The Journal of Membrane Biology

, Volume 245, Issue 8, pp 483–493 | Cite as

Pannexin 1 Ohnologs in the Teleost Lineage

  • Stephen R. BondEmail author
  • Nan Wang
  • Luc Leybaert
  • Christian C. NausEmail author


Advances in genomic analysis indicate that the early chordate lineage underwent two whole-genome duplication events in fairly rapid succession around 400–600 million years ago, and that a third duplication event punctuated the radiation of ray-finned fishes (teleosts) around 320–350 million years ago. Connexin ohnologs have been disproportionately well maintained in the teleost genome following this third event, implying that gap junction proteins are amenable to neofunctionalization. A second family of gap junction–like proteins, the pannexins, is also present in chordates, but expansion of this family following the teleost whole-genome duplication has not been addressed in the literature. In the current study we report that ohnologs of panx1 are expressed by zebrafish, and orthologs of these two genes can be found in various other teleost species. The genomic locality of each gene is described, along with sequence alignments that reveal conservation of classic pannexin-specific features/motifs. The transcripts were then cloned from cDNA for in vitro analysis, and both are shown to traffic to the plasma membrane when exogenously expressed. Furthermore, electrophysiological recordings show differences in the biophysical properties between the channels formed by these two proteins. Our results indicate that both copies of the ancestral teleost panx1 gene were conserved following the last whole-genome duplication event and, following conventional zebrafish nomenclature, should now be referred to as panx1a and panx1b.


Pannexin Teleost R3 whole-genome duplication Ohnolog Neofunctionalization 



We thank Dr. Patricia Schulte for her kind assistance with obtaining zebrafish tissues and for critical review of the manuscript.

Supplementary material

Supplementary material 1 Panx1a 3D rotation (MPG 476 kb)

Supplementary material 2 Panx1a time-lapse (MPG 1214 kb)

Supplementary material 3 Panx1b 3D rotation (MPG 352 kb)

Supplementary material 4 Panx1b time-lapse (MPG 1120 kb)

Supplementary material 5 Panx2 3D rotation (MPG 724 kb)

Supplementary material 6 Panx2 time-lapse (MPG 76 kb)

Supplementary material 7 Panx3 3D rotation (MPG 322 kb)

Supplementary material 8 Panx3 time-lapse (MPG 874 kb)

232_2012_9497_MOESM9_ESM.pdf (407 kb)
Supplementary material 9 Fig. S1 Multiple pairwise alignment of Panx1 sequences. Classic pannexin/innexin-specific residues are identified, including four cysteine residues in the extracellular loops (C), a P-X-X-X-W motif in the second transmembrane domain and a positively charged residue thought to facilitate ATP gating of the channel (⊕). A cysteine residue in Panx1a (*) has also been reported to facilitate channel activity in zebrafish but is not present in any other sequence included in this study. Predicted transmembrane domains are denoted by filled boxes (PDF 406 kb)
232_2012_9497_MOESM10_ESM.pdf (39 kb)
Fig. S2 Immunoblot confirming exogenous expression of EGFP-tagged zebrafish pannexins in HeLa cells, using an anti-GFP antibody (PDF 40 kb)
232_2012_9497_MOESM11_ESM.pdf (58 kb)
Supplementary material 11 (PDF 58 kb)


  1. Bao L, Locovei S, Dahl G (2004) Pannexin membrane channels are mechanosensitive conduits for ATP. FEBS Lett 572:65–68Google Scholar
  2. Baranova A, Ivanov D, Petrash N, Pestova A, Skoblov M, Kelmanson I, Shagin D, Nazarenko S, Geraymovych E, Litvin O, Tiunova A, Born TL, Usman N, Staroverov D, Lukyanov S, Panchin Y (2004) The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins. Genomics 83:706–716PubMedCrossRefGoogle Scholar
  3. Bhalla-Gehi R, Penuela S, Churko JM, Shao Q, Laird DW (2010) Pannexin1 and pannexin3 delivery, cell surface dynamics, and cytoskeletal interactions. J Biol Chem 285:9147–9160PubMedCrossRefGoogle Scholar
  4. Boassa D, Ambrosi C, Qiu F, Dahl G, Gaietta G, Sosinsky G (2007) Pannexin1 channels contain a glycosylation site that targets the hexamer to the plasma membrane. J Biol Chem 282:31733–31743PubMedCrossRefGoogle Scholar
  5. Bond SR, Naus CC (2012) an online tool for the design of restriction-free cloning projects. Nucleic Acids Res 40:W209–W213PubMedCrossRefGoogle Scholar
  6. Bruzzone R, Hormuzdi SG, Barbe MT, Herb A, Monyer H (2003) Pannexins, a family of gap junction proteins expressed in brain. Proc Natl Acad Sci USA 100:13644–13649PubMedCrossRefGoogle Scholar
  7. Bryksin AV, Matsumura I (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques 48:463–465PubMedCrossRefGoogle Scholar
  8. Bukauskas FF, Jordan K, Bukauskiene A, Bennett MV, Lampe PD, Laird DW, Verselis VK (2000) Clustering of connexin 43-enhanced green fluorescent protein gap junction channels and functional coupling in living cells. Proc Natl Acad Sci USA 97:2556–2561PubMedCrossRefGoogle Scholar
  9. Bukauskas FF, Bukauskiene A, Bennett MV, Verselis VK (2001) Gating properties of gap junction channels assembled from connexin43 and connexin43 fused with green fluorescent protein. Biophys J 81:137–152PubMedCrossRefGoogle Scholar
  10. Bunse S, Schmidt M, Hoffmann S, Engelhardt K, Zoidl G, Dermietzel R (2011) Single cysteines in the extracellular and transmembrane regions modulate pannexin 1 channel function. J Membr Biol 244:21–33PubMedCrossRefGoogle Scholar
  11. Carnarius C, Kreir M, Krick M, Methfessel C, Moehrle V, Valerius O, Bruggemann A, Steinem C, Fertig N (2012) Green fluorescent protein changes the conductance of connexin 43 (Cx43) hemichannels reconstituted in planar lipid bilayers. J Biol Chem 287:2877–2886PubMedCrossRefGoogle Scholar
  12. Catchen JM, Conery JS, Postlethwait JH (2009) Automated identification of conserved synteny after whole-genome duplication. Genome Res 19:1497–1505PubMedCrossRefGoogle Scholar
  13. Celetti SJ, Cowan KN, Penuela S, Shao Q, Churko J, Laird DW (2010) Implications of pannexin 1 and pannexin 3 for keratinocyte differentiation. J Cell Sci 123:1363–1372PubMedCrossRefGoogle Scholar
  14. Cifuentes M, Grandont L, Moore G, Chevre AM, Jenczewski E (2010) Genetic regulation of meiosis in polyploid species: new insights into an old question. New Phytol 186:29–36PubMedCrossRefGoogle Scholar
  15. Clair C, Combettes L, Pierre F, Sansonetti P, Tran Van Nhieu G (2008) Extracellular-loop peptide antibodies reveal a predominant hemichannel organization of connexins in polarized intestinal cells. Exp Cell Res 314:1250–1265PubMedCrossRefGoogle Scholar
  16. Dehal P, Boore JL (2005) Two rounds of whole genome duplication in the ancestral vertebrate. PLoS Biol 3:e314PubMedCrossRefGoogle Scholar
  17. Drummond AJ, A.B., Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A 2009. Geneious v4.8.
  18. Dvoriantchikova G, Ivanov D, Panchin Y, Shestopalov VI (2006) Expression of pannexin family of proteins in the retina. FEBS Lett 580:2178–2182PubMedCrossRefGoogle Scholar
  19. Elfgang C, Eckert R, Lichtenberg-Frate H, Butterweck A, Traub O, Klein RA, Hulser DF, Willecke K (1995) Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J Cell Biol 129:805–817PubMedCrossRefGoogle Scholar
  20. Felsenstein J (1985) Confidence limits on phylogenies—an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  21. Force A, Lynch M, Pickett FB, Amores A, Yan YL, Postlethwait J (1999) Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151:1531–1545PubMedGoogle Scholar
  22. Fushiki D, Hamada Y, Yoshimura R, Endo Y (2010) Phylogenetic and bioinformatic analysis of gap junction-related proteins, innexins, pannexins and connexins. Biomed Res 31:133–142PubMedCrossRefGoogle Scholar
  23. Geumann U, Barysch SV, Hoopmann P, Jahn R, Rizzoli SO (2008) SNARE function is not involved in early endosome docking. Mol Biol Cell 19:5327–5337PubMedCrossRefGoogle Scholar
  24. Giepmans BN (2004) Gap junctions and connexin-interacting proteins. Cardiovasc Res 62:233–245PubMedCrossRefGoogle Scholar
  25. Hahn MW (2009) Distinguishing among evolutionary models for the maintenance of gene duplicates. J Hered 100:605–617PubMedCrossRefGoogle Scholar
  26. Hedges SB, Dudley J, Kumar S (2006) TimeTree: a public knowledge-base of divergence times among organisms. Bioinformatics 22:2971–2972PubMedCrossRefGoogle Scholar
  27. Iwamoto T, Nakamura T, Doyle A, Ishikawa M, de Vega S, Fukumoto S, Yamada Y (2010) Pannexin 3 regulates intracellular ATP/cAMP levels and promotes chondrocyte differentiation. J Biol Chem 285:18948–18958PubMedCrossRefGoogle Scholar
  28. Jaillon O, Aury JM, Brunet F, Petit JL, Stange-Thomann N, Mauceli E, Bouneau L, Fischer C, Ozouf-Costaz C, Bernot A, Nicaud S, Jaffe D, Fisher S, Lutfalla G, Dossat C, Segurens B, Dasilva C, Salanoubat M, Levy M, Boudet N, Castellano S, Anthouard V, Jubin C, Castelli V, Katinka M, Vacherie B, Biemont C, Skalli Z, Cattolico L, Poulain J, De Berardinis V, Cruaud C, Duprat S, Brottier P, Coutanceau JP, Gouzy J, Parra G, Lardier G, Chapple C, McKernan KJ, McEwan P, Bosak S, Kellis M, Volff JN, Guigo R, Zody MC, Mesirov J, Lindblad-Toh K, Birren B, Nusbaum C, Kahn D, Robinson-Rechavi M, Laudet V, Schachter V, Quetier F, Saurin W, Scarpelli C, Wincker P, Lander ES, Weissenbach J, Roest Crollius H (2004) Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431:946–957PubMedCrossRefGoogle Scholar
  29. Kassahn KS, Dang VT, Wilkins SJ, Perkins AC, Ragan MA (2009) Evolution of gene function and regulatory control after whole-genome duplication: comparative analyses in vertebrates. Genome Res 19:1404–1418PubMedCrossRefGoogle Scholar
  30. Lai CP, Bechberger JF, Thompson RJ, Macvicar BA, Bruzzone R, Naus CC (2007) Tumor-suppressive effects of pannexin 1 in C6 glioma cells. Cancer Res 67:1545–1554PubMedCrossRefGoogle Scholar
  31. Lai CP, Bechberger JF, Naus CC (2009) Pannexin2 as a novel growth regulator in C6 glioma cells. Oncogene 28:4402–4408PubMedCrossRefGoogle Scholar
  32. Leitch IJ, Bennett MD (2004) Genome downsizing in polyploid plants. Biol J Linn Soc 82:651–663CrossRefGoogle Scholar
  33. Lynch M, O’Hely M, Walsh B, Force A (2001) The probability of preservation of a newly arisen gene duplicate. Genetics 159:1789–1804PubMedGoogle Scholar
  34. Ma W, Hui H, Pelegrin P, Surprenant A (2009) Pharmacological characterization of pannexin-1 currents expressed in mammalian cells. J Pharmacol Exp Ther 328:409–418PubMedCrossRefGoogle Scholar
  35. Mayo C, Ren R, Rich C, Stepp MA, Trinkaus-Randall V (2008) Regulation by P2X7: epithelial migration and stromal organization in the cornea. Invest Ophthalmol Vis Sci 49:4384–4391PubMedCrossRefGoogle Scholar
  36. Panchin YV (2005) Evolution of gap junction proteins—the pannexin alternative. J Exp Biol 208:1415–1419PubMedCrossRefGoogle Scholar
  37. Panchin Y, Kelmanson I, Matz M, Lukyanov K, Usman N, Lukyanov S (2000) A ubiquitous family of putative gap junction molecules. Curr Biol 10:R473–R474PubMedCrossRefGoogle Scholar
  38. Penuela S, Celetti SJ, Bhalla R, Shao Q, Laird DW (2008) Diverse subcellular distribution profiles of pannexin 1 and pannexin 3. Cell Commun Adhes 15:133–142PubMedCrossRefGoogle Scholar
  39. Penuela, S., Gehi, R., Laird, D.W. 2012. The biochemistry and function of pannexin channels. Biochim Biophys Acta (in press)Google Scholar
  40. Phelan P (2005) Innexins: members of an evolutionarily conserved family of gap-junction proteins. Biochim Biophys Acta 1711:225–245PubMedCrossRefGoogle Scholar
  41. Prochnow N, Hoffmann S, Dermietzel R, Zoidl G (2009a) Replacement of a single cysteine in the fourth transmembrane region of zebrafish pannexin 1 alters hemichannel gating behavior. Exp Brain Res 199:255–264PubMedCrossRefGoogle Scholar
  42. Prochnow N, Hoffmann S, Vroman R, Klooster J, Bunse S, Kamermans M, Dermietzel R, Zoidl G (2009b) Pannexin1 in the outer retina of the zebrafish, Danio rerio. Neuroscience 162:1039–1054PubMedCrossRefGoogle Scholar
  43. Putnam NH, Butts T, Ferrier DE, Furlong RF, Hellsten U, Kawashima T, Robinson-Rechavi M, Shoguchi E, Terry A, Yu JK, Benito-Gutierrez EL, Dubchak I, Garcia-Fernandez J, Gibson-Brown JJ, Grigoriev IV, Horton AC, de Jong PJ, Jurka J, Kapitonov VV, Kohara Y, Kuroki Y, Lindquist E, Lucas S, Osoegawa K, Pennacchio LA, Salamov AA, Satou Y, Sauka-Spengler T, Schmutz J, Shin IT, Toyoda A, Bronner-Fraser M, Fujiyama A, Holland LZ, Holland PW, Satoh N, Rokhsar DS (2008) The amphioxus genome and the evolution of the chordate karyotype. Nature 453:1064–1071PubMedCrossRefGoogle Scholar
  44. Qiu F, Dahl GP (2009) A permeant regulating its permeation pore: inhibition of pannexin 1 channels by ATP. Am J Physiol Cell Physiol 296:C250–C255PubMedCrossRefGoogle Scholar
  45. Roth C, Rastogi S, Arvestad L, Dittmar K, Light S, Ekman D, Liberles DA (2007) Evolution after gene duplication: models, mechanisms, sequences, systems, and organisms. J Exp Zool B Mol Dev Evol 308:58–73PubMedCrossRefGoogle Scholar
  46. Semple C, Wolfe KH (1999) Gene duplication and gene conversion in the Caenorhabditis elegans genome. J Mol Evol 48:555–564PubMedCrossRefGoogle Scholar
  47. Sharp LL, Zhou J, Blair DF (1995) Features of MotA proton channel structure revealed by tryptophan-scanning mutagenesis. Proc Natl Acad Sci USA 92:7946–7950PubMedCrossRefGoogle Scholar
  48. Shestopalov VI, Panchin Y (2008) Pannexins and gap junction protein diversity. Cell Mol Life Sci 65:376–394PubMedCrossRefGoogle Scholar
  49. Sosinsky GE, Boassa D, Dermietzel R, Duffy HS, Laird DW, Macvicar B, Naus CC, Penuela S, Scemes E, Spray DC, Thompson RJ, Zhao HB, Dahl G (2011) Pannexin channels are not gap junction hemichannels. Channels (Austin) 5:193–197CrossRefGoogle Scholar
  50. Swayne LA, Sorbara CD, Bennett SA (2010) Pannexin 2 is expressed by postnatal hippocampal neural progenitors and modulates neuronal commitment. J Biol Chem 285:24977–24986PubMedCrossRefGoogle Scholar
  51. Wagner A (2002) Asymmetric functional divergence of duplicate genes in yeast. Mol Biol Evol 19:1760–1768PubMedCrossRefGoogle Scholar
  52. Willecke K, Eiberger J, Degen J, Eckardt D, Romualdi A, Guldenagel M, Deutsch U, Sohl G (2002) Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem 383:725–737PubMedCrossRefGoogle Scholar
  53. Yen MR, Saier MH Jr (2007) Gap junctional proteins of animals: the innexin/pannexin superfamily. Prog Biophys Mol Biol 94:5–14PubMedCrossRefGoogle Scholar
  54. Zappala A, Cicero D, Serapide MF, Paz C, Catania MV, Falchi M, Parenti R, Panto MR, La Delia F, Cicirata F (2006) Expression of pannexin1 in the CNS of adult mouse: cellular localization and effect of 4-aminopyridine-induced seizures. Neuroscience 141:167–178PubMedCrossRefGoogle Scholar
  55. Zappala A, Li Volti G, Serapide MF, Pellitteri R, Falchi M, La Delia F, Cicirata V, Cicirata F (2007) Expression of pannexin2 protein in healthy and ischemized brain of adult rats. Neuroscience 148:653–667PubMedCrossRefGoogle Scholar
  56. Zoidl G, Petrasch-Parwez E, Ray A, Meier C, Bunse S, Habbes HW, Dahl G, Dermietzel R (2007) Localization of the pannexin1 protein at postsynaptic sites in the cerebral cortex and hippocampus. Neuroscience 146:9–16PubMedCrossRefGoogle Scholar
  57. Zoidl G, Kremer M, Zoidl C, Bunse S, Dermietzel R (2008) Molecular diversity of connexin and pannexin genes in the retina of the zebrafish Danio rerio. Cell Commun Adhes 15:169–183PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Cellular and Physiological Science, Life Sciences InstituteUniversity of British ColumbiaVancouverCanada
  2. 2.Department of Basic Medical Sciences–Physiology Group, Faculty of Medicine and Health SciencesGhent UniversityGhentBelgium

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