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

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

^*Department of Neuroscience, Albert Einstein College of Medicine, New York, NY 10461; ^§Fred Hutchinson Cancer Research Center, Seattle, WA, 98109; and ^‡Department of Anatomy and Cell Biology, University of Western Ontario, London, ON, Canada N6A 5C1

Contributed by Michael V. L. Bennett

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.

Footnotes

↵ † To whom reprint requests should be addressed. E-mail: fbukausk{at}aecom.yu.edu.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.050588497.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.050588497
Abbreviations:

Cx,

connexin;

EGFP,

enhanced green fluorescent protein;

GJ,

gap junction;

N2A,

Neuro-2a;

DAPI,

4′,6-diamidino-2-phenylindole;

wt,

wild type;

ROI,

region of interest