Oculodentodigital dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells

Albert Lai, Dung Nghi Le, William A. Paznekas, Wes D. Gifford, Ethylin Wang Jabs, Andrew C. Charles

Research output: Contribution to journalArticle

Abstract

Oculodentodigital dysplasia (ODDD) is a rare developmental disorder characterized by craniofacial and limb abnormalities. Over 35 separate mutations in human connexin43 (Cx43) causing ODDD have been identified. Several mutations are also associated with central nervous system involvement, including white-matter changes detected by magnetic resonance imaging. As Cx43 is abundantly expressed in astrocytes, we hypothesized that the mutant Cx43 proteins that produce neurological dysfunction have abnormal functional characteristics in astrocytes. To understand how ODDD-associated mutations affect Cx43 signaling in cells of glial origin, we conducted studies in rat C6 glioma cells, a communication-deficient glial cell line that expresses low levels of Cx43. We generated stable cell lines expressing enhanced yellow fluorescent protein (eYFP)-tagged human Cx43 constructs encoding wild-type and six eYFP-tagged mutant Cx43 mutants: Y17S, G21R, A40V, F52dup, L90V and I130T. Of these, Y17S, L90V and I130T are associated with neurological abnormalities. We found that all mutants could be detected on the cell surface. Y17S, G21R, A40V, L90V and I130T formed triton-resistant plaques representing gap junctions, although the relative ability to form plaques was decreased in these mutants compared with the wild type. F52dup, formed dramatically reduced numbers of plaques. Propidium iodide uptake experiments demonstrated that all mutants were associated with reduced connexin hemichannel function compared with wild type. Scrape-loading experiments performed on the same stable cell lines showed reduced gap junctional dye transfer in all mutants compared with the wild type. These studies demonstrated that ODDD-associated Cx43 mutations result in non-functional connexin hemichannels and gap junction functions in a glial cell line regardless of whether the particular mutant is associated with neurological dysfunction.

Original languageEnglish (US)
Pages (from-to)532-541
Number of pages10
JournalJournal of Cell Science
Volume119
Issue number3
DOIs
StatePublished - Feb 1 2006

Fingerprint

Connexin 43
Connexins
Gap Junctions
Glioma
Mutation
Neuroglia
Cell Line
Mutant Proteins
Astrocytes
Craniofacial Abnormalities
Oculodentodigital Dysplasia
Propidium
Cell Communication
Coloring Agents
Extremities
Central Nervous System
Magnetic Resonance Imaging

Keywords

  • Connexin43
  • Gap junction
  • Glia
  • Hemichannel
  • Oculodentodigital dysplasia (ODDD)

ASJC Scopus subject areas

  • Cell Biology

Cite this

Oculodentodigital dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells. / Lai, Albert; Le, Dung Nghi; Paznekas, William A.; Gifford, Wes D.; Jabs, Ethylin Wang; Charles, Andrew C.

In: Journal of Cell Science, Vol. 119, No. 3, 01.02.2006, p. 532-541.

Research output: Contribution to journalArticle

Lai, Albert ; Le, Dung Nghi ; Paznekas, William A. ; Gifford, Wes D. ; Jabs, Ethylin Wang ; Charles, Andrew C. / Oculodentodigital dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells. In: Journal of Cell Science. 2006 ; Vol. 119, No. 3. pp. 532-541.
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T1 - Oculodentodigital dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells

AU - Lai, Albert

AU - Le, Dung Nghi

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AU - Gifford, Wes D.

AU - Jabs, Ethylin Wang

AU - Charles, Andrew C.

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AB - Oculodentodigital dysplasia (ODDD) is a rare developmental disorder characterized by craniofacial and limb abnormalities. Over 35 separate mutations in human connexin43 (Cx43) causing ODDD have been identified. Several mutations are also associated with central nervous system involvement, including white-matter changes detected by magnetic resonance imaging. As Cx43 is abundantly expressed in astrocytes, we hypothesized that the mutant Cx43 proteins that produce neurological dysfunction have abnormal functional characteristics in astrocytes. To understand how ODDD-associated mutations affect Cx43 signaling in cells of glial origin, we conducted studies in rat C6 glioma cells, a communication-deficient glial cell line that expresses low levels of Cx43. We generated stable cell lines expressing enhanced yellow fluorescent protein (eYFP)-tagged human Cx43 constructs encoding wild-type and six eYFP-tagged mutant Cx43 mutants: Y17S, G21R, A40V, F52dup, L90V and I130T. Of these, Y17S, L90V and I130T are associated with neurological abnormalities. We found that all mutants could be detected on the cell surface. Y17S, G21R, A40V, L90V and I130T formed triton-resistant plaques representing gap junctions, although the relative ability to form plaques was decreased in these mutants compared with the wild type. F52dup, formed dramatically reduced numbers of plaques. Propidium iodide uptake experiments demonstrated that all mutants were associated with reduced connexin hemichannel function compared with wild type. Scrape-loading experiments performed on the same stable cell lines showed reduced gap junctional dye transfer in all mutants compared with the wild type. These studies demonstrated that ODDD-associated Cx43 mutations result in non-functional connexin hemichannels and gap junction functions in a glial cell line regardless of whether the particular mutant is associated with neurological dysfunction.

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