Functional Domain Mapping and Selective Trans-dominant Effects Exhibited by Cx26 Disease-causing Mutations

Abstract

Mutations in Cx26 are a major cause of autosomal dominant and recessive forms of sensorineural deafness. Some mutations in Cx26 are associated not only with deafness but also with skin disease. We examined the subcellular localization and function of two green fluorescent protein (GFP)-tagged Cx26 point mutants that exhibit both phenotypes, G59A-GFP and D66H-GFP. D66H-GFP was retained within the brefeldin A-insensitive trans-Golgi network, whereas a population of G59A-GFP was transported to the cell surface. Neither G59A nor D66H formed gap junctions that were permeable to small fluorescent dyes, suggesting they are loss-of-function mutations. When co-expressed with wild-type Cx26, both G59A and D66H exerted dominant-negative effects on Cx26 function. G59A also exerted a trans-dominant negative effect on co-expressed wild type Cx32 and Cx43, whereas D66H exerted a trans-dominant negative effect on Cx43 but not Cx32. We propose that the severity of the skin disease is dependent on the specific nature of the Cx26 mutation and the trans-dominant selectivity of the Cx26 mutants on co-expressed connexins. Additional systematic mutations at residue D66, in which the overall charge of this motif was altered, suggested that the first extracellular loop is critical for Cx26 transport to the cell surface as well as function of the resulting gap junction channels.

Serine protease hepsin regulates hepatocyte size and hemodynamic retention of tumor cells by hepatocyte growth factor signaling in mice

“…26 Moreover, these EGFinduced modifications of Cx43 may be caused by the MAPK pathway. 26 Because both Cx32 27 and Cx26 28 proteins turn over with a short half-life (1.5-5.0 hours), similar to that of Cx43, 29 and because Cx32 and Cx43 have comparable responses to proteasome inhibitors, 30 it is possible that similar signaling pathways or post-transcriptional modification mechanisms may be involved in the down-regulation of the levels of Cx32 and Cx26 by HGF/c-Met. Nevertheless, other regulatory mechanisms may exist, because many of the modifications of Cx43 are the result of the phosphorylation of the carboxyl terminus, 26 and Cx32 and Cx26 contain short carboxyl termini with fewer potential phosphorylation sites than Cx43.…”

Oculodentodigital Dysplasia-causing Connexin43 Mutants Are Non-functional and Exhibit Dominant Effects on Wild-type Connexin43

“…In some cases, these could be considered gain-of-function mutations. The third class of mutants could involve altered intracellular transport and assembly as with some Cx32 mutants associated with Charcot-Marie-Tooth disease (32) or Cx26 mutations linked to deafness and skin disease (33)(34)(35). This class of mutants may in fact be functional if transported to the cell surface in association with wild-type proteins, not unlike mutations of the chloride channel responsible for cystic fibrosis (36).…”

Functional Characterization of aGJA1Frameshift Mutation Causing Oculodentodigital Dysplasia and Palmoplantar Keratoderma

“…For cell infection, REKs at 30 -40% confluence were subjected to two rounds of retroviral infection with retroviral particles encoding GFP-tagged fs260, T259, G138R or GFP as described previously (26). Infected cells were passed at least twice, evaluated to ensure at least 90% expression efficiency, and subjected to Western blot analysis.…”