Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein.
Gap junctions allow direct intercellular coupling between many cells including those in the blood vessel wall. They are formed by a group of related proteins called connexins, containing conserved transmembrane and extracellular domains, but unique cytoplasmic regions that may confer connexin-specific physiological properties. We used polymerase chain reaction amplification and cDNA library screening to clone DNA encoding a human gap junction protein, connexin37 (Cx37). The derived human Cx37 polypeptide contains 333 amino acids, with a predicted molecular mass of 37,238 D. RNA blots demonstrate that Cx37 is expressed in multiple organs and tissues (including heart, uterus, ovary, and blood vessel endothelium) and in primary cultures of vascular endothelial cells. Cx37 mRNA is coexpressed with connexin43 at similar levels in some endothelial cells, but at much lower levels in others. To demonstrate that Cx37 could form functional channels, we stably transfected communication-deficient Neuro2A cells with the Cx37 cDNA. The induced intercellular channels were studied by the double whole cell patch clamp technique. These channels were reversibly inhibited by the uncoupling agent, heptanol (2 mM). The expressed Cx37 channels exhibited multiple conductance levels and showed a pronounced voltage dependence. These electrophysiological characteristics are similar to, but distinct from, those of previously characterized connexins.
Reed
K E
KE
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
Westphale
E M
EM
Larson
D M
DM
Wang
H Z
HZ
Veenstra
R D
RD
Beyer
E C
EC
D000438:Alcohols / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D017630:Connexins; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004553:Electric Conductivity; D004730:Endothelium, Vascular / Q000502:physiology*; D015723:Gene Library; D019850:Heptanol; D006801:Humans; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured; D000097006:Gap Junction alpha-4 Protein
D000438:Alcohols; D017630:Connexins; D004247:DNA; D019850:Heptanol; D007473:Ion Channels; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000097006:Gap Junction alpha-4 Protein