Figure 6.
Cx47 mutants do not efficiently form heterotypic channels. A–C, HeLa cells stably expressing Cx47 mutants (P87S, Y269D, or M283T) or Cx43 were transiently transfected to express DsRed (DsRed+) and mixed with DsRed− cells in a ratio of 1 to 20. After 24 h, cells were immunostained as indicated and counterstained for DAPI. One of the two possible pairings for each combination is illustrated. The DsRed+ cell is pseudocolored blue in the first and third rows, and indicated by an asterisk in the fourth row. Note that only M283T/Cx43 (C) pairings have overlapping puncta at the border of the DsRed signal (arrowhead) similar to Cx47/Cx43 pairings (Fig. 1), whereas pairing cells expressing Cx43 with those expressing other mutants do not. D, Quantitative summary of three independent experiments such as illustrated in A–C. The asterisk denotes the DsRed+ cell. Each dot represents the number of overlapping puncta determined for 1 DsRed+ cell. In each column, the horizontal bar denotes the mean, the vertical bar represents the 95% confidence interval, and the total number of DsRed+ cells is shown in parentheses. Both pairings of M283T/Cx43 (43*/M283T and M283T*/43) have overlapping puncta similar to pairings of Cx47/Cx43 (43*/47 and 47*/43) (data from Fig. 1). E, Neuro2A cells were transiently transfected with a pIRES2-EGFP or a pIRES2-DsRed bicistronic expression vector that also contained Cx43, Cx47, P87S, Y269D, or M283T. After 24 h, different combinations of red and green cell pairs were generated by mixing the transfected cells at a 1 to 1 ratio, and assessed by dual whole-cell patch clamping 24–48 h later. For each combination listed on the x-axis, the mean and SE of the Gj and number of pairs tested is shown. Using Kruskal–Wallis test followed by Dunn's multiple comparison test, the pairs containing mutant Cx47 have a Gj significantly different from that of WT Cx47/Cx43 channels (data from Fig. 2) (p < 0.001, P87S/Cx43; p < 0.05, Y269D/Cx43; p < 0.01, M283T/Cx43), but not control pairs (43/CTR or 47/CTR)(data from Fig. 2). Scale bar: 10 μm.