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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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The effect of a CK2 inhibitor on the accumulation of sodium channels at the AIS of cultured hippocampal neurons. Cultured hippocampal neurons were treated with either DMAT (50 μM) or with DMSO (control cells) either from 2 to 4 DIV (A and B) or from 9 to 10 DIV (C and D). They were subsequently immunostained for Nav1 channels (green), ankyrin G (red), and MAP2 (blue). (B and D) Quantification of Nav1 and ankyrin G staining intensity. Fluorescence intensity was normalized by taking as 100% the staining intensity measured in control cells. The results are from three independent experiments, the number of quantified AIS ranged from 70 to 230. Error bars indicate mean ± SEM. t test: ***, P < 0.0001. Bars, 10 μm.
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fig7: The effect of a CK2 inhibitor on the accumulation of sodium channels at the AIS of cultured hippocampal neurons. Cultured hippocampal neurons were treated with either DMAT (50 μM) or with DMSO (control cells) either from 2 to 4 DIV (A and B) or from 9 to 10 DIV (C and D). They were subsequently immunostained for Nav1 channels (green), ankyrin G (red), and MAP2 (blue). (B and D) Quantification of Nav1 and ankyrin G staining intensity. Fluorescence intensity was normalized by taking as 100% the staining intensity measured in control cells. The results are from three independent experiments, the number of quantified AIS ranged from 70 to 230. Error bars indicate mean ± SEM. t test: ***, P < 0.0001. Bars, 10 μm.

Mentions: To further demonstrate that CK2 is involved in the accumulation of sodium channels at the AIS, we evaluated the effect of 2-dimethylamino-4,5,6,7-tetrabromo1H-benzimidazole (DMAT), a CK2 inhibitor (Pagano et al., 2004), in cultured hippocampal neurons (Fig. 7). The immunostaining of ankyrin G and Nav.1 channels was quantified in neurons treated with either DMAT or DMSO (control cells) at two stages of maturation. It has been shown previously that the percentage of cells with an AIS increases between 2 and 4 d in vitro (DIV; Yang et al., 2007; Martin et al., 2008). The inhibition of CK2 activity in immature cells (from 2 to 4 DIV) induced an increase in the number of cells negative for ankyrin G staining as compared with what was observed in control cells. In the other cell population (ankyrin G–positive), we observed a decrease in the staining intensity of ankyrin G and Nav.1 channels (Fig. 7, A and B). At 9 DIV, all the cells present an AIS (Yang et al., 2007; Martin et al., 2008). The addition of DMAT in the culture medium of neurons at 9 DIV during 30 h induced a decrease in ankyrin G and Nav.1 channels staining (Fig. 7, C and D). These results showed that inhibition of CK2 activity perturbs the accumulation of sodium channels and ankyrin G at two different stages of AIS organization.


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)

The effect of a CK2 inhibitor on the accumulation of sodium channels at the AIS of cultured hippocampal neurons. Cultured hippocampal neurons were treated with either DMAT (50 μM) or with DMSO (control cells) either from 2 to 4 DIV (A and B) or from 9 to 10 DIV (C and D). They were subsequently immunostained for Nav1 channels (green), ankyrin G (red), and MAP2 (blue). (B and D) Quantification of Nav1 and ankyrin G staining intensity. Fluorescence intensity was normalized by taking as 100% the staining intensity measured in control cells. The results are from three independent experiments, the number of quantified AIS ranged from 70 to 230. Error bars indicate mean ± SEM. t test: ***, P < 0.0001. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2600743&req=5

fig7: The effect of a CK2 inhibitor on the accumulation of sodium channels at the AIS of cultured hippocampal neurons. Cultured hippocampal neurons were treated with either DMAT (50 μM) or with DMSO (control cells) either from 2 to 4 DIV (A and B) or from 9 to 10 DIV (C and D). They were subsequently immunostained for Nav1 channels (green), ankyrin G (red), and MAP2 (blue). (B and D) Quantification of Nav1 and ankyrin G staining intensity. Fluorescence intensity was normalized by taking as 100% the staining intensity measured in control cells. The results are from three independent experiments, the number of quantified AIS ranged from 70 to 230. Error bars indicate mean ± SEM. t test: ***, P < 0.0001. Bars, 10 μm.
Mentions: To further demonstrate that CK2 is involved in the accumulation of sodium channels at the AIS, we evaluated the effect of 2-dimethylamino-4,5,6,7-tetrabromo1H-benzimidazole (DMAT), a CK2 inhibitor (Pagano et al., 2004), in cultured hippocampal neurons (Fig. 7). The immunostaining of ankyrin G and Nav.1 channels was quantified in neurons treated with either DMAT or DMSO (control cells) at two stages of maturation. It has been shown previously that the percentage of cells with an AIS increases between 2 and 4 d in vitro (DIV; Yang et al., 2007; Martin et al., 2008). The inhibition of CK2 activity in immature cells (from 2 to 4 DIV) induced an increase in the number of cells negative for ankyrin G staining as compared with what was observed in control cells. In the other cell population (ankyrin G–positive), we observed a decrease in the staining intensity of ankyrin G and Nav.1 channels (Fig. 7, A and B). At 9 DIV, all the cells present an AIS (Yang et al., 2007; Martin et al., 2008). The addition of DMAT in the culture medium of neurons at 9 DIV during 30 h induced a decrease in ankyrin G and Nav.1 channels staining (Fig. 7, C and D). These results showed that inhibition of CK2 activity perturbs the accumulation of sodium channels and ankyrin G at two different stages of AIS organization.

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