ALPHA CONOTOXIN MII--SELECTIVE NICOTINIC RECEPTOR PROBE

描述（由申请人提供）： 尼古丁通过不同的烟碱家族引起的行为效应 乙酰胆碱受体（nAChR）和尼古丁诱发的多巴胺释放被认为是 在尼古丁的建立和维持中发挥重要作用 依赖α-芋螺毒素MII（aCtxMII），一种分离自捕食性芋螺的毒素 蜗牛Conus magus，有效地和选择性地阻断a3 b2-nAChR表达， 爪蟾卵母细胞和部分抑制尼古丁刺激的多巴胺释放 从大鼠纹状体突触体中分离出来。在最初的筹资期间， grant aCtxMII已被用于鉴定小鼠脑中nAChR的亚群。它 证实aCtxMII抑制一些但不是全部尼古丁刺激的 小鼠纹状体多巴胺释放。正如预期的那样，b2无效突变体保留了 几乎没有aCtxMII结合或尼古丁刺激的释放。令人惊讶的是， b3无效突变消除了大多数高亲和力aCtxMII结合， aCtxMII敏感性多巴胺释放。同样令人惊讶的是， a3无效突变对aCtxMII结合的影响。因此，许多原住民 与aCtxMII相互作用的nAChR不是a3 b2亚型。实验 在目前的建议中概述的将使用配体结合和功能 分析，以进一步研究nAChR的多样性。1)aCtxMII灵敏度 nAChR介导的纹状体、中脑核、额叶皮质的多巴胺释放 和嗅觉结节的小鼠，已经突变，以消除 特异性nAChR亚基（最初为a7、a5、b2、b3和b4）的表达将被 评价以获得关于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结合的调节 并通过使用野生型和无效突变体的慢性尼古丁处理发挥作用 小鼠，重点是那些影响nAChRs的突变体， aCtxMII也将进行检查。3)最近的结构特性 鉴定了aCtxPWl，其在结构上与aCtxMII显著不同，但 显示相似的药理学特征，将进行评价，以确定是否 这种新毒素将被证明是上级或补充试剂。拟议 研究将进一步深入了解三种小说的性质， nAChR对aCtxMII的抑制敏感，可能导致更好的理解 尼古丁的作用的基础

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