Translational Research: Basic Mechanisms

Abstract

Abstract 1 Arthur J. Barela, 2 Salina P. Waddy, 1 Jay G. Lickfett, 3 Jessica Hunter, 3 Aimee Anido, 2 Sandra L. Helmers, 1 Alan L. Goldin, and 3 Andrew Escayg ( 1 Microbiology & Molecular Genetics, University of California, Irvine, CA ; 2 Neurology, Emory University, Atlanta, GA ; and 3 Human Genetics, Emory University, Atlanta, GA ) Rationale: Generalized epilepsy with febrile seizures plus (GEFS+) is a familial seizure disorder that is characterized by febrile seizures persisting beyond six years of age and a variety of afebrile epilepsy subtypes. To date, mutations in three voltage-gated sodium channel genes, SCN1A, SCN2A, SCN1B, and the GABAA receptor subunit GABRG2 have been identified in GEFS+ families. We identified a 33-member, 4-generation family with a clinical presentation consistent with GEFS+ from the Emory University Epilepsy Clinic. Methods: The coding exons of SCN1A, SCN2A, SCN1B and GABRG2 were PCR amplified from genomic DNA of the affected proband. The PCR products were screened by conformation sensitive gel electrophoresis and interesting variants were tested by direct sequencing of independent PCR products. The identified SCN1A mutation was constructed in a cDNA clone encoding the orthologous rat Nav1.1 sodium channel and the electrophysiological properties were characterized in the absence and presence of the β1 subunit in Xenopus oocytes. A computational model was used to analyze how the effects of the mutation might alter action potential generation in a neuron. Results: The nucleotide substitution C2575T was identified in exon 14 of SCN1A. Co-segregation of this mutation was observed in five additional affected members. No mutations were observed in SCN2A, SCN1B and GABRG2. The substitution neutralized a positively charged arginine by replacement with cysteine (R859C) in the S4 voltage sensor of domain II. R859 is evolutionarily conserved in all mammalian sodium channels as well as in sodium channels from lower organisms. The R859C mutation caused a ∼6 mV positive shift in the voltage-dependence of activation in both the absence and presence β1. The mutation did not alter the voltage-dependence of inactivation, nor did it affect the kinetics of fast inactivation or recovery from fast inactivation. Mutant channels entered into the slow inactivated state at a comparable rate to that of wild-type channels, but recovery from slow inactivation was significantly slower than for wild-type channels. Computational analysis suggests that neurons expressing the mutant channels have higher thresholds for firing a single action potential and for firing multiple action potentials, along with decreased repetitive firing. Conclusions: The R859C mutation alters two aspects of sodium channel function, both of which should lead to decreased neuronal excitability. This result is in contrast to most previous sodium channel mutations that cause GEFS+, which have changes that are predicted to increase neuronal firing. (Supported by grants from NIH (NS48336, ALG), McKnight Foundation (34653, ALG), CURE (AE), March of Dimes (5-FY02-250, AE) and Emory University Research Council (2002120, AE).)