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[Beta]IV-spectrin regulates sodium channel clustering through ankyrin-G at axon initial segments and nodes of Ranvier.

Komada M, Soriano P - J. Cell Biol. (2002)

Bottom Line: In betaIV-spectrin- neurons, neither ankyrin-G nor voltage-gated sodium channels (VGSC) are correctly clustered at these sites, suggesting that impaired action potential caused by mislocalization of VGSC leads to the phenotype.Conversely, in ankyrin-G- neurons, betaIV-spectrin is not localized to these sites.These results indicate that betaIV-spectrin and ankyrin-G mutually stabilize the membrane protein cluster and the linked membrane cytoskeleton at AIS and NR.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental Biology and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. makomada@bio.titech.ac.jp

ABSTRACT
beta-Spectrin and ankyrin are major components of the membrane cytoskeleton. We have generated mice carrying a mutation in the betaIV-spectrin gene using gene trapping in embryonic stem cells. Mice homozygous for the mutation exhibit tremors and contraction of hindlimbs. betaIV-spectrin expression is mostly restricted to neurons, where it colocalizes with and binds to ankyrin-G at axon initial segments (AISs) and nodes of Ranvier (NR). In betaIV-spectrin- neurons, neither ankyrin-G nor voltage-gated sodium channels (VGSC) are correctly clustered at these sites, suggesting that impaired action potential caused by mislocalization of VGSC leads to the phenotype. Conversely, in ankyrin-G- neurons, betaIV-spectrin is not localized to these sites. These results indicate that betaIV-spectrin and ankyrin-G mutually stabilize the membrane protein cluster and the linked membrane cytoskeleton at AIS and NR.

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Characterization of βIV-spectrin and its gene products. (A) Schematic diagram of the βIVΣ1- and Σ6-spectrin isoforms. The gene trap vector was inserted between exons encoding the spectrin repeat 12 of the βIV-spectrin gene. (B) Sequence of the 5′ region of βIVΣ6-spectrin cDNA and its comparison with the corresponding region of βIVΣ1-spectrin cDNA. The sequence of Σ6 diverges upstream of the nucleotide 4079 of Σ1 (arrow). (C) Northern blot analysis of βIV-spectrin expression in brains from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice. The regions of the 5′- and 3′-probes are indicated in (A). Positions of the 28S and 18S rRNAs are indicated. (D) Western blot analysis of βIV-spectrin in brain lysates from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice. Positions of size markers are indicated in kD on the left. The sequence data are available from Genbank/EMBL/DDBJ accession no. AB055618 for βIVΣ1-spectrin and AB055619, AB055620, AB055621 for βIVΣ6-spectrin.
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fig2: Characterization of βIV-spectrin and its gene products. (A) Schematic diagram of the βIVΣ1- and Σ6-spectrin isoforms. The gene trap vector was inserted between exons encoding the spectrin repeat 12 of the βIV-spectrin gene. (B) Sequence of the 5′ region of βIVΣ6-spectrin cDNA and its comparison with the corresponding region of βIVΣ1-spectrin cDNA. The sequence of Σ6 diverges upstream of the nucleotide 4079 of Σ1 (arrow). (C) Northern blot analysis of βIV-spectrin expression in brains from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice. The regions of the 5′- and 3′-probes are indicated in (A). Positions of the 28S and 18S rRNAs are indicated. (D) Western blot analysis of βIV-spectrin in brain lysates from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice. Positions of size markers are indicated in kD on the left. The sequence data are available from Genbank/EMBL/DDBJ accession no. AB055618 for βIVΣ1-spectrin and AB055619, AB055620, AB055621 for βIVΣ6-spectrin.

Mentions: To identify the gene trapped in this mutant, the ROSA62–βgeo* fusion cDNA was cloned from ROSA62 mutant ES cells by 5′-RACE, and full-length cDNAs were subsequently cloned by screening cDNA libraries. Sequencing of the cDNAs revealed that the trapped gene encodes βIV-spectrin, for which the human homologue has been recently identified (Berghs et al., 2000; Tse et al., 2001). Two βIV-spectrin splice isoforms, Σ1 and Σ6, were cloned (Fig. 2 A). βIVΣ1-spectrin was 2561 amino acids long, consisted of an NH2-terminal actin-binding domain, 17 spectrin repeats, a variable region, and a COOH-terminal plecstrin homology (PH) domain, and had 95% overall sequence identity to its human homologue. The actin-binding domain, the entire spectrin repeats, and the PH domain of βIVΣ1-spectrin were ∼70, 40, and 55% identical in amino acid sequences, respectively, to those of βI-, βII-, and βIII-spectrins, with the highest sequence identity to βII-spectrin.


[Beta]IV-spectrin regulates sodium channel clustering through ankyrin-G at axon initial segments and nodes of Ranvier.

Komada M, Soriano P - J. Cell Biol. (2002)

Characterization of βIV-spectrin and its gene products. (A) Schematic diagram of the βIVΣ1- and Σ6-spectrin isoforms. The gene trap vector was inserted between exons encoding the spectrin repeat 12 of the βIV-spectrin gene. (B) Sequence of the 5′ region of βIVΣ6-spectrin cDNA and its comparison with the corresponding region of βIVΣ1-spectrin cDNA. The sequence of Σ6 diverges upstream of the nucleotide 4079 of Σ1 (arrow). (C) Northern blot analysis of βIV-spectrin expression in brains from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice. The regions of the 5′- and 3′-probes are indicated in (A). Positions of the 28S and 18S rRNAs are indicated. (D) Western blot analysis of βIV-spectrin in brain lysates from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice. Positions of size markers are indicated in kD on the left. The sequence data are available from Genbank/EMBL/DDBJ accession no. AB055618 for βIVΣ1-spectrin and AB055619, AB055620, AB055621 for βIVΣ6-spectrin.
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Related In: Results  -  Collection

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fig2: Characterization of βIV-spectrin and its gene products. (A) Schematic diagram of the βIVΣ1- and Σ6-spectrin isoforms. The gene trap vector was inserted between exons encoding the spectrin repeat 12 of the βIV-spectrin gene. (B) Sequence of the 5′ region of βIVΣ6-spectrin cDNA and its comparison with the corresponding region of βIVΣ1-spectrin cDNA. The sequence of Σ6 diverges upstream of the nucleotide 4079 of Σ1 (arrow). (C) Northern blot analysis of βIV-spectrin expression in brains from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice. The regions of the 5′- and 3′-probes are indicated in (A). Positions of the 28S and 18S rRNAs are indicated. (D) Western blot analysis of βIV-spectrin in brain lysates from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice. Positions of size markers are indicated in kD on the left. The sequence data are available from Genbank/EMBL/DDBJ accession no. AB055618 for βIVΣ1-spectrin and AB055619, AB055620, AB055621 for βIVΣ6-spectrin.
Mentions: To identify the gene trapped in this mutant, the ROSA62–βgeo* fusion cDNA was cloned from ROSA62 mutant ES cells by 5′-RACE, and full-length cDNAs were subsequently cloned by screening cDNA libraries. Sequencing of the cDNAs revealed that the trapped gene encodes βIV-spectrin, for which the human homologue has been recently identified (Berghs et al., 2000; Tse et al., 2001). Two βIV-spectrin splice isoforms, Σ1 and Σ6, were cloned (Fig. 2 A). βIVΣ1-spectrin was 2561 amino acids long, consisted of an NH2-terminal actin-binding domain, 17 spectrin repeats, a variable region, and a COOH-terminal plecstrin homology (PH) domain, and had 95% overall sequence identity to its human homologue. The actin-binding domain, the entire spectrin repeats, and the PH domain of βIVΣ1-spectrin were ∼70, 40, and 55% identical in amino acid sequences, respectively, to those of βI-, βII-, and βIII-spectrins, with the highest sequence identity to βII-spectrin.

Bottom Line: In betaIV-spectrin- neurons, neither ankyrin-G nor voltage-gated sodium channels (VGSC) are correctly clustered at these sites, suggesting that impaired action potential caused by mislocalization of VGSC leads to the phenotype.Conversely, in ankyrin-G- neurons, betaIV-spectrin is not localized to these sites.These results indicate that betaIV-spectrin and ankyrin-G mutually stabilize the membrane protein cluster and the linked membrane cytoskeleton at AIS and NR.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental Biology and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. makomada@bio.titech.ac.jp

ABSTRACT
beta-Spectrin and ankyrin are major components of the membrane cytoskeleton. We have generated mice carrying a mutation in the betaIV-spectrin gene using gene trapping in embryonic stem cells. Mice homozygous for the mutation exhibit tremors and contraction of hindlimbs. betaIV-spectrin expression is mostly restricted to neurons, where it colocalizes with and binds to ankyrin-G at axon initial segments (AISs) and nodes of Ranvier (NR). In betaIV-spectrin- neurons, neither ankyrin-G nor voltage-gated sodium channels (VGSC) are correctly clustered at these sites, suggesting that impaired action potential caused by mislocalization of VGSC leads to the phenotype. Conversely, in ankyrin-G- neurons, betaIV-spectrin is not localized to these sites. These results indicate that betaIV-spectrin and ankyrin-G mutually stabilize the membrane protein cluster and the linked membrane cytoskeleton at AIS and NR.

Show MeSH
Related in: MedlinePlus