Sequence-specific antibodies to connexins block intercellular calcium signaling through gap junctions

Mechanical stimulation of a single cell in primary airway epithelial cell cultures induces an intercellular Ca2+ wave that has been proposed to be mediated via gap junctions. To investigate directly the role of gap junctions in this multicellular response, the effects of intracellularly-loaded sequence-specific connexin (gap junction) antibodies on the propagation of intercellular Ca2+ waves were evaluated. Electroporation of antibodies to the cytosolic loop (Des 1, generated to amino acids 102–112 + 116–124; and Des 5, amino acids 108–119), or to the carboxyl tail (Gap 9, amino acids 264–283) of connexin 32 inhibited the propagation of intercellular Ca2+ waves. The inhibitory effect of Des 1 antibody was competitively reversed by the co-loading of a peptide derived from a similar cytosolic loop sequence (Des 5 peptide). Conversely, the inhibitory effects on intercellular Ca2+ wave propagation of Gap 9 antibody was not altered by co-loading with the Des 5 peptide. Antibodies raised to peptide sequences within the extracellular loop (Gap 11, amino acids 151–187), or the cytoplasmically located amino terminus (Gap 10, amino acids 1–21) of connexin 32 did not inhibit mechanically-induced intercellular communication. Also ineffective in perturbing intercellular communication were antibodies raised to peptide sequences of the cytosolic loops of connexin 43 (Gap 15, amino acids 131–142) or connexin 26 (Des 3, amino acids 106–119). These data suggest that mechanically-induced Ca2+ waves in airway cell cultures are propagated through gap junctions made up of connexin 32 proteins