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Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1.

Poliak S, Salomon D, Elhanany H, Sabanay H, Kiernan B, Pevny L, Stewart CL, Xu X, Chiu SY, Shrager P, Furley AJ, Peles E - J. Cell Biol. (2003)

Bottom Line: In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily.Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location.These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon-glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily. Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location. Furthermore, we show that the localization of Caspr2 and clustering of K+ channels at the juxtaparanodal region depends on the presence of TAG-1, an immunoglobulin-like cell adhesion molecule that binds Caspr2. These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon-glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane.

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Generation of Caspr2- mice. (A) Schematic map of a genomic DNA fragment containing the first exon of Caspr2 and flanking region, the targeting construct, and the resulting allele in which exon 1 was replaced by a neo gene. The expected size of the DNA fragment detected using the 3′ probe (black box) is labeled in red. (B) Genomic PCR analysis of the indicated animals using specific primer sets of exon 1 of Caspr2, neo, or a control CGT gene as indicated. (C) Southern blot analysis. Genomic DNA digested with HindIII and PvuII was hybridized to the 3′ probe. The expected 4.8-kb and/or 3.3-kb fragments were detected in wild-type (+/+), heterozygote (−/+), and homozygote (−/−) animals. (D) Western blot analysis. Brain lysates prepared from the indicated mice were subjected to immunoprecipitation (IP) and immunoblotting, or directly blotted (Total) using antibodies to Caspr2 or Caspr. (E) Teased sciatic nerves of adult wild-type (WT) or Caspr2- (−/−) mice were double labeled using antibodies to Na+ channel (red) and Caspr2 (green). Bar, 10 μm.
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fig1: Generation of Caspr2- mice. (A) Schematic map of a genomic DNA fragment containing the first exon of Caspr2 and flanking region, the targeting construct, and the resulting allele in which exon 1 was replaced by a neo gene. The expected size of the DNA fragment detected using the 3′ probe (black box) is labeled in red. (B) Genomic PCR analysis of the indicated animals using specific primer sets of exon 1 of Caspr2, neo, or a control CGT gene as indicated. (C) Southern blot analysis. Genomic DNA digested with HindIII and PvuII was hybridized to the 3′ probe. The expected 4.8-kb and/or 3.3-kb fragments were detected in wild-type (+/+), heterozygote (−/+), and homozygote (−/−) animals. (D) Western blot analysis. Brain lysates prepared from the indicated mice were subjected to immunoprecipitation (IP) and immunoblotting, or directly blotted (Total) using antibodies to Caspr2 or Caspr. (E) Teased sciatic nerves of adult wild-type (WT) or Caspr2- (−/−) mice were double labeled using antibodies to Na+ channel (red) and Caspr2 (green). Bar, 10 μm.

Mentions: Caspr2- mice were generated by a standard gene-targeting approach, resulting in the replacement of its first exon, which includes the translation initiation site and its signal sequence with a neo gene (Fig. 1 A). Southern blot and genomic PCR analyses were used to identify the targeted alleles in embryonic stem (ES) cells and subsequently in heterozygous (−/+) and homozygous (−/−) mice (Fig. 1, B and C). Immunoprecipitation and Western blot analyses demonstrated that Caspr2 protein was completely absent in the nervous system of homozygous animals (Fig. 1 D). Similarly, Caspr2 transcript was not detected by RT-PCR performed on homozygous brain RNA (unpublished data). Finally, in marked contrast to the juxtaparanodal appearance of Caspr2 in wild-type and heterozygous animals, no specific signal was obtained when sciatic (Fig. 1 E) or optic nerves (unpublished data) of homozygous mice where immunolabeled with Caspr2 antibodies. These results demonstrate that the animals we have generated are complete Caspr2 s.


Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1.

Poliak S, Salomon D, Elhanany H, Sabanay H, Kiernan B, Pevny L, Stewart CL, Xu X, Chiu SY, Shrager P, Furley AJ, Peles E - J. Cell Biol. (2003)

Generation of Caspr2- mice. (A) Schematic map of a genomic DNA fragment containing the first exon of Caspr2 and flanking region, the targeting construct, and the resulting allele in which exon 1 was replaced by a neo gene. The expected size of the DNA fragment detected using the 3′ probe (black box) is labeled in red. (B) Genomic PCR analysis of the indicated animals using specific primer sets of exon 1 of Caspr2, neo, or a control CGT gene as indicated. (C) Southern blot analysis. Genomic DNA digested with HindIII and PvuII was hybridized to the 3′ probe. The expected 4.8-kb and/or 3.3-kb fragments were detected in wild-type (+/+), heterozygote (−/+), and homozygote (−/−) animals. (D) Western blot analysis. Brain lysates prepared from the indicated mice were subjected to immunoprecipitation (IP) and immunoblotting, or directly blotted (Total) using antibodies to Caspr2 or Caspr. (E) Teased sciatic nerves of adult wild-type (WT) or Caspr2- (−/−) mice were double labeled using antibodies to Na+ channel (red) and Caspr2 (green). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172860&req=5

fig1: Generation of Caspr2- mice. (A) Schematic map of a genomic DNA fragment containing the first exon of Caspr2 and flanking region, the targeting construct, and the resulting allele in which exon 1 was replaced by a neo gene. The expected size of the DNA fragment detected using the 3′ probe (black box) is labeled in red. (B) Genomic PCR analysis of the indicated animals using specific primer sets of exon 1 of Caspr2, neo, or a control CGT gene as indicated. (C) Southern blot analysis. Genomic DNA digested with HindIII and PvuII was hybridized to the 3′ probe. The expected 4.8-kb and/or 3.3-kb fragments were detected in wild-type (+/+), heterozygote (−/+), and homozygote (−/−) animals. (D) Western blot analysis. Brain lysates prepared from the indicated mice were subjected to immunoprecipitation (IP) and immunoblotting, or directly blotted (Total) using antibodies to Caspr2 or Caspr. (E) Teased sciatic nerves of adult wild-type (WT) or Caspr2- (−/−) mice were double labeled using antibodies to Na+ channel (red) and Caspr2 (green). Bar, 10 μm.
Mentions: Caspr2- mice were generated by a standard gene-targeting approach, resulting in the replacement of its first exon, which includes the translation initiation site and its signal sequence with a neo gene (Fig. 1 A). Southern blot and genomic PCR analyses were used to identify the targeted alleles in embryonic stem (ES) cells and subsequently in heterozygous (−/+) and homozygous (−/−) mice (Fig. 1, B and C). Immunoprecipitation and Western blot analyses demonstrated that Caspr2 protein was completely absent in the nervous system of homozygous animals (Fig. 1 D). Similarly, Caspr2 transcript was not detected by RT-PCR performed on homozygous brain RNA (unpublished data). Finally, in marked contrast to the juxtaparanodal appearance of Caspr2 in wild-type and heterozygous animals, no specific signal was obtained when sciatic (Fig. 1 E) or optic nerves (unpublished data) of homozygous mice where immunolabeled with Caspr2 antibodies. These results demonstrate that the animals we have generated are complete Caspr2 s.

Bottom Line: In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily.Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location.These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon-glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily. Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location. Furthermore, we show that the localization of Caspr2 and clustering of K+ channels at the juxtaparanodal region depends on the presence of TAG-1, an immunoglobulin-like cell adhesion molecule that binds Caspr2. These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon-glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane.

Show MeSH
Related in: MedlinePlus