The overall goal of this research is to identify lead-protein interactions that directly contribute to the neurologic deficits produced by this potent neurotoxicant. Of central importance is the observation that lead-PKC interactions, which display an extremely high affinity, substantially enhance the secretary activity that is induced by lead ions (Pb2+) alone. It is our working hypothesis that Pb2+ directly interacts with some, if not all, PKC isoforms and that one outcome of toxicological significance may be the orderly recruitment of synaptic vesicles and, consequently, increased extent of Pb2+-induced glutamate exocytosis. Previous investigations have provided us with clues regarding a mechanism by which PKC-Pb2+ interactions could generate the mechanochemical force required to translocate synaptic vesicles from their presumed storage sites, within the axonal cytoskeleton, to plasma membrane docking sites. The combination of biochemical, molecular, and genetic methodologies that has been assembled for the proposed research will provide a powerful test of the following five hypotheses (1) that Pb2+ interacts with multiple protein binding sites to coordinate the activation of PKC, initiation of glutamate exocytosis, and mobilization of synaptic vesicles, 2) that Pb2+ stimulates PKC phosphotransferase activity by physically interacting with these proteins, 3) that actin is a PKCepsilon anchoring protein, 4) that actin is also a PKCepsilon, chaperon that secures PKCE in a catalytically active conformation, and 5) that Pb2+ and PKC interactions prime SVs for transport by augmenting the actin-activated Mg-ATPase activity of myosin. The proposed research will contribute to the understanding of how low levels of lead exposure may promote the use-independent release of glutamate, a physiologically inappropriate activity that can be expected to have a devastating effect on the establishment of synaptic connections early in development.
