The Gap Junction: A Site of Cell to Cell Communication

1. The literature relating to the structure and function of gap junctions has been reviewed. Particular attention has been paid to papers providing evidence for the identity of the junctional proteins and to the limited information on the molecular structure of the junctional channels as this structure determines junctional permeability, which will in turn determine the possible functions of gap junctions. 2. SDS PAGE analysis of gap junction enriched fractions isolated from mouse liver shows only one major protein component, apparent Mr 16000 (16K). Application of Hopp and Woods (1981) alogrithm to the known sequence of the 16K protein (amino acids 2-28) showed that the N-terminal region was the most hydrophilic. 3. The N-terminal amino acid of the 16K protein was shown by chemical analysis to be asparagine. 4. A nonapeptide was synthesized with the sequence corresponding to the N-terminal octapeptide of the 16K protein (Asn-Pro-Glu-Tyr--Ser-Ser-Phe-Phe) and a cysteine in position 9.5. The nonapeptide was coupled through the cysteine to keyhole limpet haemacyanin and the conjugate used to raise polyclonal, site specific antisera in four rabbits. 6. An immunoglobulin (IgG) fraction was separated from the site specific antiserum. The antibodies which bound to the peptide were affinity purified. 7. The site specific antisera produced were characterized using four different techniques. All four sera had the same reactivities. (i) immunoblotting of SDS PAGE separated protein from mouse liver gap junctional preparations and of electroeluted 16K protein, which showed the antibodies to the peptide recognize an antigenic site on the 16K protein. (ii) microimmunodiffusion, based on the Ouchterlony technique, of SDS solubilized protein from mouse liver gap junction preparations and electroeluted 16K protein. (iii) agglutination of isolated gap junctions (the agglutination reaction was completely inhibited by the free peptide at a concentration of 5 mM), which showed that the antigenic site is accessible in intact junctions. (iv) ultrastructural localization using Protein-A gold and isolated mouse liver gap junctions, which showed the antigenic site is present on morphologically identifiable gap junctions. 8. Gap junctions were isolated from three different mouse tissues (brain, heart, and kidney). Analysis of these preparations by SDS PAGE showed the presence in each of one major Coomassie staining band, Mr 16000. The junctional proteins isolated from these different tissues were shown by the microimmunodiffusion assay to react with the site specific antiserum. 9. Gap junctions were isolated from tissues of different species (chicken liver, Xenopus liver, and Nephrops norvegicus hepatopancreas). The chicken and Xenopus preparations showed that one major Coomassie staining band, Mr 16000 when analysed by SDS PAGE. The preparations from Nephrops showed one major band, Mr 18000. Microimmunodiffusion analyses showed that the site specific antiserum recognized the gap junctional proteins isolated from all three species. The isolated gap junctions from Nephrops hepatopancreas also showed agglutination when incubated with the site specific antiserum. 10. The structural organization of the 16K protein within the membrane was examined. It was shown that the N-terminus of the 16K protein (and the cross-reactive site of the 18K protein) was located on the cytoplasmic face of gap junctions. Treatment of intact junctions with pronase showed the presence of a further loop (or loops) of the 16K and 18K proteins on the cytoplasmic face of both the mouse liver and Nephrops hepatopancreas junctions. The protein fragments remaining after pronase treatment of intact junctions were sufficient to maintain the characteristic morphology of the gap junction. 11. Peptide mapping studies have been used in an attempt to determine which peptides are cytoplasmically located and more specifically to determine the orientation of the N-terminal peptide. 12. In collaboration with Dr. E. Kam, the site specific antiserum has been iontophoretically injected into Buffalo rat liver cells and shown to block cell to cell transfer of the fluorescent dye Lucifer Yellow. 13. The significance of the observations made in the course of this work are discussed and related to other published work on the protein constituents of gap junctions