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A synaptic laminin–calcium channel interaction organizes active zones in motor nerve terminals

Nature volume 432, pages 580–587 (2004)Cite this article

Abstract

Synapse formation requires the differentiation of a functional nerve terminal opposite a specialized postsynaptic membrane. Here, we show that laminin β2, a component of the synaptic cleft at the neuromuscular junction, binds directly to calcium channels that are required for neurotransmitter release from motor nerve terminals. This interaction leads to clustering of channels, which in turn recruit other presynaptic components. Perturbation of this interaction in vivo results in disassembly of neurotransmitter release sites, resembling defects previously observed in an autoimmune neuromuscular disorder, Lambert–Eaton myasthenic syndrome. These results identify an extracellular ligand of the voltage-gated calcium channel as well as a new laminin receptor. They also suggest a model for the development of nerve terminals, and provide clues to the pathogenesis of a synaptic disease.

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Figure 1: Laminin 9 interacts with pore-forming (CaV) subunits of calcium channels.
Figure 2: A binding site for VGCC on laminin β2.
Figure 3: A binding site for laminin β2 on the VGCC.
Figure 4: Laminin β2 promotes presynaptic differentiation by clustering VGCCs.
Figure 5: A new light microscopic assay for active zones used to monitor defects in laminin β2 and CaV2.1 mutant NMJs.
Figure 6: Blocking laminin β2–VGCC interactions in vivo disassembles active zones.

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Acknowledgements

We thank H. Umemori for laminin–alkaline phosphatase fusion constructs; J. Haubenreich, M. Wagenbach, E. Ottersburg, S. Makishima and L. Schwarz for assistance; J. M. Cunningham for electron microscopy; C. Yokoyama for discussions; H. Shin and R. W. Tsien for CaV2.1 mutant mice; R. E. Westenbroek and P. Greengard for antibodies; W. A. Catterall, T. P. Snutch and K. G. Beam for VGCC plasmids; W. Yuan and J. Nerbonne for physiological recordings and the Mouse Genetics Core at Washington University for animal husbandry. This work was supported by grants from the National Institutes of Health to S.S.C. and J.R.S.

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Authors and Affiliations

  1. Department of Anatomy and Neurobiology, Washington University School of Medicine, St Louis, Missouri, 63110

    Hiroshi Nishimune & Joshua R. Sanes

  2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, 02138, USA

    Hiroshi Nishimune & Joshua R. Sanes

  3. Department of Physiology and Biophysics, University of Washington, Seattle, Washington, 98195, USA

    Steven S. Carlson

Authors
  1. Hiroshi Nishimune
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  2. Joshua R. Sanes
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Correspondence to Joshua R. Sanes.

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Supplementary information

Supplementary Figure 1

Characterization of purified Torpedo laminin-9 by SDS-PAGE electrophoresis and immunoblotting. (PDF 113 kb)

Supplementary Figure 2

Inhibition of laminin-VGCC interaction by soluble laminin fragments. (PDF 43 kb)

Supplementary Figure 3

Characterization of antibody to the 11th extracellular loop of VGCC. (PDF 232 kb)

Supplementary Figure 4

Direct interaction of laminin β2- and VGCC-derived peptides measured in solution or by bead-binding. (PDF 50 kb)

Supplementary Figure 5

Inhibition of synaptic vesicle clustering in cultured motoneurons by VGCC peptide. (PDF 32 kb)

Supplementary Figure 6

Histological analysis of Cav 2.1 mutant neuromuscular junction. (PDF 5273 kb)

Supplementary Figure 7

Vesicle number and polarity in NMJs from mice injected with Cav 2.1- or Cav 1.2-derived peptides. (PDF 31 kb)

Supplementary Figure Legends (RTF 14 kb)

Supplementary Methods

Additional methods used in the experiments. (RTF 17 kb)

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Nishimune, H., Sanes, J. & Carlson, S. A synaptic laminin–calcium channel interaction organizes active zones in motor nerve terminals. Nature 432, 580–587 (2004). https://doi.org/10.1038/nature03112

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