Pannexin1 and Pannexin2 Channels Show Quaternary Similarities to Connexons and Different Oligomerization Numbers from Each Other |
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Authors: | Cinzia Ambrosi Oliver Gassmann Jennifer N Pranskevich Daniela Boassa Amy Smock Junjie Wang Gerhard Dahl Claudia Steinem Gina E Sosinsky |
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Institution: | From the ‡National Center for Microscopy and Imaging Research and ;the ‖Department of Neurosciences, University of California San Diego, La Jolla, California 92093-0608.;the §Institute for Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, 37077 Göttingen, Germany, and ;the ¶Department of Physiology and Biophysics, University of Miami, School of Medicine, Miami, Florida 33136 |
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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. |
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Keywords: | Connexin Electron Microscopy (EM) Gap Junctions Membrane Proteins Membrane Structure Intercellular Communication Pannexon |
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