Cloning and expression of a zebrafish SCN1B ortholog and identification of a species-specific splice variant

Abstract Background Voltage-gated Na+ channel β1 (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 β1 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 α 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 β1 subunits in vivo we cloned two β1-like subunit cDNAs from D. rerio. Results Two β1-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 β1. 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 β1 subunits. In contrast to mammalian β1, however, neither zebrafish subunit produces a complete shift to the fast gating mode and neither subunit produces complete channel inactivation or recovery from inactivation. Conclusion These data add to our understanding of structure-function relationships in Na+ channel β1 subunits and establish zebrafish as an ideal system in which to determine the contribution of scn1ba to electrical excitability in vivo.http://deepblue.lib.umich.edu/bitstream/2027.42/112585/1/12864_2007_Article_939.pd

Electrophysiological evidence of enhanced performance monitoring in recently abstinent alcoholic men

RATIONALE: Chronic alcoholism is associated with mild to moderate cognitive impairment. Under certain conditions, impairment can be ameliorated by invoking compensatory processes. OBJECTIVE: To identify electrophysiological mechanisms of such compensation that would be required to resolve response conflict. METHODS: 14 abstinent alcoholic men and 14 similarly aged control men performed a variation of the Eriksen flanker task during an electroencephalography (EEG) recording to examine whether alcoholics could achieve and maintain control-level performance and whether EEG markers could identify evidence for the action of compensatory processes in the alcoholics. Monitoring processes engaged following a response were indexed by the correct related negativity (CRN) and error related negativity (ERN), two medial-frontal negative event-related potentials. RESULTS: The alcoholics were able to perform at control levels on accuracy and reaction time (RT). Alcoholics generated larger ERN amplitudes following incorrect responses and larger CRNs following correct responses than controls. Both groups showed evidence of post-error slowing. Larger CRN amplitudes in the alcoholics were related to longer RTs. Also observed in the alcoholics was an association between smaller CRN amplitudes and length of sobriety, suggesting a normalization of monitoring activity with extended abstinence. CONCLUSIONS: To the extent that greater amplitude of these electrophysiological markers of performance monitoring indexes greater resource allocation and performance compensation, the larger amplitudes observed in the alcoholic than control group support the view that elevated performance monitoring enables abstinent alcoholics to overcome response conflict, as was evident in their control-level performance

Identification and characterization of sodium ion channel beta1-like subunits from zebrafish.

Voltage gated Na+ channels are critical for action potential generation and propagation in excitable cells. Na+ channels are composed of a pore forming alpha subunit and one or more accessory beta subunits. Mammalian beta subunits increase channel cell surface expression, modulate the voltage dependence of Na+ current, accelerate the rate of channel recovery from inactivation, and function as cell adhesion molecules (CAMs). This thesis reports the cloning of three beta1 ( SCN1B) orthologs from zebrafish. The first ortholog, scn1ba , is expressed as two splice variants, Scn1ba_tv1 and Scn1ba_tv2. Both subunits are expressed in brain, retina, skeletal muscle, olfactory pits, and the lateral line system. However, only Scn1b_tv1 is expressed in optic nerve. Scn1ba_tv1 contains a conserved C-terminal tyrosine that corresponds to tyrosine 181 in mammalian Scn1b, which dictates subunit localization in cardiac myocytes. We predict that localization differences in the two scn1ba variants result from the presence/absence of this tyrosine. Both variants of scn1ba modulate Na+ current, thus, we predict that they are functionally homologous to Scn1b in vivo. The second ortholog, scn1bb, is primarily expressed in non-excitable cell types that are not predicted to express pore-forming a subunits. Scn1bb is expressed in glia in the brain, optic nerve, and spinal cord. It is also expressed in supporting cells in lateral line neuromasts and the olfactory system, and in epithelial cells in the inner ear and lining the pronephric duct. Even though Scn1bb and the pore-forming alpha subunits do not have overlapping expression patterns in vivo, Scn1bb retains the ability to modulate Na+ currents in vitro . We predict that Scn1bb functions primarily as a CAM in vivo. scn1ba2 is the third ortholog identified in this study, and its localization is unknown to date. scn1ba2 modulates Na+ current when expressed in a heterologous system. In contrast to all of the other beta subunits identified, scn1ba2 does not produce hyperpolarizing shifts in the voltage dependence of current inactivation, suggesting that its in vivo function may be significantly different from the other orthologs identified. With the identification of SCN1B orthologs, we are now poised to study the physiological function of beta1-like subunits in zebrafish.Ph.D.Biological SciencesNeurosciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126684/2/3276153.pd

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-7

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p>acetylated α-tubulin. : Anti Scn1ba_tv1 (green), anti-acetylated α-tubulin (red). : Anti-Scn1ba_tv2 (green), anti-acetylated α-tubulin (red). Images were viewed with an Olympus FluoView 500 confocal microscope at 100× magnification with 5× additional zoom. Scale bar: 50 μm

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-11

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p>that are identical are indicted in red, strongly similar substitutions are indicated by (:), and weakly similar amino acids are indicated by (.). Identical resides in exon 5 of Scn1ba_tv1 and Scn1b are indicated in green. The two cysteine residues predicted to form the Ig loop are indicated in blue. The conserved regions that form the A/A' face of the Ig loop, sites of interaction with theα subunit [17], are underlined. Tyrosine-181 in Scn1b and the corresponding residues in Scn1ba_tv1 and Scn1ba_tv2 are highlighted in yellow. Predicted sites of N-linked glycosylation are indicated by ▼. These sites were determined using NetNGlyc 1.0 [61]. Transmembrane segments are indicated as boxes. Peptides used for antibody generation are underlined in blue. Predicted β-sheets in the Ig loop domain, based on the crystal structure of myelin P[62], are shown with labeled arrows and correspond to the ribbon diagram included in the lower panel. Lower panel: Proposed three-dimensional structure of the Ig domain of β1 using the crystal structure of myelin Po (PDB 1NEU) as a template. The figure was created with the KiNG Viewer program via the RCSB Protein Data Bank web site [63]. β strands corresponding to the arrows in the upper panel are labeled A through G. . Schematic showing the genomic organization of zebrafish . The positions of introns 1 through 5 (I1 – I5) are indicated. Positions of primers used for RT-PCR in panel D are indicated. The C-terminal alternate splice domains contained in and are encoded by exon 5. . Model of alternative splicing of . Exons 4 and 5 (boxes) and intron 4 (line) are illustrated. The splice acceptor sequence at the beginning of exon 5 is indicated by and the internal alternate splice acceptor site in exon 5 is indicated by a dashed line and by ▼. The location of stop codons in the resulting mRNAs are indicated. Drawings are not to scale. Consensus splice acceptor sequence [22] and the acceptor sequences found in exon 5 are indicated in the lower portion of the panel. P: pyrimidine. P: purine. Lower case: intronic sequence. Upper case: exonic sequence. The "T" indicated by the red arrow in the internal, alternate acceptor is rare and significantly weakens the site [22]. . RT-PCR from whole fish RNA demonstrating that both splice variants of are expressed in the mRNA pool. The upper band corresponds to and the lower band corresponds to . Translations of the resulting alternate C-terminal splice products are shown below. The sequence highlighted in green is found in Scn1ba_tv1 and corresponds to the green portion of exon 5 illustrated in panel C. The sequence highlighted in turquoise is found in Scn1ba_tv2 and corresponds to the turquoise portion of exon 5 illustrated in panel C

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-4

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p> α-tubulin (red). OP: olfactory pit. Scale bar: 50 μm

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-6

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p> α-tubulin (red). Anti-Scn1ba_tv2 stains the layers of the retina, including the ganglion cell layer (GCL), inner plexiform layer (IPL), outer plexiform layer (OPL), outer limiting membrane (OLM), and photoreceptor cell layer (PR). Staining appears to be absent in the inner nuclear layer (INL) and in the optic nerve (on). Anti-Scn1ba_tv1 stains all the layers of the retina including the inner nuclear layer, where it shows robust staining. In contrast to anti-Scn1ba_tv2, anti-Scn1ba_tv1 labels optic nerve. Scale bar: 50 μm

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-9

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p>ti-Scn1ba_tv1 produced two different staining patterns; staining at the t-tubules of striated muscle (A, D, and G), and punctate staining along the longitudinal edge of the muscle cells (arrowheads in B). Staining with anti-Scn1ba_tv2 labeled the t-tubule system and did not appear to label to muscle surface. Anti-Scn1ba_tv1 staining did not colocalize with α-BTX (D – F and H – I), suggesting that Scn1ba_tv1 is not expressed at neuromuscular junctions (arrows). Scale bar: 10 μm

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-2

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p>ane 2: Chinese hamster lung 1610 cells transiently transfected with cDNA; Lane 3: Chinese hamster lung 1610 cells transiently transfected with cDNA; Lane 4: 5 μg rat brain membranes. Arrows indicate immunoreactive bands at ~30 kD in the transfected cells and at ~30 kD and ~38 kD in rat brain. Western blot probed with anti-Scn1ba_tv1. Lane 1: 5 μg rat brain membranes; Lane 2: 15 μg zebrafish brain membranes; Lane 3: 5 μg rat brain membranes probed with anti-Scn1ba_tv1 that had been preadsorbed to the immunizing peptide ("pre"); Lane 4: 15 μg zebrafish (zf) brain membranes probed with anti-Scn1ba_tv1 that had been preadsorbed to the immunizing peptide. Arrows indicate immunoreactive bands at ~30 kD and ~38 kD in both species. . Western blot probed with anti-Scn1ba_tv2. Lane 1: mock transfected Chinese hamster lung 1610 cells; Lane 2: Chinese hamster lung 1610 cells transiently transfected with cDNA; Lane 3: Chinese hamster lung 1610 cells transiently transfected with ; Lane 4: 5 μg rat brain membranes. Arrow indicates immunoreactive band at ~30 kD. Western blot probed with anti-Scn1ba_tv2. Lane 1: 15 μg zebrafish brain membranes; Lane 2: 15 μg zebrafish brain membranes probed with anti-Scn1ba_tv2 that had been preadsorbed to the immunizing peptide ("pre"). Arrow shows immunoreactive band at ~38 kD

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-10

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p> shows the mean current elicited by depolarization to 0 mV from a holding potential of -80 mV in oocytes injected with the indicated combinations of α and β subunits. . Current Density. Coexpression of , , or cRNA with cRNA results in increased current amplitude compared to α alone. Individual peak current amplitudes for each condition were measured and normalized to the mean current amplitude of for each experiment to account for variability between different oocyte preparations. . Representative Nacurrent traces for alone (upper left), plus (upper right), plus (lower left), and plus (lower right) . Current-voltage relationships for the families of Nacurrents shown in panel C. . Voltage dependence of activation. Coexpression of with (●), (△), or (▽) results in hyperpolarizing shifts in the voltage dependence of activation compared to the expression of alone (■). Coexpression of with results in a significantly greater hyperpolarizing shift than coexpression with . . Voltage dependence of inactivation. Coexpression of with , , or resulted in hyperpolarizing shifts in the voltage dependence of inactivation compared to alone. The effects of and on the voltage-dependence of inactivation are indistinguishable from each other. . Zebrafish β subunits speed recovery from inactivation. Coexpression of with (●), (△), or (▽) results in a dramatic increase in the rate of recovery from inactivation compared with alone (■). Zebrafish (■) expressed alone has a very slow rate of recovery, and a full recovery was never achieved during the duration of the experiment