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.