A functional domain on the alpha-latrotoxin molecule, distinct from the binding site, involved in catecholamine...

Seven monoclonal antibodies (mAbs) have been produced against alpha-latrotoxin (alpha-Latx), the toxin component of black widow spider venom that stimulates release of neurotransmitters from PC12 cells. These mAbs were characterized by an enzyme-linked immunosorbent assay and by neutralization analysis of the secretagogue properties of the toxin. The production of a panel of mAbs, possibly directed against different epitopes of alpha-Latx, provides a useful set of reagents to dissect the molecular regions of the toxin having different functions and to describe steps of its mode of action in responsive cells. Attention was focused on one of these mAbs (4C4.1), which inhibits in a dose-dependent fashion both toxin-stimulated and crude venom stimulated dopamine release from PC12 cells, prevents toxin-induced 45Ca2+ accumulation in PC12, alters toxin-dependent phosphoinositide breakdown, and prevents toxin-induced channel formation in artificial lipid bilayers. Since, within certain experimental conditions, mAb 4C4.1 is able to recognize the toxin bound to cells, we conclude that its effects were not a consequence of a direct interference with binding. On the basis of kinetic analysis of mAb interference on toxin action, expressed as accumulation of inositol phosphates and transmitter secretion, we suggest that the described effects result primarily from the blockade of an event immediately successive to binding and central for the full expression of toxin action. The availability of mAb 4C4.1 now makes possible the molecular characterization of the toxin moiety responsible for such an event.