TY - JOUR
T1 - Mouse lens connexin23 (Gje1) does not form functional gap junction channels but causes enhanced ATP release from HeLa cells
AU - Sonntag, Stephan
AU - Söhl, Goran
AU - Dobrowolski, Radoslaw
AU - Zhang, Jiong
AU - Theis, Martin
AU - Winterhager, Elke
AU - Bukauskas, Feliksas F.
AU - Willecke, Klaus
N1 - Funding Information:
We would like to thank Prof. Dr. Antonia Joussen and Dr. Norbert Kociok from the Center of Ophthalmology, University of Cologne, for providing tissue biopsies from a human eye. We further thank Joana Fischer and Petra Kußmann for excellent technical assistance. We also thank Alexandra Gellhaus, PhD, Institute of Molecular Biology, and Melanie Friederichs, PhD, ZMB-Developmental Biology, University of Duisburg-Essen, for help with in situ hybridizations. This work was supported by a grant of the German Research Association (Wi 270/22-5,6) and SFB 645, Projekt B2, to K. Willecke and by NIH Grants R01NS036706 and RO1HL084464 to F.F. Bukauskas.
PY - 2009/2
Y1 - 2009/2
N2 - In the mouse genome, 20 connexin genes have been detected that code for proteins of high sequence identity in the two extracellular loops, especially six conserved cysteine residues. The mouse connexin23 (Cx23) gene (Gje1) differs from all other connexin genes in vertebrates, since it codes for a protein that contains only 4 instead of 6 cysteine residues in the extracellular loops. Recently, two zebrafish connexin genes (Cx23a and Cx23b) have been identified, and a mouse mutant in the Gje1 gene has been described that exhibits a developmental defect in the lens. Here, we have compared the Cx23 gene in different mammalian species and found no transcripts in cDNA libraries of primates. Furthermore, all primate genomes analyzed contain stop codons in the Cx23 sequence, indicating inactivation of the orthologous primate GJE1 gene. No Cx23 mRNA was found in human eye. In order to analyze the properties of mouse Cx23 channels, we isolated HeLa cell clones stably expressing wild-type mCx23 or mCx23 fused to eGFP. Cells expressing Cx23-eGFP demonstrated its insertion in the plasma membrane but no punctate staining in contacting membranes characteristic for junctional plaques. In addition, we tested whether Cx23 forms functional gap junction channels electrophysiologically in cell pairs as well as by microinjection of neurobiotin and found that mouse Cx23 did not form gap junction channels in HeLa cells. However, there was a significant release of ATP from different Cx23 HeLa cell clones, even in the presence of normal culture medium with high calcium ion concentration, suggesting a hemichannel-based function of Cx23. Therefore, Cx23 seems to share functional properties with pannexin (hemi) channels rather than gap junction channels of other connexins.
AB - In the mouse genome, 20 connexin genes have been detected that code for proteins of high sequence identity in the two extracellular loops, especially six conserved cysteine residues. The mouse connexin23 (Cx23) gene (Gje1) differs from all other connexin genes in vertebrates, since it codes for a protein that contains only 4 instead of 6 cysteine residues in the extracellular loops. Recently, two zebrafish connexin genes (Cx23a and Cx23b) have been identified, and a mouse mutant in the Gje1 gene has been described that exhibits a developmental defect in the lens. Here, we have compared the Cx23 gene in different mammalian species and found no transcripts in cDNA libraries of primates. Furthermore, all primate genomes analyzed contain stop codons in the Cx23 sequence, indicating inactivation of the orthologous primate GJE1 gene. No Cx23 mRNA was found in human eye. In order to analyze the properties of mouse Cx23 channels, we isolated HeLa cell clones stably expressing wild-type mCx23 or mCx23 fused to eGFP. Cells expressing Cx23-eGFP demonstrated its insertion in the plasma membrane but no punctate staining in contacting membranes characteristic for junctional plaques. In addition, we tested whether Cx23 forms functional gap junction channels electrophysiologically in cell pairs as well as by microinjection of neurobiotin and found that mouse Cx23 did not form gap junction channels in HeLa cells. However, there was a significant release of ATP from different Cx23 HeLa cell clones, even in the presence of normal culture medium with high calcium ion concentration, suggesting a hemichannel-based function of Cx23. Therefore, Cx23 seems to share functional properties with pannexin (hemi) channels rather than gap junction channels of other connexins.
KW - Cx23
KW - Hemichannels
KW - Pannexins
KW - Pseudogene
UR - http://www.scopus.com/inward/record.url?scp=57649110620&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=57649110620&partnerID=8YFLogxK
U2 - 10.1016/j.ejcb.2008.08.004
DO - 10.1016/j.ejcb.2008.08.004
M3 - Article
C2 - 18849090
AN - SCOPUS:57649110620
SN - 0171-9335
VL - 88
SP - 65
EP - 77
JO - European Journal of Cell Biology
JF - European Journal of Cell Biology
IS - 2
ER -