Interaction with ankG restricts Kv-Nav diffusion at the AIS of mature neurons. (A) Surface distribution of Kv-Nav, Kv-Nav 4SA (4SA), and Kv-Nav EA4SA (EA4SA) in DIV 10 hippocampal neurons. Cultured hippocampal neurons were transfected with the indicated constructs. Kv-Nav and mutants were detected, and neurons were stained as explained in Fig. 3. Bars, 10 µm. (B–E) SPT of QD-labeled Kv-Nav and mutants in DIV 10 neurons. (B) Cumulative frequencies of the instantaneous diffusion coefficients of Kv-Nav, 4SA, and EA4SA. Trajectories for Kv-Nav (n = 97), 4SA (n = 75), and EA4SA (n = 36) were analyzed from six, five, and five independent experiments, respectively. KS: *, P < 0.05 and ***, P < 0.001. (C) Histogram of the mean values ± SEM for the immobile population percentage of Kv-Nav, 4SA, and EA4SA. MW: *, P < 0.05. (D) MDC (25–75% IQR) of the mobile population of Kv-Nav, 4SA, and EA4SA. (E) SPT on Kv-Nav at the AIS of DIV 10 neurons acutely treated with 50 µM DMAT or vehicle. MDC (25–75% IQR) before and after drug addition. Analysis was performed as explained in Fig. 5. For DMAT condition, n = 11 trajectories from four independent experiments. For vehicle condition, n = 10 trajectories from three independent experiments. WSR: *, P < 0.05. (F–I) Analysis of the diffusion properties of GFP–Kv-Nav and mutants in DIV 10 neurons by FRAP. n = 22, 27, and 17 bleached regions that were analyzed from three independent experiments for Kv-Nav, EA4SA, and 4SA, respectively. (F) Representative examples of GFP–Kv-Nav (left) and GFP-EA4S4A (right) fluorescence at the AIS before photobleaching, immediately after photobleaching, and 65 s later. Bar, 1 µm. (G) Plot of the normalized mean fluorescence intensity ± SEM for GFP–Kv-Nav and mutants versus time before and after photobleaching. (H) Histogram of the mean values ± SEM for the immobile population percentage of indicated constructs. MW: ***, P < 0.001. (I) Histogram of the median values ± IQR (25–75%) for the half-recovery time constant of indicated constructs.