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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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Abrogation of E1111 of the Nav1.2 ankyrin-binding motif did not impair ion channel segregation at the AIS of hippocampal neurons. Cell surface distribution of Kv2.1 (A), Kv2.1-ΔCter (B), Kv2.1-Nav1.2 (C), and Kv2.1-Nav1.2 E1111A mutant (D–F) in cultured hippocampal neurons. The addition of the Nav1.2 ankyrin-binding motif to the C terminus of Kv2.1-ΔCter segregated Kv2.1 at the AIS (C, arrow). Kv2.1-Nav1.2 compartmentalization at the AIS was not impaired by the Nav1.2 E1111A mutation (D and F, arrow). The indicated constructs were immunodetected with an antibody to myc (green) before cell permeabilization; the somatodendritic domain was subsequently identified by MAP2 staining. Bars, 10 μm.
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fig1: Abrogation of E1111 of the Nav1.2 ankyrin-binding motif did not impair ion channel segregation at the AIS of hippocampal neurons. Cell surface distribution of Kv2.1 (A), Kv2.1-ΔCter (B), Kv2.1-Nav1.2 (C), and Kv2.1-Nav1.2 E1111A mutant (D–F) in cultured hippocampal neurons. The addition of the Nav1.2 ankyrin-binding motif to the C terminus of Kv2.1-ΔCter segregated Kv2.1 at the AIS (C, arrow). Kv2.1-Nav1.2 compartmentalization at the AIS was not impaired by the Nav1.2 E1111A mutation (D and F, arrow). The indicated constructs were immunodetected with an antibody to myc (green) before cell permeabilization; the somatodendritic domain was subsequently identified by MAP2 staining. Bars, 10 μm.

Mentions: When expressed by transfection in cultured hippocampal neurons, the potassium channel Kv2.1 was clustered at the cell surface on soma and on proximal dendrites identified by microtubule-associated protein 2 (MAP2) staining (Fig. 1 A), as described previously (Lim et al., 2000). The deletion of the C terminus of Kv2.1 (Kv2.1-ΔCter) induced a loss of compartmentalization (Fig. 1 B). The addition of a segment encompassing the AIS motif of sodium channel Nav1.2 (amino acids 1,080–1,203) to the C terminus of Kv2.1 (Kv2.1-Nav1.2) was sufficient to redirect Kv2.1 to the AIS (Fig. 1 C). This was observed at the cell surface of 70.9 ± 10.9% (n = 609) of cells expressing Kv2.1-Nav1.2, in line with our previous study (Garrido et al., 2003). To determine the critical residues involved in Kv2.1-Nav1.2 segregation, we first converted Nav1.2 E1111 into alanine, a point mutation that was sufficient to impair CD4-Nav1.2 II-III compartmentalization at the AIS (Fache et al., 2004). Because a mutation of the corresponding E residue (Nav1.5 E1053K) altered the trafficking of sodium channel Nav1.5 in cardiomyocytes (Mohler et al., 2004), we differentially stained the surface and total channel populations. Surface staining was observed in 58.3% (n = 230) of the cells expressing the Kv2.1-Nav1.2 E1111A mutant and in 90.4% (n = 240) of the cells expressing Kv2.1-Nav1.2. Strikingly, Kv2.1-Nav1.2 E1111A was correctly localized at the AIS in 52.7 ± 13.9% (n = 178) of the cells displaying surface staining (Fig. 1, D–F, and Fig. 2 E). After cell permeabilization, this mutant was visualized not only in the soma and throughout the dendrites but also in the ER identified by calreticulin costaining (unpublished data). In contrast, Kv2.1-Nav1.2 was only occasionally detected in the ER (unpublished data). These findings indicate that the abrogation of Nav1.2 E1111 altered Kv2.1-Nav1.2 trafficking, presumably by favoring retention in the ER, but did not impair its ability to be targeted to the AIS. In view of these observations, we surmised that, in addition to E1111, other residues of the ankyrin-binding motif contribute to Kv2.1-Nav1.2 segregation at the AIS.


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)

Abrogation of E1111 of the Nav1.2 ankyrin-binding motif did not impair ion channel segregation at the AIS of hippocampal neurons. Cell surface distribution of Kv2.1 (A), Kv2.1-ΔCter (B), Kv2.1-Nav1.2 (C), and Kv2.1-Nav1.2 E1111A mutant (D–F) in cultured hippocampal neurons. The addition of the Nav1.2 ankyrin-binding motif to the C terminus of Kv2.1-ΔCter segregated Kv2.1 at the AIS (C, arrow). Kv2.1-Nav1.2 compartmentalization at the AIS was not impaired by the Nav1.2 E1111A mutation (D and F, arrow). The indicated constructs were immunodetected with an antibody to myc (green) before cell permeabilization; the somatodendritic domain was subsequently identified by MAP2 staining. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2600743&req=5

fig1: Abrogation of E1111 of the Nav1.2 ankyrin-binding motif did not impair ion channel segregation at the AIS of hippocampal neurons. Cell surface distribution of Kv2.1 (A), Kv2.1-ΔCter (B), Kv2.1-Nav1.2 (C), and Kv2.1-Nav1.2 E1111A mutant (D–F) in cultured hippocampal neurons. The addition of the Nav1.2 ankyrin-binding motif to the C terminus of Kv2.1-ΔCter segregated Kv2.1 at the AIS (C, arrow). Kv2.1-Nav1.2 compartmentalization at the AIS was not impaired by the Nav1.2 E1111A mutation (D and F, arrow). The indicated constructs were immunodetected with an antibody to myc (green) before cell permeabilization; the somatodendritic domain was subsequently identified by MAP2 staining. Bars, 10 μm.
Mentions: When expressed by transfection in cultured hippocampal neurons, the potassium channel Kv2.1 was clustered at the cell surface on soma and on proximal dendrites identified by microtubule-associated protein 2 (MAP2) staining (Fig. 1 A), as described previously (Lim et al., 2000). The deletion of the C terminus of Kv2.1 (Kv2.1-ΔCter) induced a loss of compartmentalization (Fig. 1 B). The addition of a segment encompassing the AIS motif of sodium channel Nav1.2 (amino acids 1,080–1,203) to the C terminus of Kv2.1 (Kv2.1-Nav1.2) was sufficient to redirect Kv2.1 to the AIS (Fig. 1 C). This was observed at the cell surface of 70.9 ± 10.9% (n = 609) of cells expressing Kv2.1-Nav1.2, in line with our previous study (Garrido et al., 2003). To determine the critical residues involved in Kv2.1-Nav1.2 segregation, we first converted Nav1.2 E1111 into alanine, a point mutation that was sufficient to impair CD4-Nav1.2 II-III compartmentalization at the AIS (Fache et al., 2004). Because a mutation of the corresponding E residue (Nav1.5 E1053K) altered the trafficking of sodium channel Nav1.5 in cardiomyocytes (Mohler et al., 2004), we differentially stained the surface and total channel populations. Surface staining was observed in 58.3% (n = 230) of the cells expressing the Kv2.1-Nav1.2 E1111A mutant and in 90.4% (n = 240) of the cells expressing Kv2.1-Nav1.2. Strikingly, Kv2.1-Nav1.2 E1111A was correctly localized at the AIS in 52.7 ± 13.9% (n = 178) of the cells displaying surface staining (Fig. 1, D–F, and Fig. 2 E). After cell permeabilization, this mutant was visualized not only in the soma and throughout the dendrites but also in the ER identified by calreticulin costaining (unpublished data). In contrast, Kv2.1-Nav1.2 was only occasionally detected in the ER (unpublished data). These findings indicate that the abrogation of Nav1.2 E1111 altered Kv2.1-Nav1.2 trafficking, presumably by favoring retention in the ER, but did not impair its ability to be targeted to the AIS. In view of these observations, we surmised that, in addition to E1111, other residues of the ankyrin-binding motif contribute to Kv2.1-Nav1.2 segregation at the AIS.

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