DISRUPTION OF CALMODULIN/GAP 43 BINDING BY LEAD

阐明Pb 2+神经毒性机制的工作现在集中在确定在pM Pb 2+浓度下发生的与Pb 2+诱导的行为变化和功能缺陷相关的生化或解剖学变化。 蛋白激酶C（PKC）是目前已知的在如此低浓度下受Pb 2+影响的对信号转导重要的唯一酶。 另一个对钙调素依赖性信号转导很重要的pb 2+诱导的细胞变化是静息细胞内Ca 2+浓度从约100 nM增加到200 nM。 目前，PKC刺激或静息Ca 2+浓度增加（可能与Pb 2+毒性有关）都没有已知的生化或解剖学后遗症。 该项目旨在采取下一步措施，并证明Pb 2+通过引起钙调素（CaM）从PKC敏感和Ca 2+敏感的CaM结合位点（包括CaM储库蛋白）重新分布来破坏神经元中钙调素依赖性信号转导的平衡。 所讨论的CaM储库是蛋白质，如GAP-43（神经调节素），其以Cat+可降解的方式在IQ结构域结合CaM。 当它们被PKC磷酸化时，钙调素与包括神经调节素在内的几种IQ结构域蛋白的结合被阻断。 钙调素储库蛋白是最丰富的神经元和神经调素是特别重要的铅毒性，因为它主要位于神经生长锥和突触前末梢，是必不可少的正常神经生长和突触传递。 钙调素和钙调素与神经调节素的相互作用也是必要的适当的神经生长锥的发展和突触传递。 待检验的假设是，低水平铅暴露导致钙调素从膜相关的神经调节素结合位点重新分配到神经元细胞的胞质溶胶中。 它进一步假设，这两个铅2+依赖性刺激PKC与随后的磷酸化的神经调节素和铅2+依赖性增加静息细胞内钙离子浓度是因果关系上的重要钙调素的重新分配远离神经调节素和膜。 这项研究计划的长期目标是确定IQ结构域钙调素结合蛋白作为铅毒性靶点的重要性，并阐明PKC刺激和静息细胞内钙波动作为铅暴露人群中发育缺陷的基础的重要机制。

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.