Mutation of a new sodium channel gene, Scn8a, in the mouse mutant 'motor endplate disease'.

The mouse neurological mutant 'motor endplate disease' (med) is characterized by early onset progressive paralysis of the hind limbs, severe muscle atrophy, degeneration of Purkinje cells and juvenile lethality. We have isolated a voltage-gated sodium channel gene, Scn8a, from the flanking region of a transgene-induced allele of med. Scn8a is expressed in brain and spinal cord but not in skeletal muscle or heart, and encodes a predicted protein of 1,732 amino acids. An intragenic deletion at the transgene insertion site results in loss of expression. Scn8a is closely related to other sodium channel alpha subunits, with greatest similarity to a brain transcript from the pufferfish Fugu rubripes. The human homologue, SCN8A, maps to chromosome 12q13 and is a candidate gene for inherited neurodegenerative disease.

A gain-of-function mutation in the sodium channel gene Scn2a results in seizures and behavioral abnormalities.

The GAL879-881QQQ mutation in the cytoplasmic S4-S5 linker of domain 2 of the rat brain IIA sodium channel (Na(v)1.2) results in slowed inactivation and increased persistent current when expressed in Xenopus oocytes. The neuron-specific enolase promoter was used to direct in vivo expression of the mutated channel in transgenic mice. Three transgenic lines exhibited seizures, and line Q54 was characterized in detail. The seizures in these mice began at two months of age and were accompanied by behavioral arrest and stereotyped repetitive behaviors. Continuous electroencephalogram monitoring detected focal seizure activity in the hippocampus, which in some instances generalized to involve the cortex. Hippocampal CA1 neurons isolated from presymptomatic Q54 mice exhibited increased persistent sodium current which may underlie hyperexcitability in the hippocampus. During the progression of the disorder there was extensive cell loss and gliosis within the hippocampus in areas CA1, CA2, CA3 and the hilus. The lifespan of Q54 mice was shortened and only 25% of the mice survived beyond six months of age. Four independent transgenic lines expressing the wild-type sodium channel were examined and did not exhibit any abnormalities. The transgenic Q54 mice provide a genetic model that will be useful for testing the effect of pharmacological intervention on progression of seizures caused by sodium channel dysfunction. The human ortholog, SCN2A, is a candidate gene for seizure disorders mapped to chromosome 2q22-24.

Alternative splicing of the sodium channel SCN8A predicts a truncated two-domain protein in fetal brain and non-neuronal cells.

The voltage-gated sodium channel alpha subunit SCN8A is one of the most abundant sodium channels in neurons from brain and spinal cord. We have identified two alternatively spliced exons, 18N and 18A, that encode transmembrane segments S3 and S4 in domain III. Exon 18N is expressed in fetal brain and non-neuronal tissues. Transcripts with exon 18N have a conserved in-frame stop codon that predicts the synthesis of a truncated, two-domain protein similar to the fetal form of the muscle calcium channel. The proportion of transcripts containing exon 18N is highest in mouse fetal brain between E12.5 and P1.5; at later ages transcripts containing exon 18A predominate. This developmental program is recapitulated in P19 cells during retinoic acid-induced neuronal differentiation. Non-neuronal tissues contain a low level of SCN8A transcripts containing exon 18N. SCN8A thus provides a new model of differentiation specific splicing. Genomic analysis of SCN8A from human, mouse, and fish demonstrated a conserved structure in which exon 18N is located 300-500 bp upstream of exon 18A. Duplication of exon 18 thus preceded the divergence of fish and mammals. The genomic organization, developmental regulation, and coding content of exons 18N and 18A closely resemble the previously described alternate exons 5N and 5A of the neuronal sodium channel genes. Our proposal that the evolutionary origin of exons 18N and 18A was by duplication of exons 5N and 5A is consistent with other evidence that the four-domain cation channels arose by two rounds of duplication from a single-domain ancestral channel.

Ion channel mutations in mouse models of inherited neurological disease.

Analysis of the molecular defects in mouse mutants can identify candidate genes for human neurological disorders. During the past 2 years, mutations in sodium channels, calcium channels and potassium channels have been identified by positional cloning of the spontaneous mouse mutants motor endplate disease, tottering, lethargic and weaver. The phenotypes of four allelic mutations identified in the sodium channel gene Scn8a range from ataxia and muscle weakness through severe dystonia and progressive paralysis, indicating that human mutations in this gene could be associated with a variety of clinical syndromes. Mutations of the calcium channel subunits beta 4 in the lethargic mouse and alpha 1A in the tottering mouse have specific effects on cerebellar function. Targeted mutation of ligand-gated ion channels has also been used to generate new models of neurological disease. We will review these recent achievements and their implications for human neurological disease. The mouse studies indicate that mutations in ion channel genes are likely to be responsible for a broad spectrum of clinical phenotypes in human neurological disorders.

oto is a homeotic locus with a role in anteroposterior development that is partially redundant with Lim1.

Genetic control of mammalian head development involves mechanisms that are shared with trunk development as well as mechanisms that are independent. For example, mutations in the nodal gene disrupt axis formation and head development while mutations in the Otx2 or Lim1 genes block head development without disrupting development of the trunk. We show here that the oto mutation on mouse chromosome 1 defines a locus with a critical role in anterior development. The oto mutation disrupts development of the telencephalic and optic vesicles, the pharyngeal endoderm and the first branchial arch. Also, oto embryos have dose-dependent, posterior homeotic transformations throughout the axial skeleton. To further dissect the role of the oto locus in head development, we crossed mice carrying oto and Lim1 mutations. Interactions between the two mutations indicate that the role of oto in the regulation of head development is partially redundant with that of Lim1. The phenotype of oto embryos points to an early and critical role for oto in the development of forebrain subregions. Transformations of the vertebrae in oto embryos reveal a Lim1-independent role in the establishment of positional information in the trunk.

Functional analysis of the mouse Scn8a sodium channel.

The mouse Scn8a sodium channel and its ortholog Na6 in the rat are abundantly expressed in the CNS. Mutations in mouse Scn8a result in neurological disorders, including paralysis, ataxia, and dystonia. In addition, Scn8a has been observed to mediate unique persistent and resurgent currents in cerebellar Purkinje cells (Raman et al., 1997). To examine the functional characteristics of this channel, we constructed a full-length cDNA clone encoding the mouse Scn8a sodium channel and expressed it in Xenopus oocytes. The electrophysiological properties of the Scn8a channels were compared with those of the Rat1 and Rat2 sodium channels. Scn8a channels were sensitive to tetrodotoxin at a level comparable to that of Rat1 or Rat2. Scn8a channels inactivated more rapidly and showed differences in their voltage-dependent properties compared with Rat1 and Rat2 when only the alpha subunits were expressed. Coexpression of the beta1 and beta2 subunits modulated the properties of Scn8a channels, but to a lesser extent than for the Rat1 or Rat2 channels. Therefore, all three channels showed similar voltage dependence and inactivation kinetics in the presence of the beta subunits. Scn8a channels coexpressed with the beta subunits exhibited a persistent current that became larger with increasing depolarization, which was not observed for either Rat1 or Rat2 channels. The unique persistent current observed for Scn8a channels is consistent with the hypothesis that this channel is responsible for distinct sodium conductances underlying repetitive firing of action potentials in Purkinje neurons.

Insertional mutation of the motor endplate disease (med) locus on mouse chromosome 15.

Homozygous transgenic mice from line A4 have an early-onset progressive neuromuscular disorder characterized by paralysis of the rear limbs, muscle atrophy, and lethality by 4 weeks of age. The transgene insertion site was mapped to distal chromosome 15 close to the locus motor endplate disease (med). The sequence of mouse DNA flanking the insertion site junctions was determined. A small (< 20 kb) deletion was detected at the insertion site, with no evidence of additional rearrangement of the chromosomal DNA. Noncomplementation of the transgene-induced mutation and med was demonstrated in a cross with medJ/+mice. The new allele is designated medTgNA4Bs (medtg). The homologous human locus MED was assigned to chromosome 12. Synaptotagmin 1 and contactin 1 were eliminated as candidate genes for the med mutation. The transgene-induced allele provides molecular access to the med gene, whose function is required for synaptic transmission at the neuromuscular junction and long-term survival of cerebellar Purkinje cells.

Exon organization, coding sequence, physical mapping, and polymorphic intragenic markers for the human neuronal sodium channel gene SCN8A.

The voltage-gated sodium channel SCN8A is associated with inherited neurological disorders in the mouse that include ataxia, dystonia, severe muscle weakness, and paralysis. We report the complete coding sequence and exon organization of the human SCN8A gene. The predicted 1980 amino acid residues are distributed among 28 exons, including two pairs of alternatively spliced exons. The SCN8A protein is evolutionarily conserved, with 98.5% amino acid sequence identity between human and mouse. Consensus sites for phosphorylation of serine/threonine and tyrosine residues are present in cyoplasmic loop domains. The polymorphic (CA)n microsatellite marker D12S2211, with PIC = 0.68, was isolated from intron 10C of SCN8A. Single nucleotide polymorphisms in intron 19 and exon 22 were also identified. We localized SCN8A to chromosome band 12q13.1 by physical mapping on a YAC contig. The cDNA clone CSC-1 was reported by others to be a cardiac-specific sodium channel, but sequence comparison demonstrates that it is derived from exon 24 of human SCN8A. The genetic information described here will be useful in evaluating SCN8A as a candidate gene for human neurological disease.