Antibody perturbation analysis of gap-junction permeability in rat cardiac myocytes

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 domain of Cx43 could be involved in regulating junctional permeability, and that calcium modulates the effect of antibodies on junctional permeability.