Fig 2: Paraformaldehyde (PFA) concentration was crucial for Na+ channel immunostaining. (A–C, left column) Conventional fixative (4% PFA) revealed no specific labeling except a few immunosignals of NaV1.6 and AnkG with extremely low signal-to-noise ratio in the prefrontal cortex of rats. (A–C, right column), light fixation (0.5% PFA and 0.5% sucrose) resulted in robust stainings of all antibodies examined (NaV1.1, Pan-NaV, NaV1.2, NaV1.6, and AnkG, color-coded). The voxel depth is 4 µm in (A), 3 µm in (B), 5 µm in (C). Scale bar, 10 µm. Pia locates to the right of the images.

Fig 3: The Kv7.3 C-terminal tail mediates efficient targeting to the AIS. (A) Illustration of the CD4 chimera strategy in which a truncated form of the single-pass transmembrane protein CD4 is fused to the C-terminal tail of human Kv7.3. The C-terminus of Kv7.3 contains four a-helical domains, here depicted as cylinders, as well as an ankyrin G (ankG) binding motif, which is depicted as a circle. (B) 10 DIV rat hippocampal neurons transiently expressing CD4, CD4-Kv7.3CT or CD4-Kv7.3CT(ETD-AAA). The CD4-Kv7.3CT(ETD-AAA) construct contains point mutations in three essential amino acids of the ankG binding motif (ETD sequence replaced by alanines). Neurons were labeled with CD4 antibody both before and after permeabilization to distinguish surface (red) and total (green) population of CD4-Kv7.3 chimeras. MAP2 was included as a marker of soma and dendrites. White arrowheads point to the AIS positioned in the proximal part of a MAP2 negative neurite. (C) Mean CD4 surface fluorescence in the AIS (0–30 µm) versus a dendrite (20 µm), “AIS/Dendrite” ratio for the indicated constructs. Number of neurons analyzed: CD4 (n = 14), Kv7.3CT (n = 20), and Kv7.3CT(ETD-AAA) (n = 16). All data represent the mean (SD). Significances indicated as compared to CT. (D) Mean CD4 surface fluorescence in the AIS (0–30 µm) versus the distal axon (60–90 µm), “AIS/Distal axon” ratio for the indicated constructs. Number of neurons analyzed: CD4 (n = 15), Kv7.3CT (n = 19) and Kv7.3CT(ETD-AAA) (n = 19) from 3 to 4 independent experiments. All data represent the mean (SD). Significances indicated as compared to CT. Scale bar, 10 µm.

Fig 4: Helix D regulates the AIS density and cell surface dynamics of Kv7.2/Kv7.3 full-length channels by a mechanism additive to that of the ankG binding motif. (A) Illustration of the SEP-TAC-Kv7.3 full-length construct. (B) Confocal images of 10 DIV hippocampal neurons co-expressing full-length Kv7.2 and the indicated full-length SEP-TAC-Kv7.3 chimeras. Neurons were stained with GFP antibody both before and after permeabilization to distinguish surface (red) and total (green) population of the chimera. AnkG was included as marker of the AIS. The white boxes highlight the AIS, which is magnified below. Scale bars, 10 µm. (C) AIS/Dendrite ratios for the indicated constructs. Number of neurons analyzed: Kv7.2 + SEP-TAC-Kv7.3 (n = 29), Kv7.2 + SEP-TAC-Kv7.3?helixD (n = 30); Kv7.2 + SEP-TAC-Kv7.3(ETD-AAA) (n = 29), Kv7.2 + SEP-TAC-Kv7.3?helixD + (ETD-AAA) (n = 23). Significances as compared to Kv7.2 + SEP-TAC-Kv7.3. (D) The AIS density of the surface GFP signal in neurons expressing the indicated constructs. All data sets were normalized to the mean of Kv7.2 + SEP-TAC-Kv7.3, which was set to 1. Number of neurons analyzed: Kv7.2 + SEP-TAC-Kv7.3 (n = 30), Kv7.2 + SEP-TAC-Kv7.3?helixD (n = 31); Kv7.2 + SEP-TAC-Kv7.3(ETD-AAA) (n = 30), Kv7.2 + SEP-TAC-Kv7.3?helixD + (ETD-AAA) (n = 26). (E) FRAP curves showing the quantified fluorescence recovery (F/F0) over the time course of 120 s for SEP-TAC-Kv7.3 and the ankG binding mutant SEP-TAC-Kv7.3(ETD-AAA) in neurons co-expressing wild-type Kv7.2. Number of cells analyzed: Kv7.3 (n = 25), Kv7.3(ETD-AAA) (n = 24). (F) FRAP curves showing the quantified fluorescence recovery (F/F0) over the time course of 120 s for SEP-TAC-Kv7.3?helixD and the double-mutant Kv7.3?helixD + (ETD-AAA) in neurons co-expressing wild-type Kv7.2. SEP-TAC-Kv7.3 is included again for reference. Number of cells analyzed: Kv7.3?helixD (n = 24), and Kv7.3?helixD + (ETD-AAA) (n = 23). (G) The relative recovery of fluorescence at 120 s (F120/F0) for the indicated constructs, calculated as the mean of the last 5 frames from each FRAP series. Number of cells analyzed: Kv7.3 (n = 25), Kv7.3(ETD-AAA) (n = 24). Kv7.3?helixD (n = 24), and Kv7.3?helixD + (ETD-AAA) (n = 23). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: Polarized distribution of NaV1.1 and NaV1.6 at the AIS of PV neurons.(A) Triple staining using antibodies for PV (blue), AnkG (red), and NaV1.2 (green) revealed the absence of NaV1.2 at the AIS of PV neuron (arrowheads). Note that neighboring PV-negative AIS (presumably from PCs, asterisks) show strong immunosignals for NaV1.2. (B) Triple staining for PV, AnkG (green), and NaV1.6 (red). Note that distal regions of AIS were heavily stained for NaV1.6 (arrowheads). Neighboring axons (asterisks) also showed strong immunosignals. (C) Triple staining for PV, NaV1.6, and NaV1.1 shows polarized distribution of these subtypes at the AIS. (D) Plots of the averaged fluorescence intensity (± s.e.m., see Materials and Methods) as a function of distance from soma at the AIS. Data were obtained from triple-staining experiments similar to (C). Images are projections of confocal z stacks. Scale bars represent 10 µm. Error bars represent s.e.m.