A mutant connexin50 with enhanced hemichannel function leads to cell death.
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.
Minogue
Peter J
PJ
Department of Pediatrics, Section of Hematology/Oncology, University of Chicago, Chicago, Illinois 60637, USA.
Tong
Jun-Jie
JJ
Arora
Anita
A
Russell-Eggitt
Isabelle
I
Hunt
David M
DM
Moore
Anthony T
AT
Ebihara
Lisa
L
Beyer
Eric C
EC
Berthoud
Viviana M
VM
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
D017630:Connexins; D004441:Ecdysterone; D005136:Eye Proteins; D000099264:Connexin 50; C008999:ponasterone A
Am J Physiol. 1999 Jun;276(6):C1443-6
10362609
Biophys J. 1995 May;68(5):1796-803
7612821
Anal Biochem. 1976 May 7;72:248-54
942051
Invest Ophthalmol Vis Sci. 1999 Jul;40(8):1844-50
10393059
J Cell Biol. 1991 Nov;115(4):1077-89
1659572
Biophys J. 2002 Apr;82(4):2016-31
11916859
Mol Biol Cell. 1992 Jul;3(7):711-20
1325220
Development. 2002 Jan;129(1):167-74
11782410
Genes Dev. 1988 Sep;2(9):1168-78
3192078
Am J Physiol Cell Physiol. 2007 Jul;293(1):C337-45
17428836
Methods Enzymol. 1992;207:376-80
1382193
J Gen Physiol. 1993 Jul;102(1):59-74
7690837
Biophys J. 2004 Oct;87(4):2397-406
15454438
J Biol Chem. 2005 Dec 9;280(49):40788-95
16204255
Invest Ophthalmol Vis Sci. 1994 Sep;35(10):3747-58
8088962
J Med Genet. 2006 Jan;43(1):e2
16397066
Antioxid Redox Signal. 2009 Feb;11(2):339-53
18831679
Pflugers Arch. 2000 Jul;440(3):366-79
10954323
Ciba Found Symp. 1984;106:132-52
6096095
J Immunol Methods. 1992 Aug 30;153(1-2):57-65
1517601
J Gen Physiol. 2006 Jan;127(1):67-75
16380444
J Membr Biol. 1990 Jun;116(2):163-75
2166164
J Biol Chem. 2009 Feb 13;284(7):4484-93
19074140
Invest Ophthalmol Vis Sci. 2007 Dec;48(12):5630-7
18055813
Laryngoscope. 2006 Dec;116(12):2205-10
17146396
Semin Cell Dev Biol. 2008 Apr;19(2):134-49
18035564
Hum Mutat. 2009 May;30(5):E603-11
19306328
Science. 1987 Dec 11;238(4833):1563-5
3685993
J Med Genet. 2008 Mar;45(3):155-60
18006672
Eur J Cell Biol. 2003 May;82(5):209-21
12800976
Surv Ophthalmol. 2004 May-Jun;49(3):300-15
15110667
Am J Physiol Cell Physiol. 2000 Sep;279(3):C596-602
10942709
J Cell Sci. 2007 Dec 1;120(Pt 23):4107-16
18003700
Biophys J. 1999 Jan;76(1 Pt 1):198-206
9876134
J Cell Biol. 1989 Feb;108(2):595-605
2537319
Invest Ophthalmol Vis Sci. 2008 Jun;49(6):2549-56
18326694
Semin Cell Biol. 1992 Feb;3(1):49-58
1320431
J Gen Physiol. 1999 May;113(5):721-42
10228184
Science. 2002 Jan 11;295(5553):319-20
11786642