ANTIBODIES DIRECTED TO THE CALMODULIN-BINDING DOMAIN OF B-50 (GAP-43)INHIBIT CA2-INDUCED DOPAMINE RELEASE FROM PERMEATED SYNAPTOSOMES()

Protein B-50 (GAP-43) is an atypical calmodulin-binding protein with a higher affinity for calmodulin in the absence than in the presence of Ca2+. In the mature synapse B-50 has been implicated in the release o f noradrenaline and neuropeptide cholecystokinin-8. Using the cross-li nker disuccinimidyl suberate we demonstrate in native synaptosomal pla sma membranes that B-50 retains its ability to interact with calmoduli n in a Ca2+-dependent manner at physiological salt concentrations. The importance of the calmodulin-binding domain and the protein kinase C phosphorylation site of B-50 in the regulation of neurotransmitter rel ease was studied by introducing monoclonal anti-B-50 antibodies NM2 an d NM6 into streptolysin-O-permeated synaptosomes. NM2 antibodies, whic h interfere with the calmodulin-binding and phosphorylation properties at the N-terminus of B-50, inhibited the endogenous, Ca2+-induced dop amine release from permeated synaptosomes, whereas NM6 antibodies dire cted to a C-terminal domain of B-50 were without effect. We conclude t hat the N-terminal domain of the B-50 protein plays an important role in the process of Ca2+-induced dopamine release, presumably by serving as a local calmodulin store which is regulated in a Ca2+- and phospho rylation-dependent fashion.