Abstract
Connexins form a family of membrane proteins that assemble into communication channels and directly connect the cytoplasms of adjoining cells. Malfunctioning of connexin channels often cause disease, such as the mutations M34T and R75W in human connexin 26, which are associated with hereditary deafness. Another residue known to be essential for normal channel activity in the connexin is Cys-64. To obtain structural and functional insights of connexin 26, we studied the roles of these three residues by expressing mutant connexins in insect Sf9 and HeLa cells. The M34T and M34A mutants both formed gap junction plaques, but dye transfer assays showed that the M34A mutant had a significantly reduced permeability, suggesting that for proper channel function a side chain of adequate size is required at this position. We propose that Met-34 is located in the innermost helix of the channel, where it ensures a fully open channel structure via interactions with other transmembrane helices. Gap junction channels formed by the R75W and R75D mutants dissociated upon solubilization in dodecyl maltoside, whereas the R75A mutant remained hexameric. All gap junctions formed by Arg-75 mutants also showed only negligible activity in dye transfer experiments. These results suggest that residue Arg-75 plays a role in subunit interactions needed to retain a functional and stable connexin hexamer. The C64S mutant was suggested to be defective in oligomerization and/or protein folding even in the presence of wild-type connexin.
Highlights
Connexins form a family of membrane proteins that assemble into communication channels and directly connect the cytoplasms of adjoining cells
First structural studies were performed on rat liver gap junctions, mostly composed of connexin 32 (Cx32) and connexin 26 (Cx26) [9, 10], which confirmed the model of gap junction channel with six membrane-spanning subunits forming the oligomeric channel through the plasma membrane [11, 12]
Electron crystallography was used to study the three-dimensional structure of C-terminally truncated recombinant connexin 43 (Cx43) gap junction channels, and the 7.5-Å (21 Å in the vertical direction to the membrane surfaces) three-dimensional map revealed for the first time the arrangement of the ␣-helices in gap junctions [15]
Summary
Connexins form a family of membrane proteins that assemble into communication channels and directly connect the cytoplasms of adjoining cells. All gap junctions formed by Arg-75 mutants showed only negligible activity in dye transfer experiments These results suggest that residue Arg-75 plays a role in subunit interactions needed to retain a functional and stable connexin hexamer. Six connexin subunits assemble into a hexameric hemichannel, named connexon, and the docking of two connexons from adjacent membranes produces a complete gap junction channel, resulting in a narrowing of the extracellular gap between adjacent lipid bilayers to about 40 Å [8]. These characteristic structures have not yet been found in prokaryotes, indicating the necessity for more complex cell-cell communication networks in higher organisms. This residue as well as the sequence around it, is highly conserved among all connexin subtypes [29], indicating that this residue plays an important role in all gap junctions
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