N- and C-terminal interactions in KCNH channels: The spotlight on the intrinsic ligand

Tinatin I. Brelidze
2019 The Journal of General Physiology  
Human ether-à-go-go related gene (hERG) channels are widely studied potassium channels because of their importance for repolarization of the cardiac action potential (Sanguinetti and Tristani-Firouzi, 2006) . Inhibition of hERG channel currents due to inherited mutations or by drugs can lead to long-QT syndrome, a cardiac arrhythmia that may cause loss of consciousness and sudden cardiac death (Curran et al., 1995) . hERG channels belong to the KCNH family of potassium-selective, voltage-gated
more » ... on channels that also includes ether-à-go-go (EAG) and EAG-like (ELK) channels (Warmke and Ganetzky, 1994) . KCNH channels are assembled from four subunits, each of which contains a voltage sensor (VS) domain formed from the S1-S4 transmembrane segments and a central pore formed by S5-S6 (Warmke and Ganetzky, 1994; Wang and MacKinnon, 2017) . KCNH channels also contain two structured intracellular domains per subunit: the N-terminal Per-Arnt-Sym (PAS) domain and the C-terminal cyclic nucleotide-binding homology (CNBH) domain. The PAS and CNBH domains interact to confer key functional features, such as slow deactivation in hERG channels (Gustina and Trudeau, 2011; Gianulis et al., 2013) . Importantly, the interaction interface between the PAS and CNBH domains harbors many cardiac arrhythmia-associated mutations in hERG channels (Haitin et al., 2013) . A recent study by Codding and Trudeau in the Journal of General Physiology reveals that the intrinsic ligand, a conserved stretch of amino acids in the CNBH domain, is required for the PAS and CNBH domain interaction in hERG channels (Codding and Trudeau, 2018). The intrinsic ligand was discovered in the first crystal structures of the isolated CNBH domains of EAG and ELK channels (Brelidze et al., 2012; Marques-Carvalho et al., 2012) . These structures revealed that the cavity formed by antiparallel β-strands, where cyclic nucleotides would typically bind in other cyclic nucleotide-binding (CNB) domains, such as CNB domains of hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels, is occupied by a novel β-strand in KCNH channels. The novel β-strand and a few adjacent residues are conserved in KCNH channels and function as an intrinsic ligand, with two key residues of the β-strand structurally mimicking cAMP bound to HCN channels. These two residues are Y740 and L742 in zebrafish ELK (zELK) channels, Y699 and L701 in human EAG (hEAG) channels, and F860 and L862 in hERG channels. The aromatic residues are positioned where the purine ring of cAMP binds to HCN channels and the leucines where the cyclic phosphate of cAMP binds. Mutations of the intrinsic ligand affect gating (opening and closing) of KCNH channels, just like an extrinsic ligand would do (Brelidze et al., 2012 Marques-Carvalho et al., 2012; Zhao et al., 2017) . The presence of the intrinsic ligand explains the lack of cyclic nucleotide modulation in KCNH channels. Unlike HCN and cyclic nucleotide-gated (CNG) channels, which are regulated by direct binding of cyclic nucleotides to their CNB domains, KCNH channels are not directly regulated by cyclic nucleotides due to the steric hindrance of binding by the intrinsic ligand (Robertson et al., 1996; Brelidze et al., 2009) . The intrinsic ligand also interferes with the effects of flavonoids, which need to displace the intrinsic ligand in order to potentiate EAG channel currents . In their new study, Codding and Trudeau (2018) uncover a novel role of the intrinsic ligand as a structural glue that brings together the PAS and CNBH domains in KCNH channels. The investigators took advantage of the previously established FRETbased assay that reports on structural interactions between the PAS and CNBH domains (Gustina and Trudeau, 2009) and an electrophysiology-based in vitro assay that reports on functional interactions between these domains (Gustina and Trudeau, 2013) . The FRET-based assay involves coexpression of an isolated PAS domain fused to cyan fluorescent protein (CFP) and hERG channels with the deletion of the PAS domain (hERGΔPAS) with the distal C terminus fused to Citrine (Gustina and Trudeau, 2009, 2013). Any structural interaction between the PAS domain tagged with the CFP donor and the CNBH domain coupled to the Citrine acceptor leads to FRET. The
doi:10.1085/jgp.201812313 fatcat:leobobce7nbelg3sj2hzvoe53y