Clustering of connexin 43–enhanced green fluorescent protein gap junction channels and functional coupling in living cells

TLDR

The study aimed to determine how Cx43–EGFP distribution observed by fluorescence microscopy relates to electrical coupling at single‑channel resolution in living cell pairs. Communication‑incompetent cell lines were transfected with Cx43–EGFP, and coupling was measured at single‑channel resolution while fluorescence microscopy tracked channel distribution. Cx43–EGFP channels behaved like wild‑type except for reduced voltage sensitivity, clustered into plaques at cell contacts, and only plaques containing several hundred channels reliably produced coupling, indicating that clustering is required for channel opening.

Abstract

Communication-incompetent cell lines were transfected with connexin (Cx) 43 fused with enhanced green fluorescent protein (EGFP) to examine the relation between Cx distribution determined by fluorescence microscopy and electrical coupling measured at single-channel resolution in living cell pairs. Cx43–EGFP channel properties were like those of wild-type Cx43 except for reduced sensitivity to transjunctional voltage. Cx43–EGFP clustered into plaques at locations of cell–cell contact. Coupling was always absent in the absence of plaques and even in the presence of small plaques. Plaques exceeding several hundred channels always conferred coupling, but only a small fraction of channels were functional. These data indicate that clustering may be a requirement for opening of gap junction channels.