Cloning and expression of a zebrafish SCN1B ortholog and identification of a species-specific splice variant.
BACKGROUND
Voltage-gated Na+ channel beta1 (Scn1b) subunits are multi-functional proteins that play roles in current modulation, channel cell surface expression, cell adhesion, cell migration, and neurite outgrowth. We have shown previously that beta1 modulates electrical excitability in vivo using a mouse model. Scn1b null mice exhibit spontaneous seizures and ataxia, slowed action potential conduction, decreased numbers of nodes of Ranvier in myelinated axons, alterations in nodal architecture, and differences in Na+ channel alpha subunit localization. The early death of these mice at postnatal day 19, however, make them a challenging model system to study. As a first step toward development of an alternative model to investigate the physiological roles of beta1 subunits in vivo we cloned two beta1-like subunit cDNAs from D. rerio.


RESULTS
Two beta1-like subunit mRNAs from zebrafish, scn1ba_tv1 and scn1ba_tv2, arise from alternative splicing of scn1ba. The deduced amino acid sequences of Scn1ba_tv1 and Scn1ba_tv2 are identical except for their C-terminal domains. The C-terminus of Scn1ba_tv1 contains a tyrosine residue similar to that found to be critical for ankyrin association and Na+ channel modulation in mammalian beta1. In contrast, Scn1ba_tv2 contains a unique, species-specific C-terminal domain that does not contain a tyrosine. Immunohistochemical analysis shows that, while the expression patterns of Scn1ba_tv1 and Scn1ba_tv2 overlap in some areas of the brain, retina, spinal cord, and skeletal muscle, only Scn1ba_tv1 is expressed in optic nerve where its staining pattern suggests nodal expression. Both scn1ba splice forms modulate Na+ currents expressed by zebrafish scn8aa, resulting in shifts in channel gating mode, increased current amplitude, negative shifts in the voltage dependence of current activation and inactivation, and increases in the rate of recovery from inactivation, similar to the function of mammalian beta1 subunits. In contrast to mammalian beta1, however, neither zebrafish subunit produces a complete shift to the fast gating mode and neither subunit produces complete channel inactivation or recovery from inactivation.


CONCLUSIONS
These data add to our understanding of structure-function relationships in Na+ channel beta1 subunits and establish zebrafish as an ideal system in which to determine the contribution of scn1ba to electrical excitability in vivo.
Fein
Amanda J
AJ
Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109-0632, USA. ahobson@umich.edu <ahobson@umich.edu>
Meadows
Laurence S
LS
Chen
Chunling
C
Slat
Emily A
EA
Isom
Lori L
LL
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004594:Electrophysiology; D004625:Embryo, Nonmammalian; D018507:Gene Expression Regulation, Developmental; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015222:Sodium Channels / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000502:physiology; D013045:Species Specificity; D014018:Tissue Distribution; D062547:Voltage-Gated Sodium Channel beta-1 Subunit; D014982:Xenopus laevis; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D020033:Protein Isoforms; D015222:Sodium Channels; D062547:Voltage-Gated Sodium Channel beta-1 Subunit; D029961:Zebrafish Proteins; C523645:scn1ba protein, zebrafish