNG) and HCN channels, the CNBHD of KCNH encompasses the ILM, spatially replacing the cyclic nucleotides, blocking their binding site (Brelidze et al, 2012;Marques-Carvalho et al, 2012;Haitin et al, 2013). Ever since its identification (Brelidze et al, 2012;Marques-Carvalho et al, 2012), the ILM was shown to play a crucial role in KCNH channel family function (Brelidze et al, 2013;Zhao et al, 2017;Codding & Trudeau, 2019). Nevertheless, the mechanism by which it participates in channel gating modulation remains ambiguous.…”

“…Previous studies focused on the ILM itself and its effect on the interaction between the CNBHD and the eag domain (Zhao et al, 2017;Codding & Trudeau, 2019). Here, based on our structural analysis, we focused on a putative salt-bridge which deemed important for stabilization of the ILM.…”

“…4). Previously, based on electrophysiological and spectroscopic studies of intact channels, it was suggested that the interaction between the CNBHD and the eag domain plays a role in the functional regulation of KCNH channels (Dai & Zagotta, 2017;Codding & Trudeau, 2019). However, the only available high-resolution structure of the intracellular complex of KCNH channels remains the co-crystal structure of the mouse KCNH1 eag domain-CNBHD (Haitin et al, 2013).…”

“…(ii) The b-roll, featuring the canonical eight b strands fold (b1 -b8), mediating ligand binding in HCN and cyclic nucleotide-gated ion channels (Craven & Zagotta, 2006); (iii) The C-helix (helix aC), which, together with aB, flanks the b-roll and was shown to underlie ligand-induced gating enhancement of cyclic nucleotide-gated channels (Puljung & Zagotta, 2013); And, (iv) the KCNH family unique ILM, consisting of a short b strand (b9) (Fig. 1D), which is involved in the formation of the interaction interface with the N-terminal eag domain (Haitin et al, 2013;Wang & MacKinnon, 2017), and differentially affects KCNH channels activation (Zhao et al, 2017) and deactivation (Codding & Trudeau, 2019). Together, the overall fold of the CNBHD of hKCNH2 is reminiscent to that observed in other CNBD-containing proteins (Rehmann et al, 2007).…”

First steps

“…The papers published by the winners attest to the quality of work in JGP . The awards were presented in San Diego by the Society of General Physiology (SGP) President, Crina Nimigean, to Stephan Pless (Cranefield Awardee) for his work on P2X receptors (Gasparri et al, 2019), Sarah Codding (Postdoc Award) for her study of hERG channels (Codding and Trudeau, 2019), and Aaron Bozzi (Student Award) for his work on metal transporters (Bozzi et al, 2019).…”