Limits...
[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.

Show MeSH

Related in: MedlinePlus

Binding of βIV-spectrin to ankyrin. COS-7 cells were transfected with Myc-tagged βIVΣ6-spectrin (Myc-βIVΣ6) together with GFP (lanes 1), GFP-tagged 270 kD ankyrin-G (AnkG-GFP; lanes 2), or GFP-tagged 220 kD ankyrin-B (AnkB-GFP; lanes 3). Cell lysates were immunoprecipitated with anti-GFP (A and D) or anti-Myc (B and C). Immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-GFP (A and B) or anti-Myc (C and D). Positions of size markers are indicated in kD on the left.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199236&req=5

fig4: Binding of βIV-spectrin to ankyrin. COS-7 cells were transfected with Myc-tagged βIVΣ6-spectrin (Myc-βIVΣ6) together with GFP (lanes 1), GFP-tagged 270 kD ankyrin-G (AnkG-GFP; lanes 2), or GFP-tagged 220 kD ankyrin-B (AnkB-GFP; lanes 3). Cell lysates were immunoprecipitated with anti-GFP (A and D) or anti-Myc (B and C). Immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-GFP (A and B) or anti-Myc (C and D). Positions of size markers are indicated in kD on the left.

Mentions: βI- and βII-spectrins have been shown to bind ankyrin-R and ankyrin-B (Davis and Bennett, 1990; Kennedy et al., 1991). This fact, together with colocalization of βIV-spectrin with ankyrin-G at AIS and NR, suggested that βIV-spectrin might bind ankyrin-G. To test this possibility, Myc-tagged βIVΣ6-spectrin (Myc-βIVΣ6) was cotransfected with green fluorescent protein (GFP), GFP-tagged 270-kD ankyrin-G (AnkG-GFP), or GFP-tagged 220-kD ankyrin-B (AnkB-GFP) into COS-7 cells. Transfected cells were lysed and immunoprecipitated, and then immunoblotted with anti-Myc or anti-GFP antibody. Whereas Myc-βIVΣ6 was expressed at a similar level in the three transfectants (Fig. 4 C), GFP and AnkB-GFP were much more highly expressed than AnkG-GFP (Fig. 4 A). Consistent with the presence in βIVΣ6-spectrin of the spectrin repeat 15, which has been mapped as an ankyrin-binding domain in βI- and βII-spectrins (Kennedy et al., 1991), the anti-Myc antibody coprecipitated both AnkG-GFP and AnkB-GFP but not GFP alone (Fig. 4 B). However, significantly more AnkG-GFP was brought down than AnkB-GFP (Fig. 4 B). Similarly, AnkG-GFP coprecipitated Myc-βIVΣ6 more efficiently than AnkB-GFP (Fig. 4 D). These results indicate that βIV-spectrin binds to ankyrin-G with high affinity, and to ankyrin-B to a lesser extent.


[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)

Binding of βIV-spectrin to ankyrin. COS-7 cells were transfected with Myc-tagged βIVΣ6-spectrin (Myc-βIVΣ6) together with GFP (lanes 1), GFP-tagged 270 kD ankyrin-G (AnkG-GFP; lanes 2), or GFP-tagged 220 kD ankyrin-B (AnkB-GFP; lanes 3). Cell lysates were immunoprecipitated with anti-GFP (A and D) or anti-Myc (B and C). Immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-GFP (A and B) or anti-Myc (C and D). Positions of size markers are indicated in kD on the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199236&req=5

fig4: Binding of βIV-spectrin to ankyrin. COS-7 cells were transfected with Myc-tagged βIVΣ6-spectrin (Myc-βIVΣ6) together with GFP (lanes 1), GFP-tagged 270 kD ankyrin-G (AnkG-GFP; lanes 2), or GFP-tagged 220 kD ankyrin-B (AnkB-GFP; lanes 3). Cell lysates were immunoprecipitated with anti-GFP (A and D) or anti-Myc (B and C). Immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-GFP (A and B) or anti-Myc (C and D). Positions of size markers are indicated in kD on the left.
Mentions: βI- and βII-spectrins have been shown to bind ankyrin-R and ankyrin-B (Davis and Bennett, 1990; Kennedy et al., 1991). This fact, together with colocalization of βIV-spectrin with ankyrin-G at AIS and NR, suggested that βIV-spectrin might bind ankyrin-G. To test this possibility, Myc-tagged βIVΣ6-spectrin (Myc-βIVΣ6) was cotransfected with green fluorescent protein (GFP), GFP-tagged 270-kD ankyrin-G (AnkG-GFP), or GFP-tagged 220-kD ankyrin-B (AnkB-GFP) into COS-7 cells. Transfected cells were lysed and immunoprecipitated, and then immunoblotted with anti-Myc or anti-GFP antibody. Whereas Myc-βIVΣ6 was expressed at a similar level in the three transfectants (Fig. 4 C), GFP and AnkB-GFP were much more highly expressed than AnkG-GFP (Fig. 4 A). Consistent with the presence in βIVΣ6-spectrin of the spectrin repeat 15, which has been mapped as an ankyrin-binding domain in βI- and βII-spectrins (Kennedy et al., 1991), the anti-Myc antibody coprecipitated both AnkG-GFP and AnkB-GFP but not GFP alone (Fig. 4 B). However, significantly more AnkG-GFP was brought down than AnkB-GFP (Fig. 4 B). Similarly, AnkG-GFP coprecipitated Myc-βIVΣ6 more efficiently than AnkB-GFP (Fig. 4 D). These results indicate that βIV-spectrin binds to ankyrin-G with high affinity, and to ankyrin-B to a lesser extent.

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