Functional rescue of defective mutant connexons by pairing with wild-type connexons

We have compared the functional and structural integrity of gap junction ch annels assembled from a Cx45 truncation mutant with those of gap junction c hannels assembled from wild-type (wt) Cx35 and Cx43. These channel-forming proteins are constitutively expressed in HeLa cells. The truncation mutant lacks the last 26 amino acids of the COOH-terminus, including nine serine p hosphorylation sites that are associated with regulatory processes of these channels. We determined the presence of Sap junction plaques in these cell s with the immunogold freeze fracture technique, which showed that plaque f ormation is similar in all the clones investigated. Junctional permeability was probed with calcein transfer and now cytometry analyses and junctional conductance was measured in cell pairs with double whole-cell patch-clamp techniques. For homotypic pairing only the truncated mutant did not form pe rmeable channels, However, coupling was restored for heterotypic channels ( pairing wtCx45- or wtCx43- with mutant-connexons), whose junctional communi cation was not different from that of the homotypic channels. Our results i ndicate that the presence of gap junction plaques does not warrant function al coupling and that heterotypic trCx45/wtCx35 channels can be regulated by the intact wtCx45 connexons. This dominant-positive effect is also operati ve when wtCx43 are paired with trCx45 connexons.