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Protein kinase CK2 contributes to the organization of sodium channels in axonal membranes by regulating their interactions with ankyrin G.

Bréchet A, Fache MP, Brachet A, Ferracci G, Baude A, Irondelle M, Pereira S, Leterrier C, Dargent B - J. Cell Biol. (2008)

Bottom Line: We found that the ankyrin-binding motif of Na(v)1.2 that determines channel concentration at the AIS depends on a glutamate residue (E1111), but also on several serine residues (S1112, S1124, and S1126).Finally, inhibition of CK2 activity reduced sodium channel accumulation at the AIS of neurons.In conclusion, CK2 contributes to sodium channel organization by regulating their interaction with ankyrin G.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 641, Marseille F-13916, France.

ABSTRACT
In neurons, generation and propagation of action potentials requires the precise accumulation of sodium channels at the axonal initial segment (AIS) and in the nodes of Ranvier through ankyrin G scaffolding. We found that the ankyrin-binding motif of Na(v)1.2 that determines channel concentration at the AIS depends on a glutamate residue (E1111), but also on several serine residues (S1112, S1124, and S1126). We showed that phosphorylation of these residues by protein kinase CK2 (CK2) regulates Na(v) channel interaction with ankyrins. Furthermore, we observed that CK2 is highly enriched at the AIS and the nodes of Ranvier in vivo. An ion channel chimera containing the Na(v)1.2 ankyrin-binding motif perturbed endogenous sodium channel accumulation at the AIS, whereas phosphorylation-deficient chimeras did not. Finally, inhibition of CK2 activity reduced sodium channel accumulation at the AIS of neurons. In conclusion, CK2 contributes to sodium channel organization by regulating their interaction with ankyrin G.

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CK2α is concentrated in CNS and PNS nodes of Ranvier. (A and B) CK2α immunoreactivity is visible as numerous punctiform structures (B1 and B3, arrows) colabeled for ankyrin G (B2 and B3, arrows) within the alveus and cortical white matter that probably correspond to nodes. B1–B3 are enlarged views of the boxed areas in A1–A3. (C and D) Ranvier nodes are labeled for CK2α and ankyrin G (C1–C3, arrowheads) in teased fibers of the sciatic nerve. CK2α labeling is concentrated at the membrane of the nodal region (D1, arrows). CK2α immunoreactivity is also visible in the nucleus of a Schwann cell (e.g., C1 and C3, arrows). al, alveus; wm, white matter. Bars: (A) 26.8 μm; (B) 6. 71 μm; (C) 23.2 μm; (D) 3 μm.
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fig5: CK2α is concentrated in CNS and PNS nodes of Ranvier. (A and B) CK2α immunoreactivity is visible as numerous punctiform structures (B1 and B3, arrows) colabeled for ankyrin G (B2 and B3, arrows) within the alveus and cortical white matter that probably correspond to nodes. B1–B3 are enlarged views of the boxed areas in A1–A3. (C and D) Ranvier nodes are labeled for CK2α and ankyrin G (C1–C3, arrowheads) in teased fibers of the sciatic nerve. CK2α labeling is concentrated at the membrane of the nodal region (D1, arrows). CK2α immunoreactivity is also visible in the nucleus of a Schwann cell (e.g., C1 and C3, arrows). al, alveus; wm, white matter. Bars: (A) 26.8 μm; (B) 6. 71 μm; (C) 23.2 μm; (D) 3 μm.

Mentions: If CK2 has a role in ankyrin–Nav interaction, one would expect that it is present at the AIS in vivo. To test this possibility, we looked at the subcellular localization of CK2 in neurons using a polyclonal antibody directed against the catalytic α subunit. In cultured hippocampal neurons, CK2 immunostaining was concentrated in the AIS but not in the distal part of the axon (Fig. 4, A1–A3). CK2 was also visualized in nuclei (Filhol et al., 2003; Theis-Febvre et al., 2005), in soma, and in proximal dendrites. When hippocampal neurons were subjected to detergent extraction before cell fixation, CK2 immunostaining at the AIS was still observed (unpublished data), which suggests tethering to the cytoskeleton (Garrido et al., 2003; Fache et al., 2004). In vivo immunoreactivity for CK2 was further examined in the hippocampus (Fig. 4, B and C), cerebellum (Fig. 4 D), and cortex (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200805169/DC1). Initial segments were intensely immunoreactive for CK2 in all observed structures, as revealed by colabeling with ankyrin G (Figs. 4 and S1) and sodium channels (not depicted). Initial segments emanating from cell bodies of principal cells were observed in CA1 (Fig. 4, B and C), CA3, and dentate gyrus (Fig. S1, A and B). Initial segments positive for CK2 were also found in strati radiatum and oriens of CA1 and CA3, the molecular layer in the dentate gyrus, and in the hilus. Intense CK2 labeling appeared to underline the internal face of the plasma membrane of the initial segment, whereas the axoplasm displayed weak immunoreactivity, similar to ankyrin G localization (Fig. 4 B). In the hippocampus, 80% of ankyrin G–positive structures displayed a positive labeling for CK2 in the white matter (n = 637) and 88% in the gray matter (n = 784). CK2 was also detected in nuclei of neurons and putative glial cells (Fig. 4). Punctate labeling for CK2, colocalizing with ankyrin G, was observed in the alveus and white matter of forebrain and cerebellum (Fig. 5, A and B). In the cerebellum, 77% of ankyrin G–positive structures displayed a costaining with CK2 in the white matter (n = 64) and 88% in the gray matter (n = 92). Finally, CK2 immunoreactivity was present in the nodes of Ranvier of sciatic nerves (Fig. 5, C and D): 100% of the nodes of Ranvier were costained for ankyrin G and CK2 (n = 40). Collectively, these observations demonstrated that CK2 is a novel component of the AIS and of the nodes of Ranvier.


Protein kinase CK2 contributes to the organization of sodium channels in axonal membranes by regulating their interactions with ankyrin G.

Bréchet A, Fache MP, Brachet A, Ferracci G, Baude A, Irondelle M, Pereira S, Leterrier C, Dargent B - J. Cell Biol. (2008)

CK2α is concentrated in CNS and PNS nodes of Ranvier. (A and B) CK2α immunoreactivity is visible as numerous punctiform structures (B1 and B3, arrows) colabeled for ankyrin G (B2 and B3, arrows) within the alveus and cortical white matter that probably correspond to nodes. B1–B3 are enlarged views of the boxed areas in A1–A3. (C and D) Ranvier nodes are labeled for CK2α and ankyrin G (C1–C3, arrowheads) in teased fibers of the sciatic nerve. CK2α labeling is concentrated at the membrane of the nodal region (D1, arrows). CK2α immunoreactivity is also visible in the nucleus of a Schwann cell (e.g., C1 and C3, arrows). al, alveus; wm, white matter. Bars: (A) 26.8 μm; (B) 6. 71 μm; (C) 23.2 μm; (D) 3 μm.
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Related In: Results  -  Collection

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fig5: CK2α is concentrated in CNS and PNS nodes of Ranvier. (A and B) CK2α immunoreactivity is visible as numerous punctiform structures (B1 and B3, arrows) colabeled for ankyrin G (B2 and B3, arrows) within the alveus and cortical white matter that probably correspond to nodes. B1–B3 are enlarged views of the boxed areas in A1–A3. (C and D) Ranvier nodes are labeled for CK2α and ankyrin G (C1–C3, arrowheads) in teased fibers of the sciatic nerve. CK2α labeling is concentrated at the membrane of the nodal region (D1, arrows). CK2α immunoreactivity is also visible in the nucleus of a Schwann cell (e.g., C1 and C3, arrows). al, alveus; wm, white matter. Bars: (A) 26.8 μm; (B) 6. 71 μm; (C) 23.2 μm; (D) 3 μm.
Mentions: If CK2 has a role in ankyrin–Nav interaction, one would expect that it is present at the AIS in vivo. To test this possibility, we looked at the subcellular localization of CK2 in neurons using a polyclonal antibody directed against the catalytic α subunit. In cultured hippocampal neurons, CK2 immunostaining was concentrated in the AIS but not in the distal part of the axon (Fig. 4, A1–A3). CK2 was also visualized in nuclei (Filhol et al., 2003; Theis-Febvre et al., 2005), in soma, and in proximal dendrites. When hippocampal neurons were subjected to detergent extraction before cell fixation, CK2 immunostaining at the AIS was still observed (unpublished data), which suggests tethering to the cytoskeleton (Garrido et al., 2003; Fache et al., 2004). In vivo immunoreactivity for CK2 was further examined in the hippocampus (Fig. 4, B and C), cerebellum (Fig. 4 D), and cortex (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200805169/DC1). Initial segments were intensely immunoreactive for CK2 in all observed structures, as revealed by colabeling with ankyrin G (Figs. 4 and S1) and sodium channels (not depicted). Initial segments emanating from cell bodies of principal cells were observed in CA1 (Fig. 4, B and C), CA3, and dentate gyrus (Fig. S1, A and B). Initial segments positive for CK2 were also found in strati radiatum and oriens of CA1 and CA3, the molecular layer in the dentate gyrus, and in the hilus. Intense CK2 labeling appeared to underline the internal face of the plasma membrane of the initial segment, whereas the axoplasm displayed weak immunoreactivity, similar to ankyrin G localization (Fig. 4 B). In the hippocampus, 80% of ankyrin G–positive structures displayed a positive labeling for CK2 in the white matter (n = 637) and 88% in the gray matter (n = 784). CK2 was also detected in nuclei of neurons and putative glial cells (Fig. 4). Punctate labeling for CK2, colocalizing with ankyrin G, was observed in the alveus and white matter of forebrain and cerebellum (Fig. 5, A and B). In the cerebellum, 77% of ankyrin G–positive structures displayed a costaining with CK2 in the white matter (n = 64) and 88% in the gray matter (n = 92). Finally, CK2 immunoreactivity was present in the nodes of Ranvier of sciatic nerves (Fig. 5, C and D): 100% of the nodes of Ranvier were costained for ankyrin G and CK2 (n = 40). Collectively, these observations demonstrated that CK2 is a novel component of the AIS and of the nodes of Ranvier.

Bottom Line: We found that the ankyrin-binding motif of Na(v)1.2 that determines channel concentration at the AIS depends on a glutamate residue (E1111), but also on several serine residues (S1112, S1124, and S1126).Finally, inhibition of CK2 activity reduced sodium channel accumulation at the AIS of neurons.In conclusion, CK2 contributes to sodium channel organization by regulating their interaction with ankyrin G.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 641, Marseille F-13916, France.

ABSTRACT
In neurons, generation and propagation of action potentials requires the precise accumulation of sodium channels at the axonal initial segment (AIS) and in the nodes of Ranvier through ankyrin G scaffolding. We found that the ankyrin-binding motif of Na(v)1.2 that determines channel concentration at the AIS depends on a glutamate residue (E1111), but also on several serine residues (S1112, S1124, and S1126). We showed that phosphorylation of these residues by protein kinase CK2 (CK2) regulates Na(v) channel interaction with ankyrins. Furthermore, we observed that CK2 is highly enriched at the AIS and the nodes of Ranvier in vivo. An ion channel chimera containing the Na(v)1.2 ankyrin-binding motif perturbed endogenous sodium channel accumulation at the AIS, whereas phosphorylation-deficient chimeras did not. Finally, inhibition of CK2 activity reduced sodium channel accumulation at the AIS of neurons. In conclusion, CK2 contributes to sodium channel organization by regulating their interaction with ankyrin G.

Show MeSH
Related in: MedlinePlus