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. Crosslinking 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. Pannexins (Panxs),2 connexins (Cxs), and innexins belong to one superfamily (1). Panxs (Pan in ancient Greek means "whole") were given this name because of their presumptive ubiquitous presence in multicellular animals (2). Although the term "pannexin" can encompass invertebrate forms, the mammalian branch commonly known as pannexins is formed by three members: Panx1, Panx2, and Panx3. In rat, Panx1 contains 426 amino acids (ϳ48 kDa), Panx2 has 674 amino acids (ϳ70 kDa), and Panx3 has 392 amino acids (ϳ44.7 kDa). There is very high conservation of amino acid sequence within individual Panx isoforms among mammalian species compared with between Panx1, Panx2, and Panx3. Panx1 is ubiquitously expressed, whereas Panx2 has expression only in the central nervous system. Panx3 has a different pattern of expression from Panx1 and Panx2. Panx3 is found in mouse skin, osteoblasts, and specialized cartilage (3, 4).Several studies have addressed the function of pannexins in tissue, particularly with respect to the role it plays in purinergic receptor signaling. As part of this pathway, Panx1 may release large signaling molecules like ATP and arachidonic derivatives and as a result may be involved in astrocytic Ca 2ϩ wave propagation via P 2 X 7 receptors (5, 6). Panx1 has also been implicated in ischemic, excitotoxic, an...

Connexin26 (Cx26) is a member of the connexin family, the building blocks for gap junction intercellular channels. These dodecameric assemblies are involved in gap junction-mediated cellcell communication allowing the passage of ions and small molecules between two neighboring cells. Mutations in Cx26 lead to the disruption of gap junction-mediated intercellular communication with consequences such as hearing loss and skin disorders. We show here that a mutant of Cx26, M34A, forms an active hemichannel in lipid bilayer experiments. A comparison with the Cx26 wild-type is presented. Two different techniques using micro/nano-structured substrates for the formation of poresuspending lipid membranes are used. We reconstituted the Cx26 wild-type and Cx26M34A into artificial lipid bilayers and observed single channel activity for each technique, with conductance levels of around 35, 70 and 165 pS for the wildtype. The conductance levels of Cx26M34A were found at around 45 and 70 pS.