Chick connexin-56, a novel lens gap junction protein. Molecular cloning and functional expression.
We used primers corresponding to the amino-terminal sequence shared by rat connexin-46 and ovine MP70 and a consensus sequence of the second extracellular loop conserved in all connexins to amplify and subsequently clone from chick genomic DNA a new member of the connexin family of gap junction proteins, chick connexin-56. The derived chick connexin-56 polypeptide contains 510 amino acids with a predicted molecular mass of 55,857 daltons. Although identical in the first 70 amino acids to rat connexin-46, chick connexin-56 diverges significantly in length and composition in predicted cytoplasmic regions, which have previously been inferred to determine functional and regulatory specificity. We were able to detect hybridization of connexin-56 probes only to RNA derived from lens. Connexin-56 was functionally expressed by the stable transfection of communication-deficient Neuro2A cells. The connexin-56-transfected cells demonstrated intercellular coupling by transfer of microinjected 6-carboxyfluorescein. Double whole-cell patch clamp recordings demonstrated electrical coupling. The induced intercellular conductances were insensitive to uncoupling by heptanol, octanol, or acidification. This behavior of chick connexin-56 may explain previous observations of the unusual physiology of lens fiber gap junctions.
Rup
D M
DM
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
Veenstra
R D
RD
Wang
H Z
HZ
Brink
P R
PR
Beyer
E C
EC
D000595:Amino Acid Sequence; D000818:Animals; D016375:Antisense Elements (Genetics); D001483:Base Sequence; D015139:Blotting, Southern; D002645:Chickens; D003001:Cloning, Molecular; D017630:Connexins; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004553:Electric Conductivity; D005136:Eye Proteins / Q000235:genetics* / Q000502:physiology; D007365:Intercellular Junctions / Q000502:physiology*; D007473:Ion Channels / Q000502:physiology*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D016375:Antisense Elements (Genetics); D017630:Connexins; D005136:Eye Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; C078866:connexin 56; D012313:RNA; D004247:DNA