Isolation and Purification of Gap Junction Channels

• Published in: The Journal of cell biology 1991-10, Vol.115 (1), p.141-150
• Main Authors: Stauffer, Kathrin A., Kumar, Nalin M., Gilula, Norton B., Unwin, Nigel
• Format: Article
• Language: English
• Subjects: Animals; Baculoviridae; Biological and medical sciences; Cell Fractionation - methods; Cell physiology; Cells; Complementary DNA; Connexins; Detergents; Fundamental and applied biological sciences. Psychology; Gap junctions; Gels; Intercellular Junctions - ultrastructure; Liver; Macromolecular Substances; Membrane Proteins - chemistry; Membrane Proteins - isolation & purification; Membrane Proteins - ultrastructure; Microscopy, Electron; Molecular and cellular biology; Protein Binding; Rats; Rats, Inbred Strains; Recombinant Proteins; Solubility; Solubilization
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.115.1.141

This paper reports methods we have developed to solubilize gap junction channels, or connexons, from isolated gap junctions and to purify them in milligram quantities. Two sources of material are used: rat liver gap junctions and gap junctions produced by infecting insect cells with a baculovirus containing the cDNA for human liver β 1 protein (connexin 32). Complete solubilization is obtained with long chain detergents (lauryl dimethyl amineoxide, dodecyl maltoside) and requires high ionic strength and high pH as well as reducing conditions. The purification involves chromatography on hydroxylapatite and gel filtration on Superose 6. A homogeneous product is indicated by a single band on a silver-stained gel and a homogeneous population of doughnut-shaped particles under the electron microscope. These particles have hexameric symmetry. The purified connexons have a tendency to form aggregates: filaments and sheets. The filaments grow by end-to-end association of connexons and are nonpolar, suggesting that the connexons are paired as in the cell-to-cell channel. The sheets grow by lateral association of the filaments.