ALPHA CONOTOXIN MII--SELECTIVE NICOTINIC RECEPTOR PROBE

描述(由申请人提供)： 尼古丁通过一系列不同的尼古丁引起行为效应 乙酰胆碱受体(NAChR)和尼古丁引起的多巴胺释放被认为 在尼古丁的建立和维持中发挥重要作用 依赖。A-ConooxinMll(ACtxMII)，一种从捕食性锥体中分离出来的毒素 蜗牛圆锥，有效和选择性地阻断a3b2-nAChR在 非洲爪哇卵母细胞并部分抑制尼古丁刺激的多巴胺释放 来自大鼠纹状体突触小体。在最初的资助期内 Grant aCtxMII已被用于确定小鼠脑内nAChRs的亚群。它 已证实aCtxMII抑制部分但不是全部尼古丁刺激 小鼠纹状体中的多巴胺释放。正如预期的那样，b2零突变体保留了下来 几乎没有aCtxMII结合或尼古丁刺激的释放。令人惊讶的是， B3零突变消除了大多数高亲和力的aCtxMII结合 对ACtxMII敏感的多巴胺释放。同样令人惊讶的是，没有一个 A3零突变对aCtxMII结合的影响。因此，许多原住民 与aCtxMII相互作用的nAChR不是a3b2亚型。实验 在目前的提案中概述将使用配体结合和官能化 分析以进一步研究nAChR的多样性。1)aCtxMII敏感度 NAChR介导的纹状体、伏隔核、额叶皮质多巴胺释放 和小鼠的嗅觉结节，这些小鼠的嗅觉结节已经突变，以消除 特定的nAChR亚基(最初为a7、a5、b2、b3和b4)的表达将为 评估以获得有关分子组成和功能的信息 这些重要的突触前nAChRs的多样性。2)nAChR结合的调控 并通过同时使用野生型和零突变的慢性尼古丁治疗来发挥作用 小鼠，重点是那些影响与之相互作用的nAChRs的突变体 ACtxMII，也将被审查。3)新近的结构特征 鉴定出aCtxPWl，它在结构上与aCtxMII明显不同，但 显示类似的药理学配置文件，将进行评估以确定 这种新的毒素将被证明是更好的或补充的试剂。建议数 研究将提供对三个本土小说的性质的进一步洞察 NAChRs对aCtxMII的抑制敏感，并可能导致更好的理解 尼古丁的作用基础

DESCRIPTION (provided by applicant): Nicotine elicits behavioral effects through a diverse family of nicotinic acetyicholine receptors (nAChR) and nicotine evoked dopamine release is thought to play an important role in the establishment and maintenance of nicotine dependence. a-ConotoxinMll (aCtxMII), a toxin isolated from the predatory cone snail Conus magus, potently and selectively blocks a3b2-nAChR expressed in Xenopus oocytes and partially inhibits nicotine-stimulated dopamine release from rat striatal synaptosomes. During the initial funding period for this grant aCtxMII has been used to identify subsets of nAChRs in mouse brain. It was confirmed that aCtxMII inhibits some, but not all, nicotine-stimulated dopamine release in mouse striatum. As expected, b2 null mutants retained virtually no aCtxMII binding or nicotine-stimulated release. Surprisingly, the b3 null mutation eliminated most high affinity aCtxMII binding and aCtxMII-sensitive dopamine release. Equally surprising was the absence of an effect of the a3 null mutation on aCtxMII binding. Therefore, many native nAChRs that interact with aCtxMII are not the a3b2 subtype. Experiments outlined in the current proposal will use ligand binding and functional analyses to further examine the diversity of nAChR. 1) aCtxMII sensitivity of nAChR mediated dopamine release in striatum, nucleus accumbens, frontal cortex and olfactory tubercles of mice that have been mutated to eliminate the expression of specific nAChR subunits (initially a7, a5, b2, b3 and b4) will be evaluated to obtain information about the molecular composition and functional diversity of these important presynaptic nAChRs. 2) Regulation of nAChR binding and function by chronic nicotine treatment using both wild type and null mutant mice, with emphasis on those mutants that affect nAChRs that interact with aCtxMII, will also be examined. 3) Structural properties of the recently identified aCtxPWl, which differs markedly in structure from aCtxMII, but displays a similar pharmacological profile, will be evaluated to determine if this new toxin will prove to be superior or complementary reagent. The proposed studies will provide further insights into the nature of three novel, native nAChRs sensitive to inhibition by aCtxMII and may lead to better understanding of the basis of the effects of nicotine