J Membrane Biol (2012) 245:333–344 DOI 10.1007/s00232-012-9454-2 Connexin Composition in Apposed Gap Junction Hemiplaques Revealed by Matched Double-Replica Freeze-Fracture Replica Immunogold Labeling John E. Rash • Naomi Kamasawa • Kimberly G. V. Davidson • Thomas Yasumura • Alberto E. Pereda • James I. Nagy Received: 9 April 2012 / Accepted: 8 June 2012 / Published online: 4 July 2012 The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identi- fying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing mem- branes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging tech- niques. Currently, only serial thin-section transmission elec- tron microscopy and freeze-fracture replica immunogold labeling have sufﬁcient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited J. E. Rash (&) K. G. V. Davidson T. Yasumura Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA e-mail: john.rash@colostate.edu J. E. Rash Program in Molecular, Cellular, and Integrative Neurosciences, Colorado State University, Fort Collins, CO 80523, USA N. Kamasawa Electron Microscopy Core Facility, Max Planck Florida Institute, Jupiter, FL 33458, USA A. E. Pereda Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA J. I. Nagy Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, MB R3E 0J9, Canada because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difﬁcult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identiﬁcation of connexins and determination of the membrane ‘‘sidedness’’ and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons. Keywords Astrocyte Oligodendrocyte Ependymocyte Glia Neuron Abbreviations DR Double replica E-face Extraplasmic leaﬂet FRIL Freeze-fracture replica immunogold labeling P-face Protoplasmic leaﬂe RT-PCR Reverse transcriptase polymerase chain reaction Introduction and Historical Perspective Vertebrate gap junctions consist of connexin proteins that assemble as hexamers to form connexon ‘‘hemichannels’’ that link across the extracellular space, forming leakless channels that permit the direct intercellular transport of water, ions and small molecules [B450 Da (Hu and Dahl 1999)]. Of the 20 or 21 connexins expressed in mammals— named according to their molecular weight, measured in kilodaltons (Willecke et al. 2002)—more than half are 123