Fig. 8: Mutations impose reduced expression and membrane trafficking of TadNaC2 channels in vitro.

a Representative fluorescence micrographs of CHO-K1 cells co-transfected with pEGFP-TadNaC2 fusion vector (left panels) and an empty pIRES2-EBFP vector (right panels). b Plot of percent average integrated density ± standard deviation, quantifying the emitted fluorescence of pEGFP-TadNaC2 wild type (wt) and mutant channels, normalized to the average integrated density of wild-type TadNaC2 (n = 3 for each transfection condition). EBFP fluorescence was also quantified to determine transfection efficiency. Cyan-colored asterisks denote p value thresholds for Tukey post hoc means comparisons of fluorescence signals between wild-type and mutant channels after one-way ANOVAs (EGFP: p = 5.6E−11, F = 73.6; EBFP: not significant). c Top panel: Western blot of select EGFP-tagged TadNaC2 channel variants in CHO-K1 cell lysates using anti-GFP polyclonal antibodies, comparing total channel protein content (T) with membrane/surface expressed channel protein content (S) for each variant. Bottom panel: Western blot of the lower half of the membrane used in the top panel, using anti-GAPDH (top bands) and anti-EBFP (bottom bands), polyclonal antibodies. d Quantified band intensity (mean gray area) of TadNaC2 bands in (c), relative to the wild type EGFP-TadNaC2 total protein band, revealing decreased total and surface protein expression of TadNaC2 channels bearing mutations, and a near complete absence of membrane expressed variants harboring a K203 deletion, consistent with our inability to record current for this channel in vitro. Bands for each channel variant were also normalized to the intensity of EBFP present in corresponding total protein lanes.