The topological structure of connexin 26 and its distribution compared to connexin 32 in hepatic gap junctions

Jian-Ting Zhang, B. J. Nicholson

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Of the gap junction proteins characterized to date, Cx26 is unique in that it is usually expressed in conjunction with other members of the family, typically Cx32 (liver [Nicholson et al., Nature329:732-734, 1987], pancreas, kidney, and stomach [J.-T. Zhang, B.J. Nicholson, J. Cell Biol.109:3391-3410, 1989]), or Cx43 (leptomeninges [D.C. Spray et al., Brain Res.568:1-14, 1991] and pineal gland [J.C. Sáez et al., Brain Res.568:265-275,1991]). We have used specific antisera both to investigate the distribution of Cx32 and Cx26 in isolated liver gap junctions, and empirically establish the topological model of Cx26 suggested by its sequence and analogy to other connexins. Antipeptide antisera were prepared to four of the five hydrophilic domains which flank the four putative transmembrane spanning regions of Cx26. Antibodies to N-terminal residues 1-17 (αCx26-N), to residues 101-119 in the putative cytoplasmic loop (αCx26-CL), and to C-terminal residues 210-226 (αCx26-C) were all specific for Cx26. An antibody to residues 166-185 between hydrophobic domains 3 and 4 of Cx32 had affinity for both Cx26 and Cx32 (αCx32/26-E2). The antigenic sites Cx26-N, -CL and -C were each demonstrated to be cytoplasmically disposed, although the latter was conformationally hidden prior to partial proteolysis. The antigenic site for αCx32/26-E2 was only accessible after exposure of the extracellular face by separation of the junctional membranes in 8 m urea, pH 12.3. This treatment also served to reveal the region between residues 45 and 66 to Asp-N protease. The topology thus demonstrated for Cx26 is consistent with that deduced for other connexins (i.e., Cx32 and Cx43). Comparison of immunogold decorated gap junctions reacted with antibodies specific to Cx26 (αCx26-N and -CL), or to Cx32 [αCx32-CL], indicates that these connexins do not aggregate in subdomains within a junction, at least within the resolution provided by the labeling density (one antibody per 15-22 connexons). Although the presence of both connexins within a single channel could not be distinguished, possible interactions between channels is discussed.

Original languageEnglish (US)
Pages (from-to)15-29
Number of pages15
JournalJournal of Membrane Biology
Volume139
Issue number1
DOIs
StatePublished - Apr 1994
Externally publishedYes

Fingerprint

Connexins
Gap Junctions
Connexin 43
Antibodies
Liver
Immune Sera
Pineal Gland
Brain
Proteolysis
Urea
Pancreas
Stomach
Kidney
Membranes
Connexin 26
connexin 32

Keywords

  • Antibodies
  • Channels
  • Folding
  • Membrane
  • Protein

ASJC Scopus subject areas

  • Physiology
  • Cell Biology
  • Biophysics

Cite this

The topological structure of connexin 26 and its distribution compared to connexin 32 in hepatic gap junctions. / Zhang, Jian-Ting; Nicholson, B. J.

In: Journal of Membrane Biology, Vol. 139, No. 1, 04.1994, p. 15-29.

Research output: Contribution to journalArticle

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abstract = "Of the gap junction proteins characterized to date, Cx26 is unique in that it is usually expressed in conjunction with other members of the family, typically Cx32 (liver [Nicholson et al., Nature329:732-734, 1987], pancreas, kidney, and stomach [J.-T. Zhang, B.J. Nicholson, J. Cell Biol.109:3391-3410, 1989]), or Cx43 (leptomeninges [D.C. Spray et al., Brain Res.568:1-14, 1991] and pineal gland [J.C. S{\'a}ez et al., Brain Res.568:265-275,1991]). We have used specific antisera both to investigate the distribution of Cx32 and Cx26 in isolated liver gap junctions, and empirically establish the topological model of Cx26 suggested by its sequence and analogy to other connexins. Antipeptide antisera were prepared to four of the five hydrophilic domains which flank the four putative transmembrane spanning regions of Cx26. Antibodies to N-terminal residues 1-17 (αCx26-N), to residues 101-119 in the putative cytoplasmic loop (αCx26-CL), and to C-terminal residues 210-226 (αCx26-C) were all specific for Cx26. An antibody to residues 166-185 between hydrophobic domains 3 and 4 of Cx32 had affinity for both Cx26 and Cx32 (αCx32/26-E2). The antigenic sites Cx26-N, -CL and -C were each demonstrated to be cytoplasmically disposed, although the latter was conformationally hidden prior to partial proteolysis. The antigenic site for αCx32/26-E2 was only accessible after exposure of the extracellular face by separation of the junctional membranes in 8 m urea, pH 12.3. This treatment also served to reveal the region between residues 45 and 66 to Asp-N protease. The topology thus demonstrated for Cx26 is consistent with that deduced for other connexins (i.e., Cx32 and Cx43). Comparison of immunogold decorated gap junctions reacted with antibodies specific to Cx26 (αCx26-N and -CL), or to Cx32 [αCx32-CL], indicates that these connexins do not aggregate in subdomains within a junction, at least within the resolution provided by the labeling density (one antibody per 15-22 connexons). Although the presence of both connexins within a single channel could not be distinguished, possible interactions between channels is discussed.",
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