The highly conserved Gln49 and Ser50 of mammalian connexin43 are present in chick connexin43 and essential for functional gap junction channels

Cell Commun Adhes. Mar-Apr 2002;9(2):75-86. doi: 10.1080/15419060214149.

Abstract

In an attempt to compare the regulation of chick connexin43 channels to those of mammalian connexin43, we found that the nucleotide sequence reported for chick connexin43 differs from that of the chick connexin gene by two codons that had been entered as histidine49 (H49) and valine50 (V50) (accession no. M29003), but are in fact glutamine49 (Q49) and serine50 (S50). Neuro2A cells were transfected with corrected wild-type (Q49/S50) chick connexin43 (accession no. AF233738), the double-replacement Q49H/S50V connexin43, or the single replacement of Q49H or S50V. All clones had gap junctions in membrane based on immunocytochemistry and immunoblots of the triton-resistant membrane fraction. Wild-type transfectants had three conductance states with a predominant channel conductance of 85 +/- 5 pS. Cells producing the Q49H-Cx43 or the double-replacement Q49H/S50V-Cx43 protein had no detectable connexin43 channels. In contrast, cells expressing S50V-Cx43 gap junctions had channels with reduced conductances (75 +/- 8 pS) compared to wild-type controls. Low or high pH of the bathing solution had no effect on the Q49H-Cx43 channels. We conclude that glutamine49 is important for channel function, and replacement of this residue with histidine most likely distorts secondary structure of the first extracellular loop, possibly by changing the orientation of conserved cysteines, and this inhibits channel function. The S50V substitution may also cause similar but less severe structural changes.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence / genetics
  • Animals
  • Base Sequence / genetics
  • Cell Communication / physiology*
  • Chick Embryo
  • Connexin 43 / genetics
  • Connexin 43 / metabolism*
  • Eukaryotic Cells / metabolism*
  • Gap Junctions / genetics
  • Gap Junctions / metabolism*
  • Glutamine / genetics
  • Glutamine / metabolism*
  • Histidine / genetics
  • Histidine / metabolism
  • Hydrogen-Ion Concentration
  • Immunohistochemistry
  • Membrane Potentials / genetics
  • Mice
  • Molecular Sequence Data
  • Mutation / genetics
  • Protein Structure, Tertiary / genetics
  • Sequence Homology, Amino Acid
  • Serine / genetics
  • Serine / metabolism*
  • Transfection
  • Tumor Cells, Cultured

Substances

  • Connexin 43
  • Glutamine
  • Serine
  • Histidine

Associated data

  • GENBANK/AF233738
  • GENBANK/M29003