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A hierarchy of ankyrin-spectrin complexes clusters sodium channels at nodes of Ranvier

Nature Neuroscience volume 17, pages 1664–1672 (2014)Cite this article

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Abstract

The scaffolding protein ankyrin-G is required for Na+ channel clustering at axon initial segments. It is also considered essential for Na+ channel clustering at nodes of Ranvier to facilitate fast and efficient action potential propagation. However, notwithstanding these widely accepted roles, we show here that ankyrin-G is dispensable for nodal Na+ channel clustering in vivo. Unexpectedly, in the absence of ankyrin-G, erythrocyte ankyrin (ankyrin-R) and its binding partner βI spectrin substitute for and rescue nodal Na+ channel clustering. In addition, channel clustering is also rescued after loss of nodal βIV spectrin by βI spectrin and ankyrin-R. In mice lacking both ankyrin-G and ankyrin-R, Na+ channels fail to cluster at nodes. Thus, ankyrin R–βI spectrin protein complexes function as secondary reserve Na+ channel clustering machinery, and two independent ankyrin-spectrin protein complexes exist in myelinated axons to cluster Na+ channels at nodes of Ranvier.

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Figure 1: AnkG-deficient axons have Na+ channels clustered at nodes of Ranvier.
Figure 2: AnkR is found at nodes of AnkG-deficient axons and interacts with Na+ channels.
Figure 3: AnkR rescues Na+ channel clustering after loss of AnkG from adult optic nerve axons.
Figure 4: βI spectrin substitutes for βIV spectrin at AnkG-deficient nodes.
Figure 5: AnkR and βI spectrin are found at nodes of Ranvier in βIV spectrin–deficient quivering 3J mice.
Figure 6: AnkR and βI spectrin come from a pre-existing pool of proteins.
Figure 7: AnkR-deficient mice have normal PNS and CNS nodes of Ranvier.
Figure 8: Ankyrins are required for Na+ channel clustering at nodes of Ranvier.

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Acknowledgements

We thank K. Susuki for discussions. This research was supported by US National Institutes of Health grants NS044916 (MNR), NS069688 (MNR), NS49119 (ECC), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, and CURE (Citizens United for Research on Epilepsy). V.B. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, USA

    Tammy Szu-Yu Ho, Kae-Jiun Chang & Matthew N Rasband

  2. Department of Neuroscience, Baylor College of Medicine, Houston, Texas, USA

    Daniel R Zollinger, Edward C Cooper & Matthew N Rasband

  3. Department of Neurology, Baylor College of Medicine, Houston, Texas, USA

    Mingxuan Xu & Edward C Cooper

  4. Department of Pathology, Yale University, New Haven, Connecticut, USA

    Michael C Stankewich

  5. Department of Cell Biology, Duke University, Durham, North Carolina, USA

    Vann Bennett

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Contributions

M.N.R. and T.S.-Y.H. conceived the project, designed the experiments and wrote the manuscript. D.R.Z. performed the electrophysiology experiments and analyzed the data. M.N.R. performed intravitreal injections of AAV. T.S.-Y.H. performed all other experiments and analyzed the data. K.-J.C. supervised the RT-qPCR experiments. K.-J.C., M.X., E.C.C., M.C.S. and V.B. provided crucial reagents, mice and support.

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Correspondence to Matthew N Rasband.

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Integrated supplementary information

Supplementary Figure 1 Na+ channel clustering is delayed in AnkG-deficient axons.

(a) P0 Ank3F/F, Nestin-Cre; Ank3F/+, and Nestin-Cre; Ank3F/F mouse brain cortices stained with antibodies against AnkG (green) and Na+ channels (red). Scale bar, 10 µm. (b) Comparison between Ank3F/F (N=7) and Avil-Cre; Ank3F/F (N= 8) mice using the wire-hang test. All mice successfully stayed on the wire for the 60 second duration of the test. (c) Comparison between Ank3F/F (N=8) and Avil-Cre; Ank3F/F (N=8) mice using the hot-plate assay to test for thermal nociception (p=0.27, unpaired two-tailed t-test, error bars indicate +/– SEM). (d) Representative recordings of compound action potentials from P14 dorsal roots from Ank3F/F and Avil-Cre; Ank3F/F mice. (e) The ratio of the intensity for Na+ channel immunostaining to AnkB immunostaining in Ank3F/F (N=27 nodes) and Avil-Cre; Ank3F/F (N= 36 nodes) dorsal roots at P7. Error bars indicate +/– SEM. (f) Immunostaining of nodes of Ranvier from dorsal roots of P7 Ank3F/F and Avil-Cre; Ank3F/F mice using antibodies against Na+ channels (red) and AnkB (green). Scale bar, 10 µm.

Supplementary Figure 2 AnkG-deficient nodes of Ranvier remain intact in aged mice.

(a,b) Ank3F/F and Avil-Cre; Ank3F/F dorsal roots (a), and Ank3F/F and Six3-Cre; Ank3F/F optic nerve (b) immunostained using antibodies against Na+ channels (red), Caspr (blue), and AnkG (green). Nodes (arrows) of AnkG-deficient axons have Na+ channels. (c,d) Ank3F/F and Avil-Cre; Ank3F/F dorsal roots (c), and Ank3F/F and Six3-Cre; Ank3F/F optic nerve (d) immunostained using antibodies against Na+ channels (red), neurofascin (NF)/Caspr (blue), and AnkR (green). Nodes (arrows) of AnkG-deficient axons have high densities of AnkR colocalized with Na+ channels. Scale bars, 10 µm.

Supplementary Figure 3 AnkG-deficient nodes of Ranvier have Na+ channels that co-localize with AnkR and βI spectrin.

(a,b) Ank3F/F and Avil-Cre; Ank3F/F dorsal roots (a), and Ank3F/F and Six3-Cre; Ank3F/F optic nerve (b) immunostained using antibodies against Na+ channels (red), neurofascin (NF)/Caspr (blue), and AnkR (green). Nodes of AnkG-deficient axons have high densities of Na+ channels colocalized with AnkR. (c,d) Ank3F/F and Avil-Cre; Ank3F/F dorsal roots (c), and Ank3F/F and Six3-Cre; Ank3F/F optic nerve (d) immunostained using antibodies against Na+ channels (red), Caspr (blue), and βI spectrin (green). Some nodes (arrows) of AnkG-deficient axons have Na+ channels colocalized with βI spectrin. Scale bars, 10 µm.

Supplementary Figure 4 βIV spectrin is lost from many AnkG-deficient nodes.

(a,b) Ank3F/F (a) and Avil-Cre; Ank3F/F (b) dorsal roots immunostained using antibodies against AnkB (red), βIV spectrin (blue), and AnkR (green). Nodes (arrow) of AnkG-deficient axons have little or no βIV spectrin. Scale bar, 10 µm.

Supplementary Figure 5 AnkR is not detected at developing optic nerve nodes of Ranvier.

(a) Immunoblots of dorsal roots from postnatal day 14 and 4 months using antibodies against AnkR and neurofilament-m (NF-M) as a loading control. (b) Immunostaining of wild-type optic nerves using antibodies against Caspr (red), βIV spectrin (blue), and AnkR (green) at postnatal days 10, 21, and 30. Scale bar, 10 µm.

Supplementary Figure 6 Schematic representation of the mechanisms of sodium channel clustering at nodes of Ranvier.

(a) Cartoons illustrate the substitution of AnkR and βI spectrin results in the compensation for loss of AnkG and βIV spectrin in Avil-Cre; Ank3F/F, and the absence of Na+ channel clustering in AnkG/AnkR-deficient axons despite intact paranodal junctions. (b) Model illustrating the primary and secondary ankyrin/spectrin-dependent clustering mechanisms found at nodes of Ranvier. Higher affinities among nodal proteins are indicated by solid lines, while lower-affinity interactions are indicated by dashed lines.

Supplementary Figure 7 Na+ channels are clustered at nodes of Ranvier in exon 1b AnkG knockout (KO) mice.

(a, b) nodes of Ranvier labeled for Caspr (red), AnkG (blue), and Nav1.6 Na+ channels (green) in cerebellum of P90 WT (a) and exon 1b AnkG KO mice (b). Scale bar, 10 µm.

Supplementary Figure 8 Axon initial segments fail to form in AnkG-deficient retinal and dorsal root ganglion cells.

(a) Immunostaining of retinas from Ank3F/F and Six3-Cre;Ank3F/F mice using antibodies against AnkG (green), Na+ channels (red), and AnkR (blue). In the Ank3F/F retina the axon initial segment is indicated by the arrowheads (left panels). In the Six3-Cre;Ank3F/F retina the axon, with diffuse AnkR but no AnkG clustering, is indicated by arrowheads (right panels). The retinal ganglion cell is indicated by an asterisk. (b) Immunostaining of dorsal root ganglia from Ank3F/F and Avil-Cre;Ank3F/F mice using antibodies against AnkG (green), Na+ channels (red), and AnkR (blue). In the Ank3F/F dorsal root ganglion the axon initial segment is indicated by the arrowheads and a node is indicated by the arrow (left panels). The Avil-Cre;Ank3F/F dorsal root ganglia have no axon initial segments although nodes of Ranvier are intact and labeled with AnkR antibodies (arrows; right panels). Scale bars, 10 µm.

Supplementary Figure 9 Full-length pictures of the blots presented in the main figures

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Ho, TY., Zollinger, D., Chang, KJ. et al. A hierarchy of ankyrin-spectrin complexes clusters sodium channels at nodes of Ranvier. Nat Neurosci 17, 1664–1672 (2014). https://doi.org/10.1038/nn.3859

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