Human Nocturnal Frontal Lobe Epilepsy: Pharmocogenomic Profiles of Pathogenic Nicotinic Acetylcholine Receptor -Subunit Mutations outside the Ion Channel Pore Jean-Charles Hoda, Wenli Gu, Marc Friedli, Hilary A. Phillips, Sonia Bertrand, Stylianos E. Antonarakis, David Goudie, Richard Roberts, Ingrid E. Scheffer, Carla Marini, Jayesh Patel, Samuel F. Berkovic, John C. Mulley, Ortrud K. Steinlein, and Daniel Bertrand Departments of Neuroscience (J.-C.H., S.B., D.B.) and Genetics (M.F., S.E.A.), Medical Faculty, University of Geneva, Geneva, Switzerland; Department of Cytogenetics and Molecular Genetics, Centre for Medical Genetics, Women’s and Children’s Hospital, Adelaide, South Australia (H.A.P., J.C.M.); Ninewells Hospital, Dundee, Scotland (D.G.); Neurosciences Directorate, Tayside University Teaching Hospitals, Dundee, Scotland (R.R.); Department of Medicine (Neurology), University of Melbourne, Austin and Repatriation Medical Centre, Heidelberg, Melbourne, Australia (I.E.S., C.M., S.F.B.); Bristol Royal Hospital for Children, Department of Paediatric Neurology, Bristol, United Kingdom (J.P.); and Institute of Human Genetics, University Hospital, Ludwig Maximilians University Munich, Germany (W.G., O.K.S.) Received December 21, 2007; accepted April 30, 2008 ABSTRACT Certain mutations in specific parts of the neuronal nicotinic acetylcholine receptor (nAChR) subunit genes CHRNA4, CHRNB2, and probably CHRNA2, can cause autosomal dom- inant nocturnal frontal lobe epilepsy (ADNFLE). All but one of the known causative mutations are located in the second trans- membrane region (TM2), which serves as the major ion pore- forming domain of the receptor. Functional characterization of these ADNFLE mutations has shown that although each mutant exhibits specific properties, they all confer a gain of function with increased sensitivity to acetylcholine. In this work, we characterize the second and third ADNFLE-associated muta- tions that are external to TM2 but affect different amino acid residues within the third transmembrane region (TM3). The two new CHRNB2 mutations were identified in three families of Turkish Cypriot, Scottish, and English origin. These TM3 mu- tations elicit the same gain of function pathomechanism as observed for the TM2 mutations with enhanced acetylcholine sensitivity, despite their unusual localization within the gene. Electrophysiological experiments, including single channel measurements, revealed that incorporation of these new mu- tant subunits does not affect the conductance of the ionic pore but increases the probability of opening. Determination of the sensitivity to nicotine for nAChRs carrying mutations in TM2 and TM3 showed clear differences in the direction and the extent to which the window current for nicotine sensitivity was shifted for individual mutations, indicating differences in phar- macogenomic properties that are not readily correlated with increased ACh affinity. Epilepsy is one of the most prevalent neurological disor- ders in the population and represents a life long impairment that is often difficult to treat. Recent progress has been made in understanding the molecular basis for a range of geneti- cally based epilepsy syndromes (Heron et al., 2007). Autoso- mal dominant nocturnal frontal lobe epilepsy (ADNFLE) is one of the syndromes best-characterized at the molecular level (Steinlein, 2002; Mulley et al., 2003). So far, four mutations in CHRNA4, the gene encoding the 4 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) have been identified (4-S248F, 4 –776ins3, 4- S252L, and 4-T265I) (Steinlein et al., 1995, 1997; Hirose et al., 1999; Leniger et al., 2003). Likewise, three mutations have been reported for CHRNB2, the gene encoding the com- plementary 2 subunit that participates in formation of the This work was supported by the National Health and Medical Research Council of Australia (to S.B., J.M., and I.S.), Deutsche Forschungsgemein- schaft grant STE16511-1 (to O.K.), grants from the Swiss National Science Foundation 3100A0-101787/2 (to D.B.), and Deutsche Forschungsgemein- schaft Schwerpunktprogramm SPP 1226 (to D.B.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.044545. ABBREVIATIONS: ADNFLE, autosomal dominant nocturnal frontal lobe epilepsy; nAChR, nicotinic acetylcholine receptor; TM2, second trans- membrane segment; PCR, polymerase chain reaction; ACh, acetylcholine ; HEK, human embryonic kidney; CT, computed tomography; EEG, electroencephalography; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; AM, acetoxymethyl ester. 0026-895X/08/7402-379 –391$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 2 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 44545/3360502 Mol Pharmacol 74:379–391, 2008 Printed in U.S.A. 379 at ASPET Journals on November 10, 2016 molpharm.aspetjournals.org Downloaded from