Rapid determination of gap junction formation using HeLa cells microinjected with cDNAs encoding wild-type and chimeric connexins.
A procedure for rapidly determining the functionality of gap junctions constructed of recombinant connexins in communication-deficient HeLa cells is described. Nuclear microinjection of cDNA encoding wild-type connexins (Cx) 26, 32, 43, and a range of connexin-aequorin (Cx-Aeq) chimerase resulted in generation of gap junction intercellular communication channels. Expression of recombinant protein was detected in > 95% of cells 18-72 h following nuclear microinjection, and the functionality of the channels generated was determined according to their ability to transfer the fluorescent dye tracers Lucifer yellow and propidium iodide. The dye transfer results obtained correlated closely with other published studies using stably transfected cells and yet are obtained as rapidly as 18 h following microinjection of cDNA. Expression of a truncated form of Cx43 (Cx43 delta 244) by this new method indicated diminished intercellular transfer of both dyes and supports a channel-gating mechanism that postulates interaction between the carboxyl tail and the intracellular loop.
George
C H
CH
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom. georgech@cf.ac.uk
Martin
P E
PE
Evans
W H
WH
D000331:Aequorin / Q000235:genetics; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D017630:Connexins / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000502:physiology*; D015870:Gene Expression / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007473:Ion Channels / Q000502:physiology; D008845:Microinjections; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D002087:Butyrates; D017630:Connexins; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D007473:Ion Channels; D011994:Recombinant Proteins; D020148:Butyric Acid; D000331:Aequorin