The autosomal dominant skin disorder erythrokeratodermia variabilis (EKV) has been linked to mutations in the human connexin31 (hCx31) gene, which is expressed in the epidermis. We characterized and compared a pathogenic mutation resulting in replacement of amino acid glycine 12 with arginine (G12R) with wild-type hCx31 protein. HeLa cells were transfected with wild-type and mutant hCx31 cDNA, respectively, using different-constitutive and inducible-vector systems. Independent of the expression vector, wild-type and mutant hCx31 were expressed at comparative levels and localized at the plasma membranes. Mutated channels (hCx31G12R) showed higher conductance in dye coupling studies than wild type channels. Furthermore, HeLa cells died within 5 days after constitutive expression of the mutant protein. Using an inducible expression system, we demonstrated a direct correlation between survival/life span of transfected HeLa cells and expression level of the mutant protein, indicating a gain-of-function mechanism due to a defective channel closure mechanism.