Inherited disease-linked arginine76 or equivalent residue mutants in Cx50 and Cx45 showed impaired homotypic and heterotypic gap junction function, but not Cx43.

Abstract

Connexins form intercellular communication channels, known as gap junctions (GJs), found throughout different tissues and organs. Mutations in genes encoding connexins are found to be linked to various inherited diseases, but the molecular and structural mechanisms are not fully clear. The Arg76 (R76) residue in Cx50 or the equivalent residue in other connexins is fully conserved across the entire connexin family and is a hot spot for five connexin-linked inherited diseases, including congenital cataract, oculodentodigital dysplasia, and cardiac arrhythmias. To better understand the molecular and cellular mechanism of dysfunction caused by mutations of this residue, we examined the functional status and properties of GJs containing R76 mutations in Cx50 (R76H/C), Cx43 (R76H/S/C), and Cx45 (R75H) with an emphasis on heterotypic GJs in connexin-deficient model cells. All tested mutants showed a significantly decreased coupling% and conductance, except for Cx43 R76H/S. These connexin mutants also showed reduced coupling% and conductance when paired with a docking compatible connexin (Cx50/Cx46, Cx45/Cx43), and except for Cx43 R76H/S/C, all formed functional GJs with Cx45. Our homology structure models indicated that mutations of R76 or the equivalent residue in these GJs led to a loss of intra- and/or inter-connexin non-covalent interactions (salt bridges) at the sidechain of this residue, which could contribute to the observed GJ impairments underlying diseases. It remains unclear why Cx43 GJ could tolerate some variations at this residue. Supported by NSERC.