Pannexin1 and pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other

Cinzia Ambrosi, Oliver Gassmann, Jennifer N. Pranskevich, Daniela Boassa, Amy Smock, Junjie Wang, Gerhard Dahl, Claudia Steinem, Gina E. Sosinsky

Research output: Contribution to journalArticle

108 Citations (Scopus)

Abstract

Pannexins are homologous to innexins, the invertebrate gap junction family. However, mammalian pannexin1 does not form canonical gap junctions, instead forming hexameric oligomers in single plasma membranes and intracellularly. Pannexin1 acts as an ATP release channel, whereas less is known about the function of Pannexin2. We purified cellular membranes isolated from MDCK cells stably expressing rat Pannexin1 or Pannexin2 and identified pannexin channels (pannexons) in single membranes by negative stain and immunogold labeling. Protein gel and Western blot analysis confirmed Pannexin1 (Panx1) or Pannexin2 (Panx2) as the channel-forming proteins. We expressed and purified Panx1 and Panx2 using a baculovirus Sf9 expression system and obtained doughnut-like structures similar to those seen previously in purified connexin hemichannels (connexons) and mammalian membranes. Purified pannexons were comparable in size and overall appearance to Connexin46 and Connexin50 connexons. Pannexons and connexons were further analyzed by single-particle averaging for oligomer and pore diameters. The oligomer diameter increased with increasing monomer molecular mass, and we found that the measured oligomeric pore diameter for Panxs was larger than for Connexin26. Panx1 and Panx2 formed active homomeric channels in Xenopus oocytes and in vitro vesicle assays. Cross-linking and native gels of purified homomeric full-length and a C-terminal Panx2 truncation mutant showed a banding pattern more consistent with an octamer. We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other.

Original languageEnglish
Pages (from-to)24420-24431
Number of pages12
JournalJournal of Biological Chemistry
Volume285
Issue number32
DOIs
StatePublished - Aug 6 2010

Fingerprint

Oligomerization
Oligomers
Gap Junctions
Membranes
Monomers
Gels
Madin Darby Canine Kidney Cells
Connexins
Baculoviridae
Molecular mass
Invertebrates
Cell membranes
Xenopus
Labeling
Oocytes
Rats
Assays
Proteins
Coloring Agents
Adenosine Triphosphate

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Ambrosi, C., Gassmann, O., Pranskevich, J. N., Boassa, D., Smock, A., Wang, J., ... Sosinsky, G. E. (2010). Pannexin1 and pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other. Journal of Biological Chemistry, 285(32), 24420-24431. https://doi.org/10.1074/jbc.M110.115444

Pannexin1 and pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other. / Ambrosi, Cinzia; Gassmann, Oliver; Pranskevich, Jennifer N.; Boassa, Daniela; Smock, Amy; Wang, Junjie; Dahl, Gerhard; Steinem, Claudia; Sosinsky, Gina E.

In: Journal of Biological Chemistry, Vol. 285, No. 32, 06.08.2010, p. 24420-24431.

Research output: Contribution to journalArticle

Ambrosi, C, Gassmann, O, Pranskevich, JN, Boassa, D, Smock, A, Wang, J, Dahl, G, Steinem, C & Sosinsky, GE 2010, 'Pannexin1 and pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other', Journal of Biological Chemistry, vol. 285, no. 32, pp. 24420-24431. https://doi.org/10.1074/jbc.M110.115444
Ambrosi, Cinzia ; Gassmann, Oliver ; Pranskevich, Jennifer N. ; Boassa, Daniela ; Smock, Amy ; Wang, Junjie ; Dahl, Gerhard ; Steinem, Claudia ; Sosinsky, Gina E. / Pannexin1 and pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 32. pp. 24420-24431.
@article{834eb81287b24028b40e6af3bcb92966,
title = "Pannexin1 and pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other",
abstract = "Pannexins are homologous to innexins, the invertebrate gap junction family. However, mammalian pannexin1 does not form canonical gap junctions, instead forming hexameric oligomers in single plasma membranes and intracellularly. Pannexin1 acts as an ATP release channel, whereas less is known about the function of Pannexin2. We purified cellular membranes isolated from MDCK cells stably expressing rat Pannexin1 or Pannexin2 and identified pannexin channels (pannexons) in single membranes by negative stain and immunogold labeling. Protein gel and Western blot analysis confirmed Pannexin1 (Panx1) or Pannexin2 (Panx2) as the channel-forming proteins. We expressed and purified Panx1 and Panx2 using a baculovirus Sf9 expression system and obtained doughnut-like structures similar to those seen previously in purified connexin hemichannels (connexons) and mammalian membranes. Purified pannexons were comparable in size and overall appearance to Connexin46 and Connexin50 connexons. Pannexons and connexons were further analyzed by single-particle averaging for oligomer and pore diameters. The oligomer diameter increased with increasing monomer molecular mass, and we found that the measured oligomeric pore diameter for Panxs was larger than for Connexin26. Panx1 and Panx2 formed active homomeric channels in Xenopus oocytes and in vitro vesicle assays. Cross-linking and native gels of purified homomeric full-length and a C-terminal Panx2 truncation mutant showed a banding pattern more consistent with an octamer. We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other.",
author = "Cinzia Ambrosi and Oliver Gassmann and Pranskevich, {Jennifer N.} and Daniela Boassa and Amy Smock and Junjie Wang and Gerhard Dahl and Claudia Steinem and Sosinsky, {Gina E.}",
year = "2010",
month = "8",
day = "6",
doi = "10.1074/jbc.M110.115444",
language = "English",
volume = "285",
pages = "24420--24431",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "32",

}

TY - JOUR

T1 - Pannexin1 and pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other

AU - Ambrosi, Cinzia

AU - Gassmann, Oliver

AU - Pranskevich, Jennifer N.

AU - Boassa, Daniela

AU - Smock, Amy

AU - Wang, Junjie

AU - Dahl, Gerhard

AU - Steinem, Claudia

AU - Sosinsky, Gina E.

PY - 2010/8/6

Y1 - 2010/8/6

N2 - Pannexins are homologous to innexins, the invertebrate gap junction family. However, mammalian pannexin1 does not form canonical gap junctions, instead forming hexameric oligomers in single plasma membranes and intracellularly. Pannexin1 acts as an ATP release channel, whereas less is known about the function of Pannexin2. We purified cellular membranes isolated from MDCK cells stably expressing rat Pannexin1 or Pannexin2 and identified pannexin channels (pannexons) in single membranes by negative stain and immunogold labeling. Protein gel and Western blot analysis confirmed Pannexin1 (Panx1) or Pannexin2 (Panx2) as the channel-forming proteins. We expressed and purified Panx1 and Panx2 using a baculovirus Sf9 expression system and obtained doughnut-like structures similar to those seen previously in purified connexin hemichannels (connexons) and mammalian membranes. Purified pannexons were comparable in size and overall appearance to Connexin46 and Connexin50 connexons. Pannexons and connexons were further analyzed by single-particle averaging for oligomer and pore diameters. The oligomer diameter increased with increasing monomer molecular mass, and we found that the measured oligomeric pore diameter for Panxs was larger than for Connexin26. Panx1 and Panx2 formed active homomeric channels in Xenopus oocytes and in vitro vesicle assays. Cross-linking and native gels of purified homomeric full-length and a C-terminal Panx2 truncation mutant showed a banding pattern more consistent with an octamer. We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other.

AB - Pannexins are homologous to innexins, the invertebrate gap junction family. However, mammalian pannexin1 does not form canonical gap junctions, instead forming hexameric oligomers in single plasma membranes and intracellularly. Pannexin1 acts as an ATP release channel, whereas less is known about the function of Pannexin2. We purified cellular membranes isolated from MDCK cells stably expressing rat Pannexin1 or Pannexin2 and identified pannexin channels (pannexons) in single membranes by negative stain and immunogold labeling. Protein gel and Western blot analysis confirmed Pannexin1 (Panx1) or Pannexin2 (Panx2) as the channel-forming proteins. We expressed and purified Panx1 and Panx2 using a baculovirus Sf9 expression system and obtained doughnut-like structures similar to those seen previously in purified connexin hemichannels (connexons) and mammalian membranes. Purified pannexons were comparable in size and overall appearance to Connexin46 and Connexin50 connexons. Pannexons and connexons were further analyzed by single-particle averaging for oligomer and pore diameters. The oligomer diameter increased with increasing monomer molecular mass, and we found that the measured oligomeric pore diameter for Panxs was larger than for Connexin26. Panx1 and Panx2 formed active homomeric channels in Xenopus oocytes and in vitro vesicle assays. Cross-linking and native gels of purified homomeric full-length and a C-terminal Panx2 truncation mutant showed a banding pattern more consistent with an octamer. We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other.

UR - http://www.scopus.com/inward/record.url?scp=77955293473&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77955293473&partnerID=8YFLogxK

U2 - 10.1074/jbc.M110.115444

DO - 10.1074/jbc.M110.115444

M3 - Article

C2 - 20516070

AN - SCOPUS:77955293473

VL - 285

SP - 24420

EP - 24431

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 32

ER -