Interaction with ankG restricts Kv-Nav diffusion at the AIS in young neurons. (A) Accumulation of endogenous AIS components. DIV 4 hippocampal neurons were stained for ankG, Nav, and NF-186 (black). Bar, 10 µm. (B and C) Mutation of the ankyrin-binding motif perturbs polarized expression of surface Kv-Nav. Cell surface distribution of Kv-Nav (B) and Kv-Nav EA4SA (E4SA; C). DIV 4 cultured hippocampal neurons were transfected with the different constructs. Kv-Nav constructs were detected with an antibody against myc (green). The somatodendritic domain and the AIS were identified by MAP2 (blue) and ankG staining (red), respectively. Bars, 10 µm. (right) Histograms of the cell surface distribution for Kv-Nav (B) and EA4SA (C). The expression profiles of transfected (myc positive) neurons were classified into three categories: myc staining segregated at the AIS (segregated, S); distributed at the cell surface of the soma and proximal dendrites with a concentration at the AIS (concentrated, C); and uniformly distributed at the cell surface (nonpolarized, NP). 100% represents the total population of transfected neurons. Data are means ± SEM with n = 165 and 52 from four and two independent experiments for Kv-Nav and EA4SA, respectively. (D and E) Distribution (D) and cumulative frequency (E) of the instantaneous diffusion coefficients of Kv-Nav and EA4SA (n = 53 and 32 trajectories from four independent experiments for Kv-Nav and EA4SA, respectively). KS: ***, P < 0.001. (F) Histogram of the mean values ± SEM for the immobile population of Kv-Nav and EA4SA. MW: *, P < 0.05. (G) MDC (25–75% IQR) of the mobile population of Kv-Nav and EA4SA. MW: **, P < 0.01.