CALCIUM SIGNALING BY HIPPOCAMPAL NICOTINIC RECEPTORS

描述(申请人摘要)： 尼古丁是一种形成习惯的毒品，在我们的社会中被广泛使用。这个 烟碱型乙酰胆碱受体(AChRs)在人体的广泛分布 大脑表明，人类使用尼古丁的动态很可能是 要复杂一点。了解尼古丁在中枢神经系统中的作用机制 必须从阐明各种CNS-AChR的功能开始 子类型。许多乙酰胆碱受体对钙具有高度的渗透性 使它们成为调节许多重要的钙依赖事件的候选对象 通过改变细胞内钙离子浓度([Ca]i)。这个 这项建议的主要目的是研究钙信号转导特性 海马乙酰胆碱受体的一种亚型，与蛇毒毒素结合 α-银环蛇毒素(a-BGT-AChRs)。α-BGT-AChR的钙通透性 将使用直接从培养的海马神经元中确定通道 用氟-3填充电极测定钙荧光 五钾盐与全电池电压钳联用测定。 AbgT-AChRs对尼古丁作用改变[Ca]i的能力 在完整的神经元中，将通过钙成像从加载了 FLOO-3 AM钙信号的时间模式由 这些神经元上的各种胆碱能受体及其与 其他对[Ca]i有贡献的来源将被详细研究。这将是 ABGT-AChRs在维持心肌细胞钙稳态中的作用 海马神经元。AbgT-AChRs调节钙离子的能力 依赖的事件，如磷脂酶A2，CaM激酶和 钙调素敏感的腺苷环化酶将使用已建立的 生化程序和钙增加的空间要求 它们的激活决定了。这些研究将建立AbgT-AChRs为 在海马体中调节钙依赖事件的候选。 此外，钙直接调节AbgT-AChR功能的能力和 从而保持对细胞钙水平的快速而严密的控制 使用全电池电压钳测量进行检查。结构性的 AbgT-AChR功能的钙依赖调节的决定因素将是 利用alpha7基因和非洲爪哇进行定点突变检测 卵母细胞表达系统。改变假定的EF-HAND结构域的影响 用双电极电压法对基因产物的功能进行检测 钳夹技术。 这些研究将是第一次对中枢神经系统AbgT-AChRs进行详细研究 分子和细胞水平。在这个项目中获得的结果将表明 AbgT-AChRs在海马区功能调节中的作用

DESCRIPTION (Applicant's Abstract): Nicotine is a habit forming drug that is widely used in our society. The widespread distribution of the nicotinic acetylcholine receptors (AChRs) in the brain suggests that the dynamics of nicotine use in humans is likely to be complex. Understanding mechanisms underlying nicotines action in the CNS must begin with the elucidation of the functions of various CNS-AChR subtypes. The findings that many AChRs are highly permeable to calcium makes them candidates for mediating many essential calcium-dependent events in neurons by altering intracellular calcium concentrations ([Ca]i). The main aim of this proposal is to examine the calcium signalling properties of a subtype of hippocampal AChRs that bind the snake venom toxin alpha-bungarotoxin (a-Bgt-AChRs). Calcium permeability of the a-Bgt-AChR channel will be determined from cultured hippocampal neurons using direct calcium fluororescence measurements using electrodes filled with the fluo-3 pentapotassium salt coupled with whole cell voltage clamp determinations. The ability of aBgt-AChRs to alter [Ca]i in response to nicotine application in intact neurons will be examined by calcium imaging from cells loaded with fluo-3 AM. The temporal patterns of calcium signals generated by the various cholinergic receptors on these neurons and their interactions with other sources contributing to [Ca]i will be examined in detail. This will elucidate the role aBgt-AChRs play in maintaining calcium homeostasis in hippocampal neurons. The ability of aBgt-AChRs to regulate calcium dependent events like activation of phospholipase A2, CaM kinases and calmodulin sensitive adenylate cyclases will be studied using established biochemical procedures and spatial requirements of calcium increases for their activation determined. These studies will establish aBgt-AChRs as candidates for mediating calcium dependent events in the hippocampus. Further the ability of calcium to directly modulate aBgt-AChR function and thus maintain a rapid and tight control on cellular calcium levels will be examined using whole cell voltage clamp measurements. The structural determinants of calcium dependent regulation of aBgt-AChR function will be examined by site-directed mutagenesis using the alpha7 gene and the Xenopus oocyte expression system. The effects of altering a putative EF-hand domain on the function of the gene product will be examined by 2-electrode voltage clamp techniques. These studies will be the first detailed study of CNS aBgt-AChRs at the molecular and cellular level. Results obtained in this project will suggect roles for aBgt-AChRs in regulating hippocampal functions.