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beta-Spectrin is colocalized with both voltage-gated sodium channels and ankyrinG at the adult rat neuromuscular junction.

Wood SJ, Slater CR - J. Cell Biol. (1998)

Bottom Line: At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, beta-spectrin and ankyrin, which may help to maintain the high local density of VGSCs.Double antibody labeling shows that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ.These observations suggest that interactions with beta-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.

View Article: PubMed Central - PubMed

Affiliation: School of Neurosciences, The Medical School, University of Newcastle upon Tyne NE2 4HH, United Kingdom. s.j.wood@bristol.ac.uk

ABSTRACT
Voltage-gated sodium channels (VGSCs) are concentrated in the depths of the postsynaptic folds at mammalian neuromuscular junctions (NMJs) where they facilitate action potential generation during neuromuscular transmission. At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, beta-spectrin and ankyrin, which may help to maintain the high local density of VGSCs. Here we show in skeletal muscle, using immunofluorescence, that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG, the nodal isoform of ankyrin. In en face views of rat NMJs, acetylcholine receptors (AChRs), and utrophin immunolabeling are organized in distinctive linear arrays corresponding to the crests of the postsynaptic folds. In contrast, beta-spectrin, VGSCs, and ankyrinG have a punctate distribution that extends laterally beyond the AChRs, consistent with a localization in the depths of the folds. Double antibody labeling shows that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ. Furthermore, quantification of immunofluorescence in labeled transverse sections reveals that beta-spectrin is also concentrated in perijunctional regions, in parallel with an increase in labeling of VGSCs and ankyrinG, but not of dystrophin. These observations suggest that interactions with beta-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.

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Specificity of immunolabeling of VGSCs. Digitized images of transverse sections of rat soleus muscle were dual labeled  with FITC α-BgTx to identify AChRs at the NMJ and antibody  to VGSCs (AP1380); antibody to VGSCs preincubated with the  immunogenic peptide against which it was raised (AP1380/EOIII)  or no primary antibody (diluent). All were visualized with TRITC  secondary antibody. The images are printed so that the intensity  levels in each column are comparable. Bar, 20 μm.
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Figure 1: Specificity of immunolabeling of VGSCs. Digitized images of transverse sections of rat soleus muscle were dual labeled with FITC α-BgTx to identify AChRs at the NMJ and antibody to VGSCs (AP1380); antibody to VGSCs preincubated with the immunogenic peptide against which it was raised (AP1380/EOIII) or no primary antibody (diluent). All were visualized with TRITC secondary antibody. The images are printed so that the intensity levels in each column are comparable. Bar, 20 μm.

Mentions: The following primary antibodies were diluted in PBS containing 3% BSA and 0.1 M lysine as indicated. Two previously described monoclonal antibodies that recognize β-spectrin, raised against human RBC ghosts, were used. NCLSPEC2 was used in sections (1:30; Novacastra, Burlingame, CA) and RBC2/5C4 (Bewick et al., 1992) was used in teased fibers (1:2; a gift from Dr. L. Anderson, University of Newcastle upon Tyne). An affinity-purified rabbit polyclonal raised against peptides unique to the rat ankyrinG spectrin-binding domain AnkG@SpBd was used in sections at 0.01 μg/μl and in teased fibers at 0.02 μg/μl (a gift from Dr. S. Lambert, Worcester Foundation for Biomedical Research, Shrewsbury, MA) (Kordeli et al., 1995). For VGSC labeling, a rabbit polyclonal antibody AP1380 raised against a synthetic peptide EOIII, which recognizes the highly conserved segment in the intracellular III–IV loop that is present in all known vertebrate sodium channels (a gift from Dr. R. Levinson, University of Colorado School of Medicine, Denver, CO) (Dugandzija-Novakovic et al., 1995) was used at a dilution of 1:30 in sections and 1:10 for teased fibers. Control experiments where the VGSC antibody (2 μl) was preabsorbed with the peptide antigen EOIII (1 μg in 1 μl) overnight at 4°C and then applied to the tissues revealed no significant labeling above the no primary control (Fig. 1). A monoclonal antibody DY8/6C5 to a COOH-terminal epitope of dystrophin was used at 1:50 in sections and at 1:10 in teased fibers (a gift from Dr. L. Anderson) (Bewick et al., 1992). A monoclonal antibody DRP3/20C5, raised against a fusion protein containing the first 261 NH2-terminal amino acids of the utrophin sequence, was used at 1:10 in both sections and in teased fibers (a gift from Dr. L. Anderson) (also available as antibody NCLDRP2; Novacastra).


beta-Spectrin is colocalized with both voltage-gated sodium channels and ankyrinG at the adult rat neuromuscular junction.

Wood SJ, Slater CR - J. Cell Biol. (1998)

Specificity of immunolabeling of VGSCs. Digitized images of transverse sections of rat soleus muscle were dual labeled  with FITC α-BgTx to identify AChRs at the NMJ and antibody  to VGSCs (AP1380); antibody to VGSCs preincubated with the  immunogenic peptide against which it was raised (AP1380/EOIII)  or no primary antibody (diluent). All were visualized with TRITC  secondary antibody. The images are printed so that the intensity  levels in each column are comparable. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140176&req=5

Figure 1: Specificity of immunolabeling of VGSCs. Digitized images of transverse sections of rat soleus muscle were dual labeled with FITC α-BgTx to identify AChRs at the NMJ and antibody to VGSCs (AP1380); antibody to VGSCs preincubated with the immunogenic peptide against which it was raised (AP1380/EOIII) or no primary antibody (diluent). All were visualized with TRITC secondary antibody. The images are printed so that the intensity levels in each column are comparable. Bar, 20 μm.
Mentions: The following primary antibodies were diluted in PBS containing 3% BSA and 0.1 M lysine as indicated. Two previously described monoclonal antibodies that recognize β-spectrin, raised against human RBC ghosts, were used. NCLSPEC2 was used in sections (1:30; Novacastra, Burlingame, CA) and RBC2/5C4 (Bewick et al., 1992) was used in teased fibers (1:2; a gift from Dr. L. Anderson, University of Newcastle upon Tyne). An affinity-purified rabbit polyclonal raised against peptides unique to the rat ankyrinG spectrin-binding domain AnkG@SpBd was used in sections at 0.01 μg/μl and in teased fibers at 0.02 μg/μl (a gift from Dr. S. Lambert, Worcester Foundation for Biomedical Research, Shrewsbury, MA) (Kordeli et al., 1995). For VGSC labeling, a rabbit polyclonal antibody AP1380 raised against a synthetic peptide EOIII, which recognizes the highly conserved segment in the intracellular III–IV loop that is present in all known vertebrate sodium channels (a gift from Dr. R. Levinson, University of Colorado School of Medicine, Denver, CO) (Dugandzija-Novakovic et al., 1995) was used at a dilution of 1:30 in sections and 1:10 for teased fibers. Control experiments where the VGSC antibody (2 μl) was preabsorbed with the peptide antigen EOIII (1 μg in 1 μl) overnight at 4°C and then applied to the tissues revealed no significant labeling above the no primary control (Fig. 1). A monoclonal antibody DY8/6C5 to a COOH-terminal epitope of dystrophin was used at 1:50 in sections and at 1:10 in teased fibers (a gift from Dr. L. Anderson) (Bewick et al., 1992). A monoclonal antibody DRP3/20C5, raised against a fusion protein containing the first 261 NH2-terminal amino acids of the utrophin sequence, was used at 1:10 in both sections and in teased fibers (a gift from Dr. L. Anderson) (also available as antibody NCLDRP2; Novacastra).

Bottom Line: At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, beta-spectrin and ankyrin, which may help to maintain the high local density of VGSCs.Double antibody labeling shows that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ.These observations suggest that interactions with beta-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.

View Article: PubMed Central - PubMed

Affiliation: School of Neurosciences, The Medical School, University of Newcastle upon Tyne NE2 4HH, United Kingdom. s.j.wood@bristol.ac.uk

ABSTRACT
Voltage-gated sodium channels (VGSCs) are concentrated in the depths of the postsynaptic folds at mammalian neuromuscular junctions (NMJs) where they facilitate action potential generation during neuromuscular transmission. At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, beta-spectrin and ankyrin, which may help to maintain the high local density of VGSCs. Here we show in skeletal muscle, using immunofluorescence, that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG, the nodal isoform of ankyrin. In en face views of rat NMJs, acetylcholine receptors (AChRs), and utrophin immunolabeling are organized in distinctive linear arrays corresponding to the crests of the postsynaptic folds. In contrast, beta-spectrin, VGSCs, and ankyrinG have a punctate distribution that extends laterally beyond the AChRs, consistent with a localization in the depths of the folds. Double antibody labeling shows that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ. Furthermore, quantification of immunofluorescence in labeled transverse sections reveals that beta-spectrin is also concentrated in perijunctional regions, in parallel with an increase in labeling of VGSCs and ankyrinG, but not of dystrophin. These observations suggest that interactions with beta-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.

Show MeSH
Related in: MedlinePlus