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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 TAG-1–deficient mice. (A) Map of recombination strategy showing part of TAG-1 gene locus including exon 2, which encodes the ATG and signal sequence. Below is the tau-LacZ–containing targeting construct and the predicted locus after targeting. Red lines indicate HindIII fragments detected with 5′ probe (black box). Orange and blue arrows indicate PCR primers used in C and D, respectively. (B) Southern blot showing targeting of construct in ES cells with HindIII digest and 5′ probe. (C) Detection of targeted locus in wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice by PCR. Wild-type TAG-1 allele detected by orange primer pair (see A) gives and ∼450-bp product, whereas targeted allele detected by neo-specific primers gives an ∼260-bp product. (D) RT-PCR to detect TAG-1 mRNA in postnatal cerebellum using blue primer set (see A). The expected 255-bp product is detected in heterozygote but not homozygote animals. (E) Western blot of postnatal cerebellum lysates from heterozygote and homozygote mice blotted with anti-TAG-1 pAbs. TAG-1 protein is detected in heterozygote mice, but not in the mutant. (F) Double-immunofluorescence staining of teased sciatic nerves from adult wild-type (WT), or TAG-1– (−/−) mice using antibodies to Na+ channel (red) and TAG-1 (green). Bar, 10 μm.
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fig6: Generation of TAG-1–deficient mice. (A) Map of recombination strategy showing part of TAG-1 gene locus including exon 2, which encodes the ATG and signal sequence. Below is the tau-LacZ–containing targeting construct and the predicted locus after targeting. Red lines indicate HindIII fragments detected with 5′ probe (black box). Orange and blue arrows indicate PCR primers used in C and D, respectively. (B) Southern blot showing targeting of construct in ES cells with HindIII digest and 5′ probe. (C) Detection of targeted locus in wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice by PCR. Wild-type TAG-1 allele detected by orange primer pair (see A) gives and ∼450-bp product, whereas targeted allele detected by neo-specific primers gives an ∼260-bp product. (D) RT-PCR to detect TAG-1 mRNA in postnatal cerebellum using blue primer set (see A). The expected 255-bp product is detected in heterozygote but not homozygote animals. (E) Western blot of postnatal cerebellum lysates from heterozygote and homozygote mice blotted with anti-TAG-1 pAbs. TAG-1 protein is detected in heterozygote mice, but not in the mutant. (F) Double-immunofluorescence staining of teased sciatic nerves from adult wild-type (WT), or TAG-1– (−/−) mice using antibodies to Na+ channel (red) and TAG-1 (green). Bar, 10 μm.

Mentions: To determine whether TAG-1 is important for the localization of Caspr2 and K+ channel at the juxtaparanodal region in myelinated axons, we generated TAG-1–deficient mice by homologous recombination. These animals were generated by replacing exon 2, which contains the translation initiation site and the signal sequence, and four downstream exons encoding the first two Ig domains, with a tau-LacZ reporter (Fig. 6, A–C). This targeting strategy resulted in the complete absence of TAG-1 transcript or immunoreactive protein in mice homozygous for the mutant allele (Fig. 6, D–F). TAG-1 homozygotes appear with the expected Mendelian frequency, are viable, and grow to old age (>14 mo) essentially indistinguishable from their littermates under normal laboratory conditions. Standard histological examination (Nissl) of the central nervous system of these animals revealed no gross morphological abnormalities compared with the wild type (unpublished data), as has also been reported for an independent mutation of the TAG-1 gene (Fukamauchi et al., 2001). Next, we examined the distribution of Caspr2 and Kv1.1, Kv1.2, and their Kvβ2 subunit in sciatic nerves from homozygous and wild-type littermate. As depicted in Fig. 7, although in sciatic nerves from TAG-1– mice Caspr and Na+ channels were properly located at the paranodal junction and the nodes, respectively, Caspr2 was not concentrated at the juxtaparanodal region. In this mutant, weak uniform staining of Caspr2 was detected throughout the internodes, suggesting that in the absence of TAG-1, it was redistributed along the nerve. This conclusion was further supported by immunoblotting analysis, showing that although slightly reduced, Caspr2 was still present in sciatic nerve lysates from TAG-1−/− mice (Fig. 7 F, inset). Double-immunofluorescence labeling using an antibody to Caspr and Kv1.2 showed that the concentration of K+ channels at the juxtaparanodes was markedly reduced, very similar to the Caspr2 homozygotes (Fig. 7, G–L). As in Caspr2 s, Kv1.2 was still present at comparable levels in sciatic nerves of TAG-1 mutant (Fig. 7 L, inset). These results indicate that the localization of Caspr2 and K+ channels in myelinated nerves depends on the presence of TAG-1. Remarkably, in respect to the organization of the nodal environs, it appears that Caspr2- and TAG-1– mice are phenotypically identical.


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 TAG-1–deficient mice. (A) Map of recombination strategy showing part of TAG-1 gene locus including exon 2, which encodes the ATG and signal sequence. Below is the tau-LacZ–containing targeting construct and the predicted locus after targeting. Red lines indicate HindIII fragments detected with 5′ probe (black box). Orange and blue arrows indicate PCR primers used in C and D, respectively. (B) Southern blot showing targeting of construct in ES cells with HindIII digest and 5′ probe. (C) Detection of targeted locus in wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice by PCR. Wild-type TAG-1 allele detected by orange primer pair (see A) gives and ∼450-bp product, whereas targeted allele detected by neo-specific primers gives an ∼260-bp product. (D) RT-PCR to detect TAG-1 mRNA in postnatal cerebellum using blue primer set (see A). The expected 255-bp product is detected in heterozygote but not homozygote animals. (E) Western blot of postnatal cerebellum lysates from heterozygote and homozygote mice blotted with anti-TAG-1 pAbs. TAG-1 protein is detected in heterozygote mice, but not in the mutant. (F) Double-immunofluorescence staining of teased sciatic nerves from adult wild-type (WT), or TAG-1– (−/−) mice using antibodies to Na+ channel (red) and TAG-1 (green). Bar, 10 μm.
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fig6: Generation of TAG-1–deficient mice. (A) Map of recombination strategy showing part of TAG-1 gene locus including exon 2, which encodes the ATG and signal sequence. Below is the tau-LacZ–containing targeting construct and the predicted locus after targeting. Red lines indicate HindIII fragments detected with 5′ probe (black box). Orange and blue arrows indicate PCR primers used in C and D, respectively. (B) Southern blot showing targeting of construct in ES cells with HindIII digest and 5′ probe. (C) Detection of targeted locus in wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice by PCR. Wild-type TAG-1 allele detected by orange primer pair (see A) gives and ∼450-bp product, whereas targeted allele detected by neo-specific primers gives an ∼260-bp product. (D) RT-PCR to detect TAG-1 mRNA in postnatal cerebellum using blue primer set (see A). The expected 255-bp product is detected in heterozygote but not homozygote animals. (E) Western blot of postnatal cerebellum lysates from heterozygote and homozygote mice blotted with anti-TAG-1 pAbs. TAG-1 protein is detected in heterozygote mice, but not in the mutant. (F) Double-immunofluorescence staining of teased sciatic nerves from adult wild-type (WT), or TAG-1– (−/−) mice using antibodies to Na+ channel (red) and TAG-1 (green). Bar, 10 μm.
Mentions: To determine whether TAG-1 is important for the localization of Caspr2 and K+ channel at the juxtaparanodal region in myelinated axons, we generated TAG-1–deficient mice by homologous recombination. These animals were generated by replacing exon 2, which contains the translation initiation site and the signal sequence, and four downstream exons encoding the first two Ig domains, with a tau-LacZ reporter (Fig. 6, A–C). This targeting strategy resulted in the complete absence of TAG-1 transcript or immunoreactive protein in mice homozygous for the mutant allele (Fig. 6, D–F). TAG-1 homozygotes appear with the expected Mendelian frequency, are viable, and grow to old age (>14 mo) essentially indistinguishable from their littermates under normal laboratory conditions. Standard histological examination (Nissl) of the central nervous system of these animals revealed no gross morphological abnormalities compared with the wild type (unpublished data), as has also been reported for an independent mutation of the TAG-1 gene (Fukamauchi et al., 2001). Next, we examined the distribution of Caspr2 and Kv1.1, Kv1.2, and their Kvβ2 subunit in sciatic nerves from homozygous and wild-type littermate. As depicted in Fig. 7, although in sciatic nerves from TAG-1– mice Caspr and Na+ channels were properly located at the paranodal junction and the nodes, respectively, Caspr2 was not concentrated at the juxtaparanodal region. In this mutant, weak uniform staining of Caspr2 was detected throughout the internodes, suggesting that in the absence of TAG-1, it was redistributed along the nerve. This conclusion was further supported by immunoblotting analysis, showing that although slightly reduced, Caspr2 was still present in sciatic nerve lysates from TAG-1−/− mice (Fig. 7 F, inset). Double-immunofluorescence labeling using an antibody to Caspr and Kv1.2 showed that the concentration of K+ channels at the juxtaparanodes was markedly reduced, very similar to the Caspr2 homozygotes (Fig. 7, G–L). As in Caspr2 s, Kv1.2 was still present at comparable levels in sciatic nerves of TAG-1 mutant (Fig. 7 L, inset). These results indicate that the localization of Caspr2 and K+ channels in myelinated nerves depends on the presence of TAG-1. Remarkably, in respect to the organization of the nodal environs, it appears that Caspr2- and TAG-1– mice are phenotypically identical.

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