Relationship between the binding capacities of site-directed Kv-Nav mutants with their restriction of diffusion in N2A cells. (A) Sequence alignment of the ankyrin-binding domain for Kv-Nav, Kv-Nav 4SA (4SA), and Kv-Nav E4SA (E4SA) constructs. Critical residues for the interaction with ankG are represented in bold, and the mutated residues are highlighted in pink. (B–F) SPT of QD-labeled Kv-Nav with various ankyrin-binding affinities. (B and C) DIDC for EA4SA (red, B) and 4SA (gray, C). DIDC for Kv-Nav is represented in black for comparison. Trajectories for E4SA (n = 623) and 4SA (n = 419) conditions were analyzed from six and four independent experiments, respectively. (D and E) Cumulative frequencies of the instantaneous diffusion coefficients for Kv-Nav and 4SA (D) and Kv-Nav and E4SA (E). KS: ***, P < 0.001. (F) Histogram of the mean values ± SEM for the immobile population percentage of Kv-Nav, 4SA, and E4SA. MW: *, P < 0.05 and **, P < 0.01. (G) MDC (25–75% IQR) for the mobile population of Kv-Nav, 4SA, and E4SA. MW: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.