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The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains

Yancey, S. Barbara and John, Scott A. and Lal, Ratneshwar and Austin, Brian J. and Revel, Jean-Paul (1989) The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains. Journal of Cell Biology, 89 (6). pp. 2241-2254. ISSN 0021-9525. http://resolver.caltech.edu/CaltechAUTHORS:YANjcb89

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Abstract

Analysis by SDS-PAGE of gap junction fractions isolated from heart suggests that the junctions are comprised of a protein with an Mr 43,000. Antibodies against the electroeluted protein and a peptide representing the 20 amino terminal residues bind specifically on immunoblots to the 43-kD protein and to the major products arising from proteolysis during isolation. By immunocytochemistry, the protein is found in ventricle and atrium in patterns consistent with the known distribution of gap junctions. Both antibodies bind exclusively to gap junctions in fractions from heart examined by EM after gold labeling. Since only domains of the protein exposed at the cytoplasmic surface should be accessible to antibody, we conclude that the 43-kD protein is assembled in gap junctions with the amino terminus of the molecule exposed on the cytoplasmic side of the bilayer, that is, on the same side as the carboxy terminus as determined previously. By combining proteolysis experiments with data from immunoblotting, we can identify a third cytoplasmic region, a loop of some 4 kD between membrane protected domains. This loop carries an antibody binding site. The protein, if transmembrane, is therefore likely to cross the membrane four times. We have used the same antisera to ascertain if the 43-kD protein is involved in cell-cell communication. The antiserum against the amino terminus blocked dye coupling in 90% of cell pairs tested; the antiserum recognizing epitopes in the cytoplasmic loop and cytoplasmic tail blocked coupling in 75% of cell pairs tested. Preimmune serum and control antibodies (one against MIP and another binding to a cardiac G protein) had no or little effect on dye transfer. Our experimental evidence thus indicates that, in spite of the differences in amino acid sequence, the gap junction proteins in heart and liver share a general organizational plan and that there may be several domains (including the amino terminus) of the molecule that are involved in the control of junctional permeability.


Item Type:Article
Additional Information:Copyright © 1989 by The Rockefeller University Press Received for publication 6 October 1988 and in revised form 8 February 1989. The authors want to express their gratitude to Dr. D. Laird and J. Hoh for their critical review of the manuscript. We, and Dr. Yancey in particular, want to express appreciation to Dr. R. Gomer (formerly at the University of California, San Diego, and now at Rice University) for his help and advice in the production of the anti-43-kD antiserum. We also appreciate the support provided by Dr. B. Nicholson in preparing some of the antipeptide antibodies and thank Dr. T. Amatruda for providing anti-G protein antibody. A. Battaglia and J. Backstrom gave us excellent technical assistance. This research was supported by National Institutes of Health grant HL 37109 and the Markey Foundation. S. John's work on Topology of the Heart Junction Protein was supported by a Proctor and Gamble Fellowship, and R. Lars Functional Studies were made possible by contributions from the Albert Billings Ruddock Fund. Portions of this work have appeared in abstract form (John et al., 1988. Proc. Int. Congr. Cell Biol.; Lal et al. 1988. Soc. Neurosci. Abstr. 14:145; Yancey et al., 1988. J. Cell Biol. 107:555a. [Abstr.]).
Record Number:CaltechAUTHORS:YANjcb89
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:YANjcb89
Alternative URL:http://www.jcb.org/cgi/content/abstract/108/6/2241
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Deposited On:22 Sep 2006
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