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.