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Though the majority of 66del D-Cx31/EGFP was cytoplasmic (Fig.2G), some punctate staining at the plasma membrane was observed.In addition, R32W-Cx31/EGFP also did not form functional channels.Expression of R32W-Cx31/EGFP in NEB1 cells did not elevate dye transfer rates above basal levels (Figs 3E–H and 6), and little or no dye transfer was observed in fibroblasts (Fig. R32W in has been reported as a polymorphism with no proven disease association (20,21).

From the antibodies available, the R42P in Cx31 does not appear to effect the expression and localization of other epidermally expressed connexins, including Cx26, Cx30, Cx30.3 and Cx43 (data not shown).Unlike WT-Cx31/EGFP, the expression of 66del D-Cx31/EGFP did not significantly enhance the rate of dye transfer in either NEB1 cells (Figs 3I-L and 6) or NIH 3T3 fibroblasts (Fig.6), indicating that the mutant form cannot form functional channels.This suggested that these skin disease-associated mutant Cx31 proteins were inducing cell death.A slightly different cellular distribution was observed for the mutant Cx31 associated with peripheral neuropathy and hearing impairment.

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