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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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Impact of Kv2.1-Nav1.2 and of phosphorylation-deficient Kv2.1-Nav1.2 constructs on the accumulation of sodium channels (Nav1) and KCNQ2/KCNQ3 potassium channels at the AIS of cultured hippocampal neurons. (A, top and middle) Kv2.1-Nav1.2 expression perturbed Nav1 accumulation at the AIS, unlike the phosphorylation-deficient Kv2.1-Nav1.2 4SA mutant. Hippocampal neurons were transfected with either Kv2.1-Nav1.2 or phosphorylation-deficient Kv2.1-Nav1.2 mutants. Then cells were stained for myc (gray), ankyrin G (red), and sodium channels (green). (B) Quantification of Nav1 and ankyrin G staining intensity in untransfected cells (A, open arrowheads) and in transfected cells (closed arrowheads). (A, bottom) Kv2.1-Nav1.2 expression did not perturb KCNQ3 accumulation at the AIS. Cells transfected with Kv2.1-Nav1.2 were subsequently stained for myc (gray), ankyrin G (red), and KCNQ3 potassium channels (green). (C) Quantification of KCNQ3 and ankyrin G staining intensity. Fluorescence intensity measured in transfected cells, identified by myc staining, was normalized by taking as 100% the staining intensity measured in nontransfected cells (arrowheads). Numbers at the base of the bars denote the number of quantified cells. Error bars indicate mean ± SEM. Mann-Whitney test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. WT, wild type. Bars, 10 μm.
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fig6: Impact of Kv2.1-Nav1.2 and of phosphorylation-deficient Kv2.1-Nav1.2 constructs on the accumulation of sodium channels (Nav1) and KCNQ2/KCNQ3 potassium channels at the AIS of cultured hippocampal neurons. (A, top and middle) Kv2.1-Nav1.2 expression perturbed Nav1 accumulation at the AIS, unlike the phosphorylation-deficient Kv2.1-Nav1.2 4SA mutant. Hippocampal neurons were transfected with either Kv2.1-Nav1.2 or phosphorylation-deficient Kv2.1-Nav1.2 mutants. Then cells were stained for myc (gray), ankyrin G (red), and sodium channels (green). (B) Quantification of Nav1 and ankyrin G staining intensity in untransfected cells (A, open arrowheads) and in transfected cells (closed arrowheads). (A, bottom) Kv2.1-Nav1.2 expression did not perturb KCNQ3 accumulation at the AIS. Cells transfected with Kv2.1-Nav1.2 were subsequently stained for myc (gray), ankyrin G (red), and KCNQ3 potassium channels (green). (C) Quantification of KCNQ3 and ankyrin G staining intensity. Fluorescence intensity measured in transfected cells, identified by myc staining, was normalized by taking as 100% the staining intensity measured in nontransfected cells (arrowheads). Numbers at the base of the bars denote the number of quantified cells. Error bars indicate mean ± SEM. Mann-Whitney test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. WT, wild type. Bars, 10 μm.

Mentions: In addition to sodium channels, KCNQ2/KCNQ3 potassium channels are highly concentrated at the AIS both in cultured hippocampal neurons and in vivo, via an interaction with the MBD of ankyrin G (Devaux et al., 2004; Chung et al., 2006; Pan et al., 2006). We next addressed the question as to whether Kv2.1-Nav1.2 acts as a dominant negative of sodium channel and KCNQ2/KCNQ3 potassium channel accumulation at the AIS. We therefore quantified immunostaining of endogenous sodium channels, KCNQ2/KCNQ3 channels, and ankyrin G in cells expressing Kv2.1-Nav1.2 (transfected cells) and in nontransfected cells. The intensity of sodium channel staining was robustly reduced in Kv2.1-Nav1.2–positive cells as compared with that observed in nontransfected cells, whereas ankyrin G staining was unmodified (Fig. 6, A and B). In contrast, KCNQ2/KCNQ3 staining intensity detected with an antibody directed against KCNQ3 was equivalent in cells expressing Kv2.1-Nav1.2 and in nontransfected cells (Fig. 6, A and C). This strongly suggested that the presence of Kv2.1-Nav1.2 specifically disturbed the accumulation of sodium channels but not KCNQ2/KCNQ3.


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)

Impact of Kv2.1-Nav1.2 and of phosphorylation-deficient Kv2.1-Nav1.2 constructs on the accumulation of sodium channels (Nav1) and KCNQ2/KCNQ3 potassium channels at the AIS of cultured hippocampal neurons. (A, top and middle) Kv2.1-Nav1.2 expression perturbed Nav1 accumulation at the AIS, unlike the phosphorylation-deficient Kv2.1-Nav1.2 4SA mutant. Hippocampal neurons were transfected with either Kv2.1-Nav1.2 or phosphorylation-deficient Kv2.1-Nav1.2 mutants. Then cells were stained for myc (gray), ankyrin G (red), and sodium channels (green). (B) Quantification of Nav1 and ankyrin G staining intensity in untransfected cells (A, open arrowheads) and in transfected cells (closed arrowheads). (A, bottom) Kv2.1-Nav1.2 expression did not perturb KCNQ3 accumulation at the AIS. Cells transfected with Kv2.1-Nav1.2 were subsequently stained for myc (gray), ankyrin G (red), and KCNQ3 potassium channels (green). (C) Quantification of KCNQ3 and ankyrin G staining intensity. Fluorescence intensity measured in transfected cells, identified by myc staining, was normalized by taking as 100% the staining intensity measured in nontransfected cells (arrowheads). Numbers at the base of the bars denote the number of quantified cells. Error bars indicate mean ± SEM. Mann-Whitney test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. WT, wild type. Bars, 10 μm.
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fig6: Impact of Kv2.1-Nav1.2 and of phosphorylation-deficient Kv2.1-Nav1.2 constructs on the accumulation of sodium channels (Nav1) and KCNQ2/KCNQ3 potassium channels at the AIS of cultured hippocampal neurons. (A, top and middle) Kv2.1-Nav1.2 expression perturbed Nav1 accumulation at the AIS, unlike the phosphorylation-deficient Kv2.1-Nav1.2 4SA mutant. Hippocampal neurons were transfected with either Kv2.1-Nav1.2 or phosphorylation-deficient Kv2.1-Nav1.2 mutants. Then cells were stained for myc (gray), ankyrin G (red), and sodium channels (green). (B) Quantification of Nav1 and ankyrin G staining intensity in untransfected cells (A, open arrowheads) and in transfected cells (closed arrowheads). (A, bottom) Kv2.1-Nav1.2 expression did not perturb KCNQ3 accumulation at the AIS. Cells transfected with Kv2.1-Nav1.2 were subsequently stained for myc (gray), ankyrin G (red), and KCNQ3 potassium channels (green). (C) Quantification of KCNQ3 and ankyrin G staining intensity. Fluorescence intensity measured in transfected cells, identified by myc staining, was normalized by taking as 100% the staining intensity measured in nontransfected cells (arrowheads). Numbers at the base of the bars denote the number of quantified cells. Error bars indicate mean ± SEM. Mann-Whitney test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. WT, wild type. Bars, 10 μm.
Mentions: In addition to sodium channels, KCNQ2/KCNQ3 potassium channels are highly concentrated at the AIS both in cultured hippocampal neurons and in vivo, via an interaction with the MBD of ankyrin G (Devaux et al., 2004; Chung et al., 2006; Pan et al., 2006). We next addressed the question as to whether Kv2.1-Nav1.2 acts as a dominant negative of sodium channel and KCNQ2/KCNQ3 potassium channel accumulation at the AIS. We therefore quantified immunostaining of endogenous sodium channels, KCNQ2/KCNQ3 channels, and ankyrin G in cells expressing Kv2.1-Nav1.2 (transfected cells) and in nontransfected cells. The intensity of sodium channel staining was robustly reduced in Kv2.1-Nav1.2–positive cells as compared with that observed in nontransfected cells, whereas ankyrin G staining was unmodified (Fig. 6, A and B). In contrast, KCNQ2/KCNQ3 staining intensity detected with an antibody directed against KCNQ3 was equivalent in cells expressing Kv2.1-Nav1.2 and in nontransfected cells (Fig. 6, A and C). This strongly suggested that the presence of Kv2.1-Nav1.2 specifically disturbed the accumulation of sodium channels but not KCNQ2/KCNQ3.

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