ankG restricts Kv-Nav diffusion at the surface of N2A cells. (A) Schematic representation of Kv-Nav chimera in which the C terminus of Kv2.1 was substituted by a segment encompassing the ankyrin-binding motif of Nav1.2. Note the presence of the tetramerization domain T1 in the N terminus of Kv2.1 and of an extracellular myc tag. (B) Surface expression of Kv-Nav in ankG-GFP–positive cells. Surface Kv-Nav was immunodetected with an antibody to myc (red), and ankG signal corresponds to the GFP fluorescent signal on fixed cells (green). Bar, 10 µm. (C and E) Representative examples of QD trajectories corresponding to Kv-Nav in GFP (red, C)- and ankG-GFP–expressing N2A cells (black, E). (D and F) DIDC of Kv-Nav in GFP (red, D)- and ankG-GFP–expressing N2A cells (black, F). The immobile population (D ≤ 0.00075 µm².s⁻¹) was defined from the DIDC of QDs stuck on the glass coverslips (gray). Trajectories for GFP (n = 198) and ankG-GFP (n = 253) conditions were analyzed from three and four independent experiments, respectively. (G) Histogram of the mean values ± SEM for the immobile population (percentage) of Kv-Nav in GFP and ankG-GFP conditions. (H) MDC (25–75% IQR) for the mobile population of Kv-Nav in GFP and ankG-GFP conditions. MW: ***, P < 0.001.