Isolation and characterization of gap junctions from tissue culture cells.
The purification of membrane proteins in a form and amount suitable for structural or biochemical studies still remains a great challenge. Gap junctions have long been studied using electron microscopy and X-ray diffraction. However, only a limited number of proteins in the connexin family have been amenable to protein or membrane purification techniques. Molecular biology techniques for expressing large gap junctions in tissue culture cells combined with improvements in electron crystallography have shown great promise for determining the channel structure to better than 10 A resolution. Here, we have isolated two-dimensional (2D) gap junction crystals from HeLa Cx26 transfectants. This isoform has never been isolated in large fractions from tissues. We characterize these preparations by SDS-PAGE, Western blotting, negative stain electron microscopy and atomic force microscopy. In our preparations, the Cx26 is easily detected in the Western blots and we have increased expression levels so that connexin bands are visible on SDS-PAGE gels. Preliminary assessment of the samples by electron cryo-microscopy shows that these 2D crystals diffract to at least 22 A. Atomic force microscopy of these Cx26 gap junctions show exquisite surface modulation at the extracellular surface in force dissected gap junctions. We also applied our protocol to cell lines such as NRK cells that express endogenous Cx43 and NRK and HeLa cell lines transfected with exogenous connexins. While the gap junction membrane channels are recognizable in negatively stained electron micrographs, these lattices are disordered and the gap junction plaques are smaller. SDS-PAGE and Western blotting revealed expression of connexins, but at a lower level than with our HeLa Cx26 transfectants. Therefore, the purity and morphology of the gap junction plaques depends the size and abundance of the gap junctions in the cell line itself.
Hand
Galen M
GM
National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, CA, USA.
Müller
Daniel J
DJ
Nicholson
Bruce J
BJ
Engel
Andreas
A
Sosinsky
Gina E
GE
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D000072259:Connexin 26; D018031:Connexin 43 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D017630:Connexins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure*; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D017629:Gap Junctions / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection
D018031:Connexin 43; D017630:Connexins; C000607042:GJB2 protein, human; C000607044:Gjb2 protein, rat; D008164:Luminescent Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D000072259:Connexin 26; D049452:Green Fluorescent Proteins