Abstract
Gap junctions form intercellular channels that mediate metabolic and electrical signaling between neighboring cells in a tissue. Lack of an atomic resolution structure of the gap junction has made it difficult to identify interactions that stabilize its transmembrane domain. Using a recently computed model of this domain, which specifies the locations of each amino acid, we postulated the existence of several interactions and tested them experimentally. We introduced mutations within the transmembrane domain of the gap junction-forming protein connexin that were previously implicated in genetic diseases and that apparently destabilized the gap junction, as evidenced here by the absence of the protein from the sites of cell-cell apposition. The model structure helped identify positions on adjacent helices where second-site mutations restored membrane localization, revealing possible interactions between residue pairs. We thus identified two putative salt bridges and one pair involved in packing interactions in which one disease-causing mutation suppressed the effects of another. These results seem to reveal some of the physical forces that underlie the structural stability of the gap junction transmembrane domain and suggest that abrogation of such interactions bring about some of the effects of disease-causing mutations.
Highlights
Gap junction channels are formed by the docking of two hemichannels or connexons from adjacent membranes [1]
We introduced mutations within the transmembrane domain of the gap junction-forming protein connexin that were previously implicated in genetic diseases and that apparently destabilized the gap junction, as evidenced here by the absence of the protein from the sites of cell-cell apposition
Based on the intermediate resolution structure [5] and computational inference methods [15, 16], a model of canonical ␣-helices corresponding to the M1–M4 segments, which specifies the approximate positions of ␣-carbons in the TM domain, was recently proposed [17]
Summary
We followed the experimental procedures presented in Ref. 37. Cloning—Genomic DNA of each of the genes, GJB2 (Cx26) and GJB1 (Cx32), was double-digested with HindIII and KpnI and cloned into a pEGFP-N1 expression vector (Clontech, Palo Alto, CA). The amount of reagent was reduced by half and incubated mixed with an equal volume of Neowater (DoCoop Technologies, Or Yehuda, Israel) for 5 min at room temperature This mixture and plasmid DNA were incubated separately in OptiMEM for 5 min and combined for another 20 min at room temperature. HeLa cells (60 –70% confluence) were washed with OptiMEM and incubated with the combined Lipofectamine/plasmid DNA solution at 37 °C. The data presented in the paper are based on paraformaldehyde as the fixation agent, as was done, e.g. in Refs. Structural Modeling—The template structure of the TM domain of the gap junction, comprising Cx32 monomers (Protein Data Bank code 1txh) [17], was used as a starting point. Glu residues, which are reciprocally charged and are near the water-filled pore lumen, could be involved in stabilizing electrostatic interactions; it has been estimated that salt bridges embedded in water can add nearly 1 kcal/mol to protein stability [25, 26]
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