Multistate Structural Modeling and Voltage-Clamp Analysis of Epilepsy/Autism Mutation Kv10.2–R327H Demonstrate the Role Of
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16586 • The Journal of Neuroscience, October 16, 2013 • 33(42):16586–16593 Neurobiology of Disease Multistate Structural Modeling and Voltage-Clamp Analysis of Epilepsy/Autism Mutation Kv10.2–R327H Demonstrate the Role of This Residue in Stabilizing the Channel Closed State Yang Yang,1,2,3* Dmytro V. Vasylyev,1,2,3* Fadia Dib-Hajj,1,2,3 Krishna R. Veeramah,4 Michael F. Hammer,4 Sulayman D. Dib-Hajj,1,2,3 and Stephen G. Waxman1,2,3 1Department of Neurology and 2Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New Haven, Connecticut 06510, 3Rehabilitation Research Center, Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut 06516, and 4Arizona Research Laboratories Division of Biotechnology, University of Arizona, Tucson, Arizona 85721 Voltage-gated potassium channel Kv10.2 (KCNH5) is expressed in the nervous system, but its functions and involvement in human disease are poorly understood. We studied a human Kv10.2 channel mutation (R327H) recently identified in a child with epileptic encephalopathy and autistic features. Using multistate structural modeling, we demonstrate that the Arg327 residue in the S4 helix of voltage-sensingdomainhasstrongionicinteractionswithnegativelychargedresidueswithintheS1–S3helicesintheresting(closed)and early-activation state but not in the late-activation and fully-activated (open) state. The R327H mutation weakens ionic interactions between residue 327 and these negatively charged residues, thus favoring channel opening. Voltage-clamp analysis showed a strong hyperpolarizing(ϳ70mV)shiftofvoltagedependenceofactivationandanaccelerationofactivation.Ourresultsdemonstratethecritical role of the Arg327 residue in stabilizing the channel closed state and explicate for the first time the structural and functional change of a Kv10.2 channel mutation associated with neurological disease. Introduction the nervous system and its involvement in human diseases other The Kv10.2 channel (encoded by the KCNH5 gene) belongs to the than cancer are not well understood (Wulff et al., 2009). Recently, ether-a`-go-go (EAG) family within the voltage-gated potassium we identified a human Kv10.2 channel mutation (R327H) in a (Kv) channel superfamily (Saganich et al., 1999). EAG channel child with epileptic encephalopathy with seizures intractable to family members, including both Kv10.1 and Kv10.2, are overex- medical therapy and autistic features via exome sequencing pressed in cancer and have been suggested as tumor markers as (Veeramah et al., 2013). To understand how this R327H muta- well as cancer therapeutic targets (Camacho, 2006; Hemmerlein tion, which substitutes a single residue within the voltage-sensing et al., 2006; Mello de Queiroz et al., 2006; Stu¨hmer et al., 2006; helix S4, affects the structure and function of Kv10.2, we per- Huang et al., 2012). Kv10.2 is natively expressed in the nervous formed this structural and functional study. system (Saganich et al., 1999; Ludwig et al., 2000; Ju and Wray, The crystal structures of mammalian Kv channels in the open 2002; Schonherr et al., 2002); however, the functions of Kv10.2 in state have been determined (Long et al., 2007), and recently the transitions of Kv channel from the activated (open) state to rest- ing (closed) state have been studied using long (Ն200 s) all- Received May 29, 2013; revised Sept. 4, 2013; accepted Sept. 10, 2013. atom molecular dynamic simulations (Jensen et al., 2012), Author contributions: Y.Y., D.V.V., S.D.D.-H., and S.G.W. designed research; Y.Y. and D.V.V. performed research; providing six structure snapshots of the intermediate states dur- F.D.-H., K.R.V., and M.F.H. contributed unpublished reagents/analytic tools; Y.Y., D.V.V., and S.D.D.-H. analyzed data; Y.Y., D.V.V., S.D.D.-H., and S.G.W. wrote the paper. ing channel gating. Here, using multistate structural modeling, *Y.Y. and D.V.V. contributed equally to this work. we constructed a Kv10.2 voltage-sensing domain (VSD) model The authors declare no competing financial interests. containing the wild-type (WT) Arg327 or mutant His327 residue This work was supported by the Medical Research Service and Rehabilitation Research Service, Department of in all six states obtained from molecular dynamic simulations Veterans Affairs (S.G.W.). Y.Y. is supported by Connecticut Stem Cell Research Grants Program. The Center for (Jensen et al., 2012) and provide evidence showing that the rest- Neuroscience and Regeneration Research is a Collaboration of the Paralyzed Veterans of America with Yale Univer- sity. We thank Dr. Mark Estacion, Dr. Chongyang Han, and Dr. Jianying Huang for valuable comments. We thank Dr. ing and early activation state of the channel are destabilized by David E. Shaw and Dr. Morten Jensen of D. E. Shaw Research for providing coordinates of their molecular dynamics R327H mutation. This dynamic model suggests that the Arg327 simulation results and helpful discussion. residue of WT Kv10.2 has strong ionic interactions with nega- Correspondence should be addressed to Dr. Stephen G. Waxman, Neuroscience and Regeneration Research tively charged residues within the S1–S3 helices in the resting Center, Veterans Affairs Connecticut Healthcare System, 950 Campbell Avenue, Building 34, West Haven, CT 06516. E-mail: [email protected]. state and states transiting into or out of the resting state but not in DOI:10.1523/JNEUROSCI.2307-13.2013 the activated state. The R327H mutation weakens the ionic inter- Copyright © 2013 the authors 0270-6474/13/3316586-08$15.00/0 action between residue 327 and negatively charged residues of Yang, Vasylyev et al. • Kv10.2 R327H Mutation J. Neurosci., October 16, 2013 • 33(42):16586–16593 • 16587 S1–S3, thus favoring channel opening. Consistent with this pre- channel gating, and S5 and S6 as the pore-forming module (Fig. diction, voltage-clamp analysis revealed a remarkable hyperpo- 1A). Functional Kv channels are tetramers assembled from four larizing shift in the voltage dependence of activation and an independent subunits. acceleration of activation. Together, our results demonstrate the Sequence analysis shows that Arg327 is located in the critical role of the Arg327 residue in stabilizing the closed state of the S4 helix of the VSD (Fig. 1A). Within the S4 helix of VSD, Arg327, channel and explicate, for the first time, the structural and func- together with Lys325, Arg329, and Arg331 (commonly known as tional change of a Kv10.2 channel mutation associated with neu- R[K]1–R4), are positively charged residues that sense changes in rological disease. the membrane potential and produce a conformational change of the VSD, which in turn controls channel gating (Fig. 1A). Muta- Materials and Methods tion R327H, identified from a child with epilepsy and other neu- Structural modeling and residue interaction maps. The alignment between rological diseases (Veeramah et al., 2013), occurs at the critical R2 Kv10.2 and other Kv channels was generated with ClustalW2 and man- position. At physiological pH, Arg (R) is a positively charged ually refined based on previous studies (Long et al., 2007; Pathak et al., residue, and His (H) is predominantly neutral (with a relatively 2007; Lee et al., 2009; Zhang et al., 2009). Six different states of Kv1.2/ small proportion carrying positive charges). Because multiple Kv2.1 “paddle chimera” voltage-gated K ϩ channel (Protein Data Bank negatively charged residues existed in S0–S3 helices (Fig. 1B), we identification number 2R9R) obtained in a molecular dynamics simula- hypothesized that Arg327 may form ionic interaction with these tion were kindly provided by Dr. David E. Shaw, D. E. Shaw Research negatively charged residues in certain channel gating states, and (New York, NY) (Jensen et al., 2012). Modeling of the S1–S4 VSD in six mutation R327H may disrupt the ionic interaction between res- states was performed with SWISS-MODEL (Arnold et al., 2006; Bordoli idue 327 and negative charged residues, thus affecting channel et al., 2009) and refined as described previously (Yang et al., 2011, 2012, 2013). Models were further energy minimized, analyzed, and visualized gating. To test this hypothesis, we constructed a Kv10.2 VSD in Molecular Operating Environment (MOE; Chemical Computing structural model based on the crystal structure of Kv1.2/Kv2.1 Group). Ligand and receptor interaction maps were constructed using paddle chimera channel in its open state (Protein Data Bank MOE. identification number 2R9R). Our structural model of Kv10.2 Plasmid. Kv10.2 cDNA was synthesized from human fetal brain total VSD in its open state is shown in Figure 1C. Positively charged RNA (Clontech) using SuperscriptIII Reverse Transcriptase (Invitro- residues are labeled cyan (Arg327 are labeled gray), and nega- gen). The PCR product was cloned into pGEMT-easy vector (Promega) tively charged residues are labeled red (Fig. 1C). As can be seen in and then subcloned into pCDNA3mod (Cummins et al., 2001). In a Figure 1C, the S4 helix of the Kv10.2 channel is in an “outward” second step, an EcoRI–NotI fragment from pIRES2–EGFP (Clontech) position (“outward” means close to the extracellular space and was cloned into downstream of the stop codon of Kv10.2 to generate “inward” means close to the intracellular space) in this activated plasmid phKv10.2–IRES–EGFP. Sequencing of the insert and comparing it with the reference sequence NM_139318 confirmed a 988 aa open state, and the Arg327 residue is not in contact with any negatively reading frame, including one variant (c. 2233GϾA; p. A745T) within the charged residues. C terminus of the channel, which has been reported as a natural variant Because channel gating involves substantial conformational with a minor allele frequency (MAF) ϭ 0.25 (www.1000genomes.org). changes of the S1–S4 helix of the VSD, we tested the hypothesis Transfection of human embryonic kidney 293 cells. Human embryonic that Arg327 may interact with negatively charged residues in kidney 293 (HEK293) cells, 80% confluent in a 24-well plate, were trans- other intermediate states during the channel gating process.