Abstract
Oculodentodigital dysplasia, a rare condition displaying congenital craniofacial deformities and limb abnormalities, has been associated with over 20 known human connexin43 (Cx43) mutations. The localization of two of these mutants, G21R and G138R, was examined in Cx43-positive normal rat kidney cells (NRK) and Cx43-negative gap junctional intercellular communication-deficient HeLa cells. Green fluorescent protein-tagged and untagged Cx43 G21R and G138R mutants were transported to the plasma membrane and formed punctate structures reminiscent of gap junction plaques in both NRK and HeLa cells. Further localization studies revealed no significant trafficking defects as subpopulations of Cx43 mutants were found in both the Golgi apparatus and lysosomes, not unlike wild-type Cx43. Dual patch clamp functional analysis of the mutants expressed in gap junctional intercellular communication-deficient N2A cells revealed that neither G21R nor G138R formed functional gap junction channels, although they successfully reached cell-cell interfaces between cell pairs. Importantly, when either mutant was expressed in NRK cells, dye coupling experiments revealed that both mutants inhibited endogenous Cx43 function. These studies suggest that, although patients suffering from oculodentodigital dysplasia possess one wild-type Cx43 allele, it is likely that Cx43-mediated gap junctional intercellular communication is reduced below 50% because of a dominant-negative effect of mutant Cx43 on wild-type Cx43.
Highlights
Gap junctions are protein intercellular channels that link adjacent cells to allow for the exchange of small ions and molecules less than 1 kDa in size [1]
G21R is predicted to be located within the first transmembrane domain, 1 The abbreviations used are: Cx43, connexin43; GJIC, gap junctional intercellular communication; ODDD, oculodentodigital dysplasia; normal rat kidney cells (NRK), normal rat kidney cell; GFP, green fluorescent protein
Cx43 Mutants Are Transported to, and Cluster at, the Cell Surface in the Presence or Absence of Endogenous Cx43—To determine the localization patterns of G21R and G138R Cx43 mutants, cDNA constructs were engineered where wild-type and Cx43 mutants were tagged with GFP (Fig. 1) and expressed in NRK and HeLa cells
Summary
Gap junctions are protein intercellular channels that link adjacent cells to allow for the exchange of small ions and molecules less than 1 kDa in size [1]. Paznekas et al [15] report that GJA1, the gene encoding the gap junction protein Cx43, was responsible for the pleiotropic condition ODDD because of its map location These authors have further confirmed mutations in 100% of studied individuals who are affected with ODDD [15]. G138R is located within the intracellular loop, which has been shown to have gating function as proposed by the “ball and chain” model of channel regulation in which the carboxyl tail acts as a gating particle that interacts with the “receptor” encoded within the intracellular loop [21,22,23] It was the purpose of this study to examine the functional properties of select Cx43 disease-linked mutants, G21R or G138R, in an attempt to provide insight into the pathophysiology underlying the ODDD condition
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