Abstract
We have used site-directed antibodies against various segments of the connexin43 (Cx43) gap-junction protein in an attempt to explore the role of different portions of this molecule in regulating junctional permeability. The antibodies used in the present study were raised against epitopes exposed at the cytoplasmic face of the junctions, specifically the amino (AT-2) and carboxy (CT-360) termini and the cytoplasmic loop (CL-100) of Cx43. Neonatal rat cardiac myocytes, which are known to express Cx43, were microinjected with a series of anti-Cx43 antibodies, followed by Lucifer yellow. The extent of cell coupling was quantified as the percentage of instances of intercellular transfer of the dye. The effectiveness of the AT-2 and CT-360 antibodies varied strongly and differentially with the external calcium concentration. In the absence of antibody, the dye permeability was unaffected by calcium. In medium containing physiological concentrations of calcium, the antibodies inhibited dye transfer to different degrees: AT-2 and CT-360 antibodies inhibited well while the CL-100 antibody had very little effect on dye permeability. Our results indicate that several highly conserved cytoplasmic domains of Cx43 could be involved in regulating junctional permeability, and that calcium modulates the effect of antibodies on junctional permeability.
Similar content being viewed by others
References
Bennett MVL, Spray DC (1987) Intercellular communication mediated by gap junction can be controlled in many ways. In: Edelman G, Gall E, Cowan WM (eds) Synaptic function. Wiley, New York, pp 109–135
Burt J (1987) Block of intercellular communications: interactions of intercellular H+ and Ca2+. Am J Physiol 253:C607- C612
Chen L, Goings GE, Upshaw-Earley J, Page E (1989) Cardiac gap junctions and gap junction-associated vesicles: ultrastructural comparison of in situ negative staining with conventional positive staining. Circ Res 64:501–514
Christensen O (1987) Mediation of cell volume regulation by Ca2+ influx through stretch-activated channels. Nature 330: 66–68
Endo M (1985) Calcium release from sarcoplasmic reticulum. Curr Top Membr Transp 25:181–230
Fabiato A, Fabiato F (1979) Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 75:463–505
Fraser SE, Green CR, Bode H, Gilula NB (1987) Selective disruption of gap junctional communication interferes with a patterning process in Hydra. Science 273:49–55
Green FA, Costello PB (1987) Divalent cation effects on the binding of human anti-phospholipid antibodies. Biochim Biophys Acta 896:47–51
Hertzberg EL, Spray DC, Bennett MVL (1985) Reduction of gap junctional conductance by microinjection of antibodies against the 27-kDa liver gap junction polypeptide. Proc Natl Acad Sci USA 82:2412–2416
Hoh JH, John SA, Revel J-P (1991) Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31. J Biol Chem 266:6524–6531
Jongen WMF, Fitzgerald DJ, Asamoto M, Piccoli C, Slaga TJ, Gros D, Takeichi M, Yamasaki H (1991) Regulation of connexin43-mediated gap junctional intercellular communication by calcium in mouse epidermal cells is controlled by E-cadherin. J Cell Biol 114:545–555
Kanter HL, Saffitz JE, Beyer EC (1992) Cardiac myocytes express multiple gap junction proteins. Circ Res 70:438–444
Laird DW, Revel J-P (1990) Biochemical and immunochemical analysis of the arrangement of connexin43 in rat heart gap junction membranes. J Cell Sci 97:109–117
Laird DW, Puranam KL, Revel J-P (1991) Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes. Biochem J 273:67–72
Loewenstein WR (1985) Regulation of cell-to-cell communication by phosphorylation. Biochem Soc Symp Lond 50:43–58
Luttrell BM, Henniker AJ (1991) Reaction coupling of chelation and antigen binding in the calcium ion-dependent antibody binding of cyclic AMP. J Biol Chem 266:21626–21630
Matis WL, Anhalt GJ, Diaz LA, Rivitti EA, Martins CR, Berger RS (1987) Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies. J Invest Dermatol 89:302–304
Maurer P, Weingart R (1987) Cell pairs isolated from adult guinea pig and rat hearts: effects of [Ca]i on nexal membrane resistance. Pflügers Arch 409:384–402
Milks LC, Kumar NM, Houghten R, Unwin N, Gilula NB (1988) Topology of the 32-kD liver gap junction protein determined by site-directed antibody localizations. EMBO J 7:2967–2975
Morgan J, Lal R, Arnsdorf M, Cohen L (1990) Evidence that cell-cell uncoupling occurs during the asynchronous contractile behavior in the tissue culture model of calcium induced ventricular fibrillation. Clinical Res 38:987A
Musil LS, Beyer EC, Goodenough DA (1990) Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization and post-translational phosphorylation. J Membr Biol 116:163–175
Noma A, Tsuboi N (1987) Dependence of junctional conductance on proton, calcium and magnesium ions in cardiac paired cells of guinea pig. J Physiol (Lond) 382:193–211
Paschke D, Eckert R, Hulser DF (1992) High-resolution measurements of gap junctional conductance during perfusion with anti-connexin antibodies in pairs of cultured mammalian cells. Pflügers Arch 420:87–93
Peracchia C (1990) Increase in gap junction resistance with acidification in crayfish septate axons is closely related to changes in intracellular calcium but not hydrogen ion concentration. J Membr Biol 113:75–92
Randriamapita C, Giaume D, Neyton J, Trautmann A (1988) Acetylcholine-induced closure of gap junction channels in rat lacrimal glands is probably mediated by protein kinase C. Pflügers Arch 412:462–468
Revel JP, Hoh JH, John SA, Laird DW, Puranam K, Yancey SB (1992) Aspects of gap junction structure and assembly. Semin Cell Biol 3:21–28
Rose B, Simpson I, Loewenstein WR (1977) Calcium ion produces graded changes in permeability of membrane channels in cell junction. Nature 267:625–627
Saez JC, Connor JA, Spray DC, Bennett MVL (1989) Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-triphosphate, and to calcium ions. Proc Natl Acad Sci USA 86:2708–2712
Saez JC, Nairn AC, Czernik AJ, Spray DC, Hertzberg EL, Greengard P, Bennett MVL (1990) Phosphorylation of connexin32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. Eur J Biochem 192:263–273
Solntseva EI (1988) Abolition of the inhibitory effect of antibodies against S-100 proteins on the calcium current of molluscan neurons after intercellular injection of EGTA. Bull Exp Biol Med 105:646–649
Spray DC, Burt JM (1990) Structure-active relations of the cardiac gap junction channels. Am J Physiol 258:c159-c205
Takeda A, Saheki S, Shimazu T, Takeuchi N (1989) Phosphorylation of the 27-kDa gap junction protein by protein kinase C in vitro and in rat hepatocytes. J Biochem (Tokyo) 106:723–727
Unwin N (1989) The structure of ion channels in membranes of excitable cells. Neuron 3:665–676
Warner AE, Guthrie SC, Gilula NB (1984) Antibodies to gapjunctional protein selectively disrupt junctional communication in the early amphibian embryo. Nature 311:127–131
White RL, Doeller JE, Verselis VK, Wittenberg BA (1990) Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca2+. J Gen Physiol 95:1061–1075
Willecke K, Hennemann H, Dahl E, Jungbluth S, Heynkes R (1991) The diversity of connexin genes encoding gap junctional proteins. Eur J Cell Biol 56:1–7
Yancey SB (I), John SA (II), Lal R (III), Austin BJ, Revel J-P (1989) The 43-kDa polypeptide of heart gap junctions: I. Immunolocalization. II. Topology. III. Functional domains. J Cell Biol 108:2241–2254
Zagotta WN, Hoshi T, Aldrich RW (1990) Restoration of inactivation in mutants of Shaker potassium channels by peptide derived from ShB. Science 250:568–571
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lal, R., Laird, D.W. & Revel, J.P. Antibody perturbation analysis of gap-junction permeability in rat cardiac myocytes. Pflugers Arch. 422, 449–457 (1993). https://doi.org/10.1007/BF00375070
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00375070