title: A mutant connexin50 with enhanced hemichannel function leads to cell death.
abstract: OBJECTIVE
  To determine the consequences of expression of a novel connexin50 (CX50) mutant identified in a child with congenital total cataracts.
  
  
  METHODS
  The GJA8 gene was directly sequenced. Formation of functional channels was assessed by the two-microelectrode voltage-clamp
  
  
  METHODS
  Connexin protein levels and distribution were assessed by immunoblot analysis and immunofluorescence. The proportion of apoptotic cells was determined by flow cytometry.
  
  
  RESULTS
  Direct sequencing of the GJA8 gene identified a 137 G>T transition that resulted in the replacement of glycine by valine at position 46 of the coding region of CX50 (CX50G46V). Both CX50 and CX50G46V induced gap junctional currents in pairs of Xenopus oocytes. In single Xenopus oocytes, CX50G46V induced connexin hemichannel currents that were activated by removal of external calcium; their magnitudes were much higher than those in oocytes injected with similar amounts of CX50 cRNA. When expressed in HeLa cells under the control of an inducible promoter, both CX50 and CX50G46V formed gap junctional plaques. Induction of CX50G46V expression led to a decrease in the number of cells and an increase in the proportion of apoptotic cells. CX50G46V-induced cell death was prevented by high concentrations of extracellular calcium ions.
  
  
  CONCLUSIONS
  Unlike previously characterized CX50 mutants that exhibit impaired trafficking and/or lack of function, CX50G46V traffics properly to the plasma membrane and forms functional hemichannels and gap junction channels; however, it causes cell death even when expressed at minute levels. The biochemical results indirectly suggest a potential novel mechanism by which connexin mutants could lead to cataracts: cytotoxicity due to enhanced hemichannel function.
authors.affiliation: Department of Pediatrics, Section of Hematology/Oncology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Minogue
authors.forename: Jun-Jie
authors.initials: JJ
authors.lastname: Tong
authors.forename: Anita
authors.initials: A
authors.lastname: Arora
authors.forename: Isabelle
authors.initials: I
authors.lastname: Russell-Eggitt
authors.forename: David M
authors.initials: DM
authors.lastname: Hunt
authors.forename: Anthony T
authors.initials: AT
authors.lastname: Moore
authors.forename: Lisa
authors.initials: L
authors.lastname: Ebihara
authors.forename: Eric C
authors.initials: EC
authors.lastname: Beyer
authors.forename: Viviana M
authors.initials: VM
authors.lastname: Berthoud
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002386:Cataract / Q000151:congenital / Q000235:genetics* / Q000473:pathology; D018929:Cell Culture Techniques; D002675:Child, Preschool; D017630:Connexins / Q000235:genetics*; D004441:Ecdysterone / Q000031:analogs & derivatives / Q000494:pharmacology; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D017629:Gap Junctions / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014162:Transfection; D014982:Xenopus laevis; D000099264:Connexin 50
chemical_list: D017630:Connexins; D004441:Ecdysterone; D005136:Eye Proteins; D000099264:Connexin 50; C008999:ponasterone A
references.citation: Am J Physiol. 1999 Jun;276(6):C1443-6
references.pmid: 10362609
references.citation: Biophys J. 1995 May;68(5):1796-803
references.pmid: 7612821
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Invest Ophthalmol Vis Sci. 1999 Jul;40(8):1844-50
references.pmid: 10393059
references.citation: J Cell Biol. 1991 Nov;115(4):1077-89
references.pmid: 1659572
references.citation: Biophys J. 2002 Apr;82(4):2016-31
references.pmid: 11916859
references.citation: Mol Biol Cell. 1992 Jul;3(7):711-20
references.pmid: 1325220
references.citation: Development. 2002 Jan;129(1):167-74
references.pmid: 11782410
references.citation: Genes Dev. 1988 Sep;2(9):1168-78
references.pmid: 3192078
references.citation: Am J Physiol Cell Physiol. 2007 Jul;293(1):C337-45
references.pmid: 17428836
references.citation: Methods Enzymol. 1992;207:376-80
references.pmid: 1382193
references.citation: J Gen Physiol. 1993 Jul;102(1):59-74
references.pmid: 7690837
references.citation: Biophys J. 2004 Oct;87(4):2397-406
references.pmid: 15454438
references.citation: J Biol Chem. 2005 Dec 9;280(49):40788-95
references.pmid: 16204255
references.citation: Invest Ophthalmol Vis Sci. 1994 Sep;35(10):3747-58
references.pmid: 8088962
references.citation: J Med Genet. 2006 Jan;43(1):e2
references.pmid: 16397066
references.citation: Antioxid Redox Signal. 2009 Feb;11(2):339-53
references.pmid: 18831679
references.citation: Pflugers Arch. 2000 Jul;440(3):366-79
references.pmid: 10954323
references.citation: Ciba Found Symp. 1984;106:132-52
references.pmid: 6096095
references.citation: J Immunol Methods. 1992 Aug 30;153(1-2):57-65
references.pmid: 1517601
references.citation: J Gen Physiol. 2006 Jan;127(1):67-75
references.pmid: 16380444
references.citation: J Membr Biol. 1990 Jun;116(2):163-75
references.pmid: 2166164
references.citation: J Biol Chem. 2009 Feb 13;284(7):4484-93
references.pmid: 19074140
references.citation: Invest Ophthalmol Vis Sci. 2007 Dec;48(12):5630-7
references.pmid: 18055813
references.citation: Laryngoscope. 2006 Dec;116(12):2205-10
references.pmid: 17146396
references.citation: Semin Cell Dev Biol. 2008 Apr;19(2):134-49
references.pmid: 18035564
references.citation: Hum Mutat. 2009 May;30(5):E603-11
references.pmid: 19306328
references.citation: Science. 1987 Dec 11;238(4833):1563-5
references.pmid: 3685993
references.citation: J Med Genet. 2008 Mar;45(3):155-60
references.pmid: 18006672
references.citation: Eur J Cell Biol. 2003 May;82(5):209-21
references.pmid: 12800976
references.citation: Surv Ophthalmol. 2004 May-Jun;49(3):300-15
references.pmid: 15110667
references.citation: Am J Physiol Cell Physiol. 2000 Sep;279(3):C596-602
references.pmid: 10942709
references.citation: J Cell Sci. 2007 Dec 1;120(Pt 23):4107-16
references.pmid: 18003700
references.citation: Biophys J. 1999 Jan;76(1 Pt 1):198-206
references.pmid: 9876134
references.citation: J Cell Biol. 1989 Feb;108(2):595-605
references.pmid: 2537319
references.citation: Invest Ophthalmol Vis Sci. 2008 Jun;49(6):2549-56
references.pmid: 18326694
references.citation: Semin Cell Biol. 1992 Feb;3(1):49-58
references.pmid: 1320431
references.citation: J Gen Physiol. 1999 May;113(5):721-42
references.pmid: 10228184
references.citation: Science. 2002 Jan 11;295(5553):319-20
references.pmid: 11786642