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.
Highlights
Polypeptide subunits that make up gap junctions, and a recent genomic study highlighted the common features of the mouse and human Cx genes [4]
When expressed in gap junctional intercellular communication (GJIC)-deficient cell lines, the D66H-green fluorescent protein (GFP) mutant was retained within the trans-Golgi apparatus, and a population of the G59A-GFP mutant was transported to the cell surface and assembled into structures reminiscent of gap junctions
When co-expressed with Cx26, the D66H-GFP mutant was transported to the cell surface but the resulting gap junctions were impermeable to Lucifer yellow, suggesting that this mutant exerted a dominant-negative effect on wild-type Cx26 function
Summary
Polypeptide subunits that make up gap junctions, and a recent genomic study highlighted the common features of the mouse and human Cx genes [4]. Heteromeric connexons composed of Cx40/Cx43 or Cx26/Cx32 have been characterized and exhibit altered functional properties compared with their homomeric counterparts [8, 11,12,13,14] These combinations further expand the potential multitude of signals that may pass between cells and highlight the requirement to characterize the intermixing that may occur between different Cxs co-expressed within a cell population. Rouan et al [23] further hypothesized that mutant Cx26 causes skin disease by exerting trans-dominant-negative effects on other Cxs co-expressed in keratinocytes. When co-expressed with Cx26, the D66H-GFP mutant was transported to the cell surface but the resulting gap junctions were impermeable to Lucifer yellow, suggesting that this mutant exerted a dominant-negative effect on wild-type Cx26 function. Our results suggest that the severity of Cx26-linked skin diseases is probably related to selective trans-dominant interactions with other Cxs expressed within the skin
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