DISRUPTION OF CALMODULIN/GAP 43 BINDING BY LEAD

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Work to elucidate mechanisms underlying Pb2+ neurotoxicity now focuses on determining what biochemical or anatomical changes occur at the pM Pb2+ concentrations associated with Pb2+-induced behavioral changes and functional deficits. Protein Kinase C (PKC) is the only enzyme important for signal transduction which is currently known to be affected by Pb2+ at such low concentrations. One other pb2+-induced cellular change that is important for calmodulin-dependent signal transduction is a two-fold increase in the resting intracellular Ca2+ concentration from approximately 100 to 200 nM. At this time, there are no known biochemical or anatomical sequelae of either the PKC stimulation or the increase in resting Ca2+ concentration that can be linked to Pb2+ toxicities. This project is designed to take that next step and demonstrate that Pb2+ disrupts the balance of calmodulin-dependent signal transduction in neurons by causing a redistribution of calmodulin (CaM) away from PKC-sensitive and Ca2+-sensitive CaM binding sites, including CaM-reservoir proteins. The CaM reservoirs in question are proteins such as GAP-43 (neuromodulin) which bind CaM at IQ-domains in a Cat+-inhibitable manner. CaM binding to several of the IQ-domain proteins, including neuromodulin, is blocked when they are phosphorylated by PKC. The CaM-reservoir proteins are most abundant in neurons and neuromodulin is of particular importance to Pb toxicity as it is located predominantly in the nerve growth cone and presynaptic terminals and is essential for normal nerve growth and synaptic transmission. CaM and the interaction of CaM with neuromodulin are also essential for proper nerve growth cone development and for synaptic transmission. The hypothesis to be tested is that low level lead exposure causes redistribution of CaM from membrane associated neuromodulin binding sites to cytosol in neuronal cells. It is further hypothesized that both a Pb2+-dependent stimulation of PKC with a subsequent phosphorylation of neuromodulin and a Pb2+-dependent increase in resting intracellular Ca2+ concentration are causally important in the redistribution of CaM away from neuromodulin and the membrane. The long term goal of this research program is to determine the importance of IQ-domain CaM-binding proteins as targets for Pb2+ toxicity and to elucidate the mechanistic importance of PKC stimulation and resting intracellular Ca2+ fluctuations as underlying the developmental deficits seen in Pb2+ exposed populations.