DISRUPTION OF CALMODULIN/GAP 43 BINDING BY LEAD

目前，阐明Pb2+神经毒性机制的工作主要集中在确定pM Pb2+浓度与Pb2+诱导的行为改变和功能缺陷相关的生化或解剖变化。蛋白激酶C （PKC）是目前已知的唯一对信号转导重要的酶，它会受到如此低浓度Pb2+的影响。另一个pb2+诱导的细胞变化对钙调素依赖的信号转导很重要，这是静息细胞内Ca2+浓度从大约100到200 nM增加两倍。目前，没有已知的PKC刺激或静止Ca2+浓度增加可能与Pb2+毒性有关的生化或解剖学后遗症。该项目旨在采取下一步，并证明Pb2+通过引起钙调蛋白（CaM）从pkc敏感和Ca2+敏感的CaM结合位点（包括CaM库蛋白）重新分布，破坏神经元中钙调蛋白依赖的信号转导的平衡。所讨论的CaM储存库是诸如GAP-43（神经调节素）之类的蛋白质，它们以抑制Cat+的方式在iq结构域结合CaM。CaM与一些iq结构域蛋白的结合，包括神经调节蛋白，当它们被PKC磷酸化时被阻断。cam -库蛋白在神经元中最丰富，而神经调节素在铅中毒中尤为重要，因为它主要位于神经生长锥和突触前终末，是正常神经生长和突触传递所必需的。CaM和CaM与神经调节素的相互作用对于正常的神经生长锥发育和突触传递也是必不可少的。待验证的假设是，低水平的铅暴露会导致CaM从神经调节素结合位点重新分布到神经元细胞的细胞质中。进一步的假设是，Pb2+依赖性的PKC刺激和随后的神经调节素磷酸化，以及Pb2+依赖性的静息细胞内Ca2+浓度的增加，在CaM远离神经调节素和膜的重新分布中都是重要的。本研究计划的长期目标是确定iq结构域cam结合蛋白作为Pb2+毒性靶点的重要性，并阐明PKC刺激和静息细胞内Ca2+波动作为Pb2+暴露人群中发育缺陷的潜在机制重要性。

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.