Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel.
Single site mutations in connexins have provided insights about the influence specific amino acids have on gap junction synthesis, assembly, trafficking, and functionality. We have discovered a single point mutation that eliminates functionality without interfering with gap junction formation. The mutation occurs at a threonine residue located near the cytoplasmic end of the third transmembrane helix. This threonine is strictly conserved among members of the alpha- and beta-connexin subgroups but not the gamma-subgroup. In HeLa cells, connexin43 and connexin26 mutants are synthesized, traffic to the plasma membrane, and make gap junctions with the same overall appearance as wild type. We have isolated connexin26T135A gap junctions both from HeLa cells and baculovirus-infected insect Sf9 cells. By using cryoelectron microscopy and correlation averaging, difference images revealed a small but significant size change within the pore region and a slight rearrangement of the subunits between mutant and wild-type connexons expressed in Sf9 cells. Purified, detergent-solubilized mutant connexons contain both hexameric and partially disassembled structures, although wild-type connexons are almost all hexameric, suggesting that the three-dimensional mutant connexon is unstable. Mammalian cells expressing gap junction plaques composed of either connexin43T154A or connexin26T135A showed an absence of dye coupling. When expressed in Xenopus oocytes, these mutants, as well as a cysteine substitution mutant of connexin50 (connexin50T157C), failed to produce electrical coupling in homotypic and heteromeric pairings with wild type in a dominant-negative effect. This mutant may be useful as a tool for knocking down or knocking out connexin function in vitro or in vivo.
Beahm
Derek L
DL
Department of Biological Sciences, State University of New York, Buffalo, New York 14260, USA.
Oshima
Atsunori
A
Gaietta
Guido M
GM
Hand
Galen M
GM
Smock
Amy E
AE
Zucker
Shoshanna N
SN
Toloue
Masoud M
MM
Chandrasekhar
Anjana
A
Nicholson
Bruce J
BJ
Sosinsky
Gina E
GE
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D000072259:Connexin 26; D018031:Connexin 43 / Q000235:genetics; D017630:Connexins / Q000737:chemistry* / Q000235:genetics*; D020285:Cryoelectron Microscopy; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D005456:Fluorescent Dyes / Q000494:pharmacology; D017629:Gap Junctions; D005799:Genes, Dominant; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007313:Insecta; D015603:Keratinocytes / Q000378:metabolism; D008027:Light; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010802:Phylogeny; D017354:Point Mutation; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D013912:Threonine / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection; D014981:Xenopus
D018031:Connexin 43; D017630:Connexins; D018076:DNA, Complementary; D005456:Fluorescent Dyes; C000607042:GJB2 protein, human; D018075:RNA, Complementary; D000072259:Connexin 26; D013912:Threonine; D003545:Cysteine; D010100:Oxygen