We recently isolated a novel gene, SCN8A, that is a member of the mammalian voltage gated sodium channel alpha subunit gene family. We demonstrated that the mouse neurological mutant med ('motor endplate disease') is associated with mutations in SCN8A. Mice with inactivating mutations of Scn8a have neuromuscular disease characterized by failure of neuromuscular transmission, leading to paralysis and muscle atrophy, and degeneration of cerebellar Purkinje cells. Scn8a transcripts are present in brain and spinal cord, consistent with the clinical syndrome. Extrapolating from the med mouse, we predict that null alleles of human SCN8A would be lethal, while partially active alleles would be associated with syndromes that include muscle weakness and cerebellar ataxia. We have mapped the human SCN8A gene and identified markers for linkage analysis. In order to identify mutations in human patients with defects in SCN8A, we will clone and sequence the human cDNA and identify the intron/exon borders of the human gene. DNA from appropriate patients will be analyzed by SSCP, chemical mismatch, and other mutation detection techniques. We will determine the molecular basis for the three spontaneous med mutations including jolting, a viable allele with mild cerebellar ataxia. Mutations of human and mouse Scn8a identified in affected individuals will be incorporated into an expression construct to permit analysis of their pharmacological and electrophysiological properties. The role of alternatively spliced products of Scn8a will be evaluated with developmental and functional studies. The sequences of the human and mouse SCN8A promoters will be compared to identify conserved regulatory elements. Promoter function will also be assayed in transgenic mice. This work will provide thorough characterization of normal and mutated forms of a novel sodium channel and contribute to the diagnosis and treatment of neuromuscular disease.