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 of noradrenaline and neuropeptide cholecystokinin-8. Using the cross-linker disuccinimidyl suberate we demonstrate in native synaptosomal plasma membranes that B-50 retains its ability to interact with calmodulin 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 release was studied by introducing monoclonal anti-B-50 antibodies NM2 and NM6 into streptolysin-O-permeated synaptosomes. NM2 antibodies, which interfere with the calmodulin-binding and phosphorylation properties at the N-terminus of B-50, inhibited the endogenous, Ca2+-induced dopamine release from permeated synaptosomes, whereas NM6 antibodies directed to a C-terminal domain of B-50 were without effect. We conclude that 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 phosphorylation-dependent fashion.