Skin disease-associated GJB4 variants differentially influence connexin stability, cell viability and channel function.

Here we characterize seven Cx30.3 gene variants (R22H, S26Y, P61R, C86S, E99K, T130M and M190L) clinically associated with the rare skin disorder erythrokeratodermia variabilis et progressiva (EKVP) in tissue-relevant and differentiation-competent rat epidermal keratinocytes (REKs). We found that all variants, when expressed alone or together with wildtype (WT) Cx30.3, had the capacity to traffic and form gap junctions with an efficiency like WT Cx30.3. Cx30.3 was found to have a slower relative turnover than Cx43. However, turnover was more rapid for the R22H and P61R variants relative to Cx30.3. Furthermore, REKs that expressed the P61R variant exhibited reduced viability and were more permeable to fluorescent dyes, indicative of leaky hemichannels and/or the loss of membrane integrity associated with cell death. In connexin-null AD-293 cells, dual patch clamp studies revealed that the variants had either reduced (C86S) or no (S26Y and T130M) gap junction channel function. The remaining variants formed functional gap junction channels with enhanced transjunctional voltage (V)-dependent gating. Moreover, WT Cx30.3 and functional variant gap junction channels had similar unitary conductance of ∼34-42 pS, though variant channels appeared to have lower open probability than WT Cx30.3 channels at high Vs. In conclusion, EKVP-associated Cx30.3 variants each alter one or more Cx30.3 characteristics although the molecular changes identified for E99K were limited to enhanced V gating. The breadth of molecular changes identified may all be sufficient to cause EKVP, but this remains to be firmly established as more familial patients are genotyped for these variants. KEY POINTS: Here we characterize seven Cx30.3 variants (R22H, S26Y, P61R, C86S, E99K, T130M and M190L) that have been clinically associated with the rare skin disorder erythrokeratodermia variabilis et progressiva (EKVP). We discovered human Cx30.3 undergoes relatively slow turnover compared with Cx43 and exhibits kinetically slow and limited voltage gating. Wildtype Cx30.3 and all variants localized to intracellular compartments and gap junctions in rat epidermal keratinocytes. Each EKVP-associated Cx30.3 variant altered one or more Cx30.3 characteristics related to protein stability, cell viability and/or channel function. The breadth of molecular changes identified for each Cx30.3 variant may independently be sufficient to cause EKVP, but this remains to be firmly established through additional genetic and molecular analysis.