GENERALIZED EPILEPSY WITH FEBRILE SEIZURES PLUS–ASSOCIATED SODIUM CHANNEL 1 SUBUNIT MUTATIONS SEVERELY REDUCE BETA SUBUNIT–MEDIATED MODULATION OF SODIUM CHANNEL FUNCTION R. XU, a E. A. THOMAS, a E. V. GAZINA, a K. L. RICHARDS, a M. QUICK, a R. H. WALLACE, b,c L. A. HARKIN, b,c,h S. E. HERON, b,h S. F. BERKOVIC, d,e I. E. SCHEFFER, d,e,f J. C. MULLEY b,g,h AND S. PETROU a,h * a Howard Florey Institute, The University of Melbourne, Parkville, Mel- bourne, Victoria 3010, Australia b Department of Genetic Medicine, Women’s and Children’s Hospital, North Adelaide, South Australia, Australia c Department of Paediatrics, University of Adelaide, South Australia, Australia d Department of Medicine (Neurology), The University of Melbourne, Austin Health, Heidelberg, Melbourne, Australia e Department of Paediatrics, The University of Melbourne, Royal Chil- dren’s Hospital, Parkville, Melbourne, Australia f Neurosciences, Monash Medical Centre, Melbourne, Australia g School of Molecular and Biomedical Sciences, University of Adelaide, South Australia, Australia h Bionomics Ltd., Thebarton, Adelaide, South Australia, Australia Abstract—Two novel mutations (R85C and R85H) on the ex- tracellular immunoglobulin-like domain of the sodium chan- nel 1 subunit have been identified in individuals from two families with generalized epilepsy with febrile seizures plus (GEFS). The functional consequences of these two muta- tions were determined by co-expression of the human brain NaV1.2  subunit with wild type or mutant 1 subunits in human embryonic kidney (HEK)-293T cells. Patch clamp studies confirmed the regulatory role of 1 in that relative to NaV1.2 alone the NaV1.21 currents had right-shifted volt- age dependence of activation, fast and slow inactivation and reduced use dependence. In addition, the NaV1.21 current entered fast inactivation slightly faster than NaV1.2 channels alone. The 1(R85C) subunit appears to be a complete loss of function in that none of the modulating effects of the wild type 1 were observed when it was co-expressed with NaV1.2. Interestingly, the 1(R85H) subunit also failed to modulate fast kinetics, however, it shifted the voltage depen- dence of steady state slow inactivation in the same way as the wild type 1 subunit. Immunohistochemical studies re- vealed cell surface expression of the wild type 1 subunit and undetectable levels of cell surface expression for both mu- tants. The functional studies suggest association of the 1(R85H) subunit with the  subunit where its influence is limited to modulating steady state slow inactivation. In sum- mary, the mutant 1 subunits essentially fail to modulate  subunits which could increase neuronal excitability and un- derlie GEFS pathogenesis. © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: epilepsy, genetics, sodium channels, electro- physiology, beta subunit, GEFS. Generalized epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome characterized by heteroge- neous clinical phenotypes with the most common subtypes being febrile seizures and febrile seizures plus (Scheffer and Berkovic, 1997). Inheritance is complex, with some families segregating a major autosomal dominant gene (Singh et al., 1999). GEFS+ families have been identified with mutations in the genes encoding the regulatory so- dium channel 1 subunit (SCN1B), the pore-forming  subunit (SCN1A) and other ion channels (Wallace et al., 1998, 2001, 2002; Baulac et al., 2001; Harkin et al., 2002). Since the first discovery of a missense mutation in the SCN1B gene, mutation analysis has revealed an additional SCN1B gene mutation (I70_E74del) on the same extracel- lular domain which also causes febrile seizures and early onset absence epilepsy (Audenaert et al., 2003). Brain sodium channels are complexes of a large, cen- tral pore-forming  subunit of 260 kDa and one or two comparatively smaller  subunits of 30 – 40 kDa (Isom and Catterall, 1996; Isom, 2001). 1 Subunits are single trans- membrane domain glycoproteins composed of a large N- terminal extracellular and a short C-terminal intracellular domains.  Subunits are not pore forming but interact with the  subunit to modulate electrophysiological properties (Qu et al., 1995, 2001). Increases in sodium currents have been attributed to  subunit co-expression in some studies (Tammaro et al., 2002) but not in others (Meadows et al., 2002), although this latter study showed an increase in surface binding. Studies of the 1 subunit have yielded a large amount of information about the importance of the extracellular loop (Chen and Cannon, 1995; Makita et al., 1996) containing the Cys 121 and Arg 85 residues implicated in familial epilepsy.  Subunits also modulate slow inactiva- tion which has been shown to be important in cardiac and skeletal muscle physiology (Vilin et al., 1999). However, the information on the role of the 1 subunit in modulation of slow inactivation for brain sodium channels is limited. The C121W missense mutation in human brain SCN1B gene causes GEFS+ (Wallace et al., 1998, 2002) and has been particularly well studied. This mutation dis- *Corresponding author. Tel: +61-3-8344-1957; fax: +61-3-9347-0446. E-mail address: spetrou@unimelb.edu.au (S. Petrou). Abbreviations: EGFP, enhanced green fluorescent protein; GEFS+, generalized epilepsy with febrile seizures plus; HEK, human embry- onic kidney; PBS, phosphate-buffered saline; SCN1A, sodium channel 1 subunit; SCN1B, sodium channel 1 subunit; WT, wild type. Neuroscience 148 (2007) 164 –174 0306-4522/07$30.00+0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.05.038 164