Functional rescue of defective mutant connexons by pairing with wild-type connexons.
We have compared the functional and structural integrity of gap junction channels assembled from a Cx45 truncation mutant with those of gap junction channels assembled from wild-type (wt) Cx45 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 phosphorylation sites that are associated with regulatory processes of these channels. We determined the presence of gap junction plaques in these cells with the immunogold freeze fracture technique, which showed that plaque formation is similar in all the clones investigated. Junctional permeability was probed with calcein transfer and flow 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 permeable channels. However, coupling was restored for heterotypic channels (pairing wtCx45- or wtCx43- with mutant-connexons), whose junctional communication was not different from that of the homotypic channels. Our results indicate that the presence of gap junction plaques does not warrant functional coupling and that heterotypic trCx45/wtCx45 channels can be regulated by the intact wtCx45 connexons. This dominant-positive effect is also operative when wtCx43 are paired with trCx45 connexons.
Hülser
D F
DF
Abteilung Biophysik, Biologisches Institut, Universität Stuttgart, Germany. Huelser@po.uni-stuttgart.de
Rütz
M L
ML
Eckert
R
R
Traub
O
O
D018031:Connexin 43 / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D017630:Connexins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D004553:Electric Conductivity; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D005614:Freeze Fracturing; D017629:Gap Junctions / Q000648:ultrastructure; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009154:Mutation; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D018031:Connexin 43; D017630:Connexins; D005452:Fluoresceins; C082920:connexin 45; D012694:Serine; C007740:fluorexon