Figure 2. Requirement for glutamate 1111 and serine residues for Kv2.1-Nav1.2 compartmentalization at the AIS of hippocampal neurons. (A) Schematic representation of the ankyrin-binding motif of sodium channels predominantly expressed in the CNS. Note the presence of four conserved serines (black boxes) and three potential serine phosphorylation sites for CK2 (Nav1.2 S1112, S1124, and S1126). Numbers refer to the position of the amino acid residues in the corresponding Nav1 types. (B–E) Cell surface distribution of Kv2.1-Nav1.2 mutants. Cultured hippocampal neurons were transfected with the indicated constructs. 1 d after transfection, Kv2.1-Nav1.2 was detected with an antibody to myc (green) before permeabilization, and the somatodendritic domain was subsequently identified by MAP2 staining (red). Mutations of different serine residues in the ankyrin-binding motif did not perturb Kv2.1-Nav1.2 segregation at the AIS (B and C). In contrast, co-mutation of E1111 with serine residues induced a loss of Kv2.1-Nav1.2 compartmentalization (D and E). (C and E) Histograms of the cell surface distribution of Kv2.1-Nav1.2 mutants. The percentage of myc-positive neurons were classified into four categories: myc staining segregated at the AIS ([AIS]); distributed at the cell surface of the soma and proximal dendrites with an enrichment at the AIS (SD-[AIS]); enriched at the AIS with a distribution in soma, proximal dendrites, and axons (SD-[AIS]-PA); and uniformly distributed at the cell surface (non polarized; SD-A). 100% represents the total population of transfected neurons. Data are means ± SD from two to three different experiments; n denotes the total number of cells analyzed for each construct. Bars, 10 μm.
Figure 2.

Requirement for glutamate 1111 and serine residues for Kv2.1-Nav1.2 compartmentalization at the AIS of hippocampal neurons. (A) Schematic representation of the ankyrin-binding motif of sodium channels predominantly expressed in the CNS. Note the presence of four conserved serines (black boxes) and three potential serine phosphorylation sites for CK2 (Nav1.2 S1112, S1124, and S1126). Numbers refer to the position of the amino acid residues in the corresponding Nav1 types. (B–E) Cell surface distribution of Kv2.1-Nav1.2 mutants. Cultured hippocampal neurons were transfected with the indicated constructs. 1 d after transfection, Kv2.1-Nav1.2 was detected with an antibody to myc (green) before permeabilization, and the somatodendritic domain was subsequently identified by MAP2 staining (red). Mutations of different serine residues in the ankyrin-binding motif did not perturb Kv2.1-Nav1.2 segregation at the AIS (B and C). In contrast, co-mutation of E1111 with serine residues induced a loss of Kv2.1-Nav1.2 compartmentalization (D and E). (C and E) Histograms of the cell surface distribution of Kv2.1-Nav1.2 mutants. The percentage of myc-positive neurons were classified into four categories: myc staining segregated at the AIS ([AIS]); distributed at the cell surface of the soma and proximal dendrites with an enrichment at the AIS (SD-[AIS]); enriched at the AIS with a distribution in soma, proximal dendrites, and axons (SD-[AIS]-PA); and uniformly distributed at the cell surface (non polarized; SD-A). 100% represents the total population of transfected neurons. Data are means ± SD from two to three different experiments; n denotes the total number of cells analyzed for each construct. Bars, 10 μm.

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