Structure of the C-terminal region of an ERG channel and functional implications Tinatin I. Brelidzea,1, Elena C. Gianulisb, Frank DiMaioc, Matthew C. Trudeaub, and William N. Zagottaa,2 Departments of aPhysiology and Biophysics and cBiochemistry, University of Washington School of Medicine, Seattle, WA 98195; and bDepartment of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201 Edited by Richard W. Aldrich, University of Texas at Austin, Austin, TX, and approved May 31, 2013 (received for review April 11, 2013) The human ether-à-go-go–related gene (hERG) encodes a K+ of related cyclic nucleotide-gated (CNG) and hyperpolarization- channel crucial for repolarization of the cardiac action potential. activated cyclic nucleotide-modulated (HCN) channels (12, 19). EAG-related gene (ERG) channels contain a C-terminal cyclic nu- However, unlike the CNBD of CNG and HCN channels, the cleotide-binding homology domain coupled to the pore of the CNBHD of KCNH channels does not appreciably bind cyclic channel by a C-linker. Here, we report the structure of the C- nucleotides (18, 23). Therefore, paradoxically, despite the linker/cyclic nucleotide-binding homology domain of a mosquito presence of a CNBHD, the KCNH channels are not regulated ERG channel at 2.5-Å resolution. The structure reveals that the by direct binding of cyclic nucleotides (23–26).BasedonFör- region expected to form the cyclic nucleotide-binding pocket is ster resonance energy transfer (FRET) experiments, the negatively charged and is occupied by a short β-strand, referred CNBHDs and PAS domains are located in close proximity (27). to as the intrinsic ligand, explaining the lack of direct regulation Moreover, it has been proposed that the C-linker/CNBHD di- of ERG channels by cyclic nucleotides. In hERG channels, the in- rectly interacts with the PAS domain (28–30), forming an trinsic ligand harbors hereditary mutations associated with long- intersubunit interaction (28). In hERG channels, both the QT syndrome (LQTS), a potentially lethal cardiac arrhythmia. C-linker/CNBHD and PAS domain harbor genetically occur- Mutations in the intrinsic ligand affected hERG channel gating ring mutations that are linked to LQTS (11, 31–34) (reviewed in and LQTS mutations abolished hERG currents and altered traffick- ref. 32). These LQTS mutations have been found to speed up ing of hERG channels, which explains the LQT phenotype. The deactivation of hERG channels or lead to defects in protein structure also reveals a dramatically different conformation of trafficking and folding, precluding formation of functional PHARMACOLOGY the C-linker compared with the structures of the related ether-à- channels (35–43). + go-go–like K and hyperpolarization-activated cyclic nucleotide- Here, we present the crystal structure of the C-linker/CNBHD modulated channels, suggesting that the C-linker region may be of an Anopheles gambiae ERG channel (agERG) at 2.5-Å reso- highly dynamic in the KCNH, hyperpolarization-activated cyclic nu- lution. The agERG and hERG channels share 78% amino acid cleotide-modulated, and cyclic nucleotide-gated channels. identity and 90% similarity in the C-linker/CNBHD (Fig. 1A and Fig. S1). The structure of the C-terminal region of ERG chan- agERG | KCNH2 | cyclic nucleotide-binding domain | Kv11.1 channels nels reveals the structural basis of ERG channel regulation by the CNBHD. The C-linker in the ERG structure is in a dramat- he human ether-à-go-go-related gene (hERG) ion channel ically different conformation in comparison with the C-linkers in fi related zELK (18) and mHCN2 (21) channels. The diversity of Tunderlies the fast delayed recti er current (IKr) in the heart and is the major contributor to the repolarization phase of the the C-linker conformations observed in the ERG, ELK, and ventricular action potential (1–4). Disruption of the function of HCN structures suggests that the C-linker region may be highly this channel, either genetically or as an unintended consequence dynamic in the KCNH, HCN, and CNG channels. of prescription medication, causes lengthening of the ventricular Results action potential, which can lead to a condition known as long-QT syndrome (LQTS) (5–8). LQTS is characterized by a prolonged Structure of the C-Linker/CNBHD of an ERG Channel. To gain mo- QT interval on an electrocardiogram, cardiac arrhythmias, and lecular insight into the gating and regulation of ERG channels, predisposition to sudden cardiac death. hERG channel dysfunction we crystallized the C-linker/CNBHD of Anopheles gambiae is responsible for ∼45% of LQTS-related mutations identified (mosquito) ERG (agERG) channels. The C-linker/CNBHD of fi so far and the vast majority of drug-induced LQTS (9–11). agERG was identi ed as a suitable candidate for crystallization fl The EAG-related gene (ERG) channel subfamily belongs to the by using a screen based on the uorescence-detection size- + KCNH family of voltage-gated K channels, which also includes exclusion chromatography (FSEC) (44). The C-linker/CNBHD of + the ether-à-go-go (EAG) and EAG-like K (ELK) channel sub- agERG channels crystallized in the space group P3221 with a sin- families (Fig. 1A) (12). EAG and ELK channels are key regulators gle molecule in the asymmetric unit and diffracted X-rays to 2.5-Å of tumor progression (13, 14) and neuronal excitability (15, 16). resolution (Table S1). The structure was solved with molecular + fi Similar to other K selective channels, KCNH channels are as- replacement, using the structure of the CNBHD of zebra sh ELK sembled from four subunits around a central ion conducting pore. Each of the four subunits contains six membrane spanning seg- ments (S1–S6) and an intervening pore-forming loop (Fig. 1B). Author contributions: T.I.B., M.C.T., and W.N.Z. designed research; T.I.B., E.C.G., and M.C. – T. performed research; F.D. contributed new reagents/analytic tools; T.I.B., E.C.G., and The S1 S4 segments comprise a voltage sensing domain, whereas M.C.T. analyzed data; and T.I.B., M.C.T., and W.N.Z. wrote the paper. – the S5 S6 segments together with the intervening loop form a pore The authors declare no conflict of interest. domain (17). This article is a PNAS Direct Submission. A characteristic feature of the channels in the KCNH family is Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, the presence of a Per-Arnt-Sim (PAS) domain in their cyto- www.pdb.org (PDB ID code 4L11). plasmic amino-terminal region, and a cyclic nucleotide-binding 1Present address: Department of Pharmacology and Physiology, Georgetown University homology domain (CNBHD) in their cytoplasmic carboxyl-terminal Medical Center, Washington, DC 20057. region coupled to the pore of the channel by a C-linker (12, 18–22) 2To whom correspondence should be addressed. E-mail: [email protected]. (Fig. 1B). The CNBHD of KCNH channels shares general This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. architecture with the cyclic nucleotide-binding domains (CNBD) 1073/pnas.1306887110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1306887110 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 A B cyclic nucleotides, explaining, in part, the absence of direct ELK regulation of KCNH channels by cyclic nucleotides (23–26). ERG hELK1 hELK2 The β9-strand is also unique to the KCNH family and is not hERG3 mELK3 mERG2 agERG present in other CNBD-containing proteins. It is part of a short hERG1 sequence of amino acids following the αC-helix that occupies the β-roll cavity where cyclic nucleotides normally bind in canonical cyclic nucleotide-binding proteins (Fig. 2C). We refer to this re- “ ” mEAG1 gion as the intrinsic ligand, a portion of the protein that occupies mEAG2 EAG the ligand binding site whose displacement regulates the channel. The sequence and structure of the intrinsic ligand is well conserved C D in the KCNH family of channels (18, 22) (Fig. 2 A and B and Fig. Elbow C-linker S1). In agERG, residues Y727 and M729 in the intrinsic ligand B’ form a network of direct interactions with the residues in the β-roll Shoulder A’ cavity of agERG channels (Fig. 2D). Y727 is located where the purine ring of cAMP is located in the CNBD of HCN2 channels, C’ D’ whereas M729 is located where the cyclic phosphate of cAMP is in roll the HCN2 structure. A Effect of Mutations in the Intrinsic Ligand on hERG Channel Gating. B To investigate whether the intrinsic ligand regulates the gating of C ERG channels, we mutated the intrinsic ligand and measured the effects on channel function. Expression of agERG channels in Xenopus oocytes did not produce detectable voltage-activated + K currents so we mutated the intrinsic ligand in hERG chan- Fig. 1. Topology and similarity of KCNH channels and the structure of the nels. In hERG channels, the intrinsic ligand includes conserved agERG C-linker/CNBHD. (A) Phylogenetic tree of the KCNH family of ion residues F860, N861, and L862 (Fig. S1). The mutant channels channels computed with Cobalt. (B) Cartoon of two opposing subunits of were expressed in Xenopus oocytes, and currents were recorded a tetrameric KCNH channel. The pore-forming loop and S5–S6 trans- by using two-electrode voltage clamp. All of the electrophysio- membrane domains are gray. The N-terminal α-helix and PAS domain are logical studies in this section were performed in the background magenta. The elbow and shoulder regions of the C-linker are represented by of S620T mutation in the pore region that removes the C-type the red and pink cylinders, respectively. The β9-strand is represented by a green rectangle.