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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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Association of Caspr2 with TAG-1. (A) Association of TAG-1 and Caspr2 in rat brain. Immunoprecipitation (IP) from rat brain membrane lysates was performed using antibodies to TAG-1 (IC12; TAG-1 mAb or rabbit pAb; TAG-1 pAb), Kv1.2, Kv2.1, contactin, or Caspr2 as indicated, followed by immunoblotting with an antibody to Caspr2. Anti-mouse (mBeads) or protein A (rBeads) beads were used as additional controls. (B) Co-immunoprecipitation of TAG-1 and K+ channels. Rat brain membrane lysates were subjected to immunoprecipitation using the indicated antibodies, followed by blotting with an antibody to Kv1.2. Total protein extract (Total) was used to determine the location of Kv1.2 on the gel. Note that Kv1.2 was detected using two different antibodies to TAG-1. (C) Association of TAG-1 and Caspr2 in transfected cells. Lysates of HEK-293 cells expressing Caspr2 and TAG-1, Caspr2 and contactin (CNTN), or Caspr and contactin (Cells), were used for immunoprecipitation (IP) and immunoblotting using different combinations of antibodies as indicated in each panel. Note that Caspr2 associated with TAG-1, but not with contactin, which interacts with Caspr. (D) Immunofluorescence staining showing surface expression of Caspr2. COS-7 cells expressing Caspr2 were stained using an antibody against its extracellular (ECD) or intracellular (CT) region, with or without permeabilization as indicated (−Tx, without Triton X-100; +Tx, with Triton X-100). Caspr2 immunoreactivity was detected using the ECD, but not CT antibody in nonpermeabilized cells. Bar, 50 μm. (E) TAG-1 binds homophilically, but not to Caspr2. A soluble TAG-1–Fc was allowed to bind COS-7 cells expressing TAG-1, Caspr2, Caspr2 and TAG-1, or contactin as indicated. Bound Fc fusion was detected using Cy3-conjugated anti-human Fc antibody (red). Note that TAG-1–Fc only bound to TAG-1– or TAG-1/Caspr2-expressing cells, but not to cells expressing Caspr2 or contactin. The insets on the top right of each panel show staining for the corresponding transfected proteins. Bar, 50 μm. (F) Top: β-galactosidase staining of adult sciatic nerve (SN) from heterozygous TAG-1–LacZ animals. Bottom: β-galactosidase staining of adult dorsal root ganglion (DRG). Intense lacZ expression was detected in cell bodies and the track (asterisk). Bars: D and E, 50 μm; F (top), 50 μm; F (bottom), 100 μm.
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fig8: Association of Caspr2 with TAG-1. (A) Association of TAG-1 and Caspr2 in rat brain. Immunoprecipitation (IP) from rat brain membrane lysates was performed using antibodies to TAG-1 (IC12; TAG-1 mAb or rabbit pAb; TAG-1 pAb), Kv1.2, Kv2.1, contactin, or Caspr2 as indicated, followed by immunoblotting with an antibody to Caspr2. Anti-mouse (mBeads) or protein A (rBeads) beads were used as additional controls. (B) Co-immunoprecipitation of TAG-1 and K+ channels. Rat brain membrane lysates were subjected to immunoprecipitation using the indicated antibodies, followed by blotting with an antibody to Kv1.2. Total protein extract (Total) was used to determine the location of Kv1.2 on the gel. Note that Kv1.2 was detected using two different antibodies to TAG-1. (C) Association of TAG-1 and Caspr2 in transfected cells. Lysates of HEK-293 cells expressing Caspr2 and TAG-1, Caspr2 and contactin (CNTN), or Caspr and contactin (Cells), were used for immunoprecipitation (IP) and immunoblotting using different combinations of antibodies as indicated in each panel. Note that Caspr2 associated with TAG-1, but not with contactin, which interacts with Caspr. (D) Immunofluorescence staining showing surface expression of Caspr2. COS-7 cells expressing Caspr2 were stained using an antibody against its extracellular (ECD) or intracellular (CT) region, with or without permeabilization as indicated (−Tx, without Triton X-100; +Tx, with Triton X-100). Caspr2 immunoreactivity was detected using the ECD, but not CT antibody in nonpermeabilized cells. Bar, 50 μm. (E) TAG-1 binds homophilically, but not to Caspr2. A soluble TAG-1–Fc was allowed to bind COS-7 cells expressing TAG-1, Caspr2, Caspr2 and TAG-1, or contactin as indicated. Bound Fc fusion was detected using Cy3-conjugated anti-human Fc antibody (red). Note that TAG-1–Fc only bound to TAG-1– or TAG-1/Caspr2-expressing cells, but not to cells expressing Caspr2 or contactin. The insets on the top right of each panel show staining for the corresponding transfected proteins. Bar, 50 μm. (F) Top: β-galactosidase staining of adult sciatic nerve (SN) from heterozygous TAG-1–LacZ animals. Bottom: β-galactosidase staining of adult dorsal root ganglion (DRG). Intense lacZ expression was detected in cell bodies and the track (asterisk). Bars: D and E, 50 μm; F (top), 50 μm; F (bottom), 100 μm.

Mentions: The interdependent localization of Caspr2 and TAG-1 in myelinated nerves, together with the high sequence similarity of TAG-1 with contactin (48% sequence identity), which associates with Caspr (Peles et al., 1997), suggests that Caspr2 and TAG-1 may form a complex at the juxtaparanodal region. This possibility was directly tested using coimmunoprecipitation and binding experiments. As shown in Fig. 8 A, Caspr2 was specifically coimmunoprecipitated from adult rat brain membrane lysates using two different antibodies to TAG-1. In contrast, no association was detected between Caspr2 and contactin. Under the same conditions, Caspr2 was precipitated using an antibody to Kv1.2, but not with Kv2.1, as expected from previous reports (Poliak et al., 1999; Rasband et al., 2002). Furthermore, antibodies to TAG-1 and Caspr2 specifically coprecipitated Kv1.2 from rat brain (Fig. 8 B), suggesting the existence of a tripartite complex of Caspr2, TAG-1, and K+ channels. We estimated that ∼5% of Caspr2 molecules are found in association with TAG-1, which is similar to the amount we get with anti-Kv1.2 antibody. Next, we examined the interactions between Caspr2 and TAG-1 in transfected cells. As depicted in Fig. 8 C, TAG-1 antibody precipitated Caspr2 from HEK-293 cells expressing both proteins. Similarly, TAG-1 was detected in Caspr2 immunocomplexes, indicating that these two proteins are associated in these cells. No association between Caspr2 and TAG-1 was observed by mixing detergent lysates of cells independently transfected with Caspr2 and TAG-1, indicating that these proteins interact before cell lysis (unpublished data). In contrast to Caspr, which strongly associated with contactin, no association between Caspr2 and contactin was detected when coexpressed in HEK-293 cells. As previously reported for Caspr2 (Poliak et al., 1999), we could not detect any interaction between TAG-1 and Kv1.2 in transfected cells (unpublished data), suggesting that the association between TAG-1 and these channels observed in rat brain occurs indirectly, and probably requires Caspr2 and additional unknown adaptor proteins.


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)

Association of Caspr2 with TAG-1. (A) Association of TAG-1 and Caspr2 in rat brain. Immunoprecipitation (IP) from rat brain membrane lysates was performed using antibodies to TAG-1 (IC12; TAG-1 mAb or rabbit pAb; TAG-1 pAb), Kv1.2, Kv2.1, contactin, or Caspr2 as indicated, followed by immunoblotting with an antibody to Caspr2. Anti-mouse (mBeads) or protein A (rBeads) beads were used as additional controls. (B) Co-immunoprecipitation of TAG-1 and K+ channels. Rat brain membrane lysates were subjected to immunoprecipitation using the indicated antibodies, followed by blotting with an antibody to Kv1.2. Total protein extract (Total) was used to determine the location of Kv1.2 on the gel. Note that Kv1.2 was detected using two different antibodies to TAG-1. (C) Association of TAG-1 and Caspr2 in transfected cells. Lysates of HEK-293 cells expressing Caspr2 and TAG-1, Caspr2 and contactin (CNTN), or Caspr and contactin (Cells), were used for immunoprecipitation (IP) and immunoblotting using different combinations of antibodies as indicated in each panel. Note that Caspr2 associated with TAG-1, but not with contactin, which interacts with Caspr. (D) Immunofluorescence staining showing surface expression of Caspr2. COS-7 cells expressing Caspr2 were stained using an antibody against its extracellular (ECD) or intracellular (CT) region, with or without permeabilization as indicated (−Tx, without Triton X-100; +Tx, with Triton X-100). Caspr2 immunoreactivity was detected using the ECD, but not CT antibody in nonpermeabilized cells. Bar, 50 μm. (E) TAG-1 binds homophilically, but not to Caspr2. A soluble TAG-1–Fc was allowed to bind COS-7 cells expressing TAG-1, Caspr2, Caspr2 and TAG-1, or contactin as indicated. Bound Fc fusion was detected using Cy3-conjugated anti-human Fc antibody (red). Note that TAG-1–Fc only bound to TAG-1– or TAG-1/Caspr2-expressing cells, but not to cells expressing Caspr2 or contactin. The insets on the top right of each panel show staining for the corresponding transfected proteins. Bar, 50 μm. (F) Top: β-galactosidase staining of adult sciatic nerve (SN) from heterozygous TAG-1–LacZ animals. Bottom: β-galactosidase staining of adult dorsal root ganglion (DRG). Intense lacZ expression was detected in cell bodies and the track (asterisk). Bars: D and E, 50 μm; F (top), 50 μm; F (bottom), 100 μm.
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fig8: Association of Caspr2 with TAG-1. (A) Association of TAG-1 and Caspr2 in rat brain. Immunoprecipitation (IP) from rat brain membrane lysates was performed using antibodies to TAG-1 (IC12; TAG-1 mAb or rabbit pAb; TAG-1 pAb), Kv1.2, Kv2.1, contactin, or Caspr2 as indicated, followed by immunoblotting with an antibody to Caspr2. Anti-mouse (mBeads) or protein A (rBeads) beads were used as additional controls. (B) Co-immunoprecipitation of TAG-1 and K+ channels. Rat brain membrane lysates were subjected to immunoprecipitation using the indicated antibodies, followed by blotting with an antibody to Kv1.2. Total protein extract (Total) was used to determine the location of Kv1.2 on the gel. Note that Kv1.2 was detected using two different antibodies to TAG-1. (C) Association of TAG-1 and Caspr2 in transfected cells. Lysates of HEK-293 cells expressing Caspr2 and TAG-1, Caspr2 and contactin (CNTN), or Caspr and contactin (Cells), were used for immunoprecipitation (IP) and immunoblotting using different combinations of antibodies as indicated in each panel. Note that Caspr2 associated with TAG-1, but not with contactin, which interacts with Caspr. (D) Immunofluorescence staining showing surface expression of Caspr2. COS-7 cells expressing Caspr2 were stained using an antibody against its extracellular (ECD) or intracellular (CT) region, with or without permeabilization as indicated (−Tx, without Triton X-100; +Tx, with Triton X-100). Caspr2 immunoreactivity was detected using the ECD, but not CT antibody in nonpermeabilized cells. Bar, 50 μm. (E) TAG-1 binds homophilically, but not to Caspr2. A soluble TAG-1–Fc was allowed to bind COS-7 cells expressing TAG-1, Caspr2, Caspr2 and TAG-1, or contactin as indicated. Bound Fc fusion was detected using Cy3-conjugated anti-human Fc antibody (red). Note that TAG-1–Fc only bound to TAG-1– or TAG-1/Caspr2-expressing cells, but not to cells expressing Caspr2 or contactin. The insets on the top right of each panel show staining for the corresponding transfected proteins. Bar, 50 μm. (F) Top: β-galactosidase staining of adult sciatic nerve (SN) from heterozygous TAG-1–LacZ animals. Bottom: β-galactosidase staining of adult dorsal root ganglion (DRG). Intense lacZ expression was detected in cell bodies and the track (asterisk). Bars: D and E, 50 μm; F (top), 50 μm; F (bottom), 100 μm.
Mentions: The interdependent localization of Caspr2 and TAG-1 in myelinated nerves, together with the high sequence similarity of TAG-1 with contactin (48% sequence identity), which associates with Caspr (Peles et al., 1997), suggests that Caspr2 and TAG-1 may form a complex at the juxtaparanodal region. This possibility was directly tested using coimmunoprecipitation and binding experiments. As shown in Fig. 8 A, Caspr2 was specifically coimmunoprecipitated from adult rat brain membrane lysates using two different antibodies to TAG-1. In contrast, no association was detected between Caspr2 and contactin. Under the same conditions, Caspr2 was precipitated using an antibody to Kv1.2, but not with Kv2.1, as expected from previous reports (Poliak et al., 1999; Rasband et al., 2002). Furthermore, antibodies to TAG-1 and Caspr2 specifically coprecipitated Kv1.2 from rat brain (Fig. 8 B), suggesting the existence of a tripartite complex of Caspr2, TAG-1, and K+ channels. We estimated that ∼5% of Caspr2 molecules are found in association with TAG-1, which is similar to the amount we get with anti-Kv1.2 antibody. Next, we examined the interactions between Caspr2 and TAG-1 in transfected cells. As depicted in Fig. 8 C, TAG-1 antibody precipitated Caspr2 from HEK-293 cells expressing both proteins. Similarly, TAG-1 was detected in Caspr2 immunocomplexes, indicating that these two proteins are associated in these cells. No association between Caspr2 and TAG-1 was observed by mixing detergent lysates of cells independently transfected with Caspr2 and TAG-1, indicating that these proteins interact before cell lysis (unpublished data). In contrast to Caspr, which strongly associated with contactin, no association between Caspr2 and contactin was detected when coexpressed in HEK-293 cells. As previously reported for Caspr2 (Poliak et al., 1999), we could not detect any interaction between TAG-1 and Kv1.2 in transfected cells (unpublished data), suggesting that the association between TAG-1 and these channels observed in rat brain occurs indirectly, and probably requires Caspr2 and additional unknown adaptor proteins.

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