Relevance of α-Conotoxin MII Sensitive Nicotinic Receptor Subtypes to Nicotine Addiction

总结/摘要 α-芋螺毒素MII（α-CtxMII）选择性拮抗α3β2*-和α6β2*-nAChR。我们之前已经开发了 α6β2*-nAChR-选择性α-Ctx，以确定中脑边缘α6β2*-nAChR对尼古丁和其他药物的贡献 滥用表型缺乏选择性化合物，以及α3 nAChR无效突变小鼠的致死性意味着实际上 尚未进行α3β2*-nAChR研究。也没有额外的边缘α6β2*-nAChR贡献成瘾- 相关行为进行了广泛调查。因此，我们将开发α-Ctx配体， 之间，表征，并定义α3β2*-和α6β2*-nAChR的作用。内侧缰核（MH）和 脚间核（IPN）含有密集的CNS α3β2*-nAChR群体（其数量超过MH， IPN α6β2*-nAChR）。MH和IPN中的α3β4*-和α5*-nAChR支持尼古丁依赖，但神经节依赖 α3β4*-nAChR的表达和α5*-nAChR在正构结合位点的缺乏可能使这些问题 戒烟目标。确定和区分MH和IPN α3β2*-和α6β2*-nAChR的贡献 针对尼古丁滥用和成瘾表型，提出了三个具体目标：1）进一步发现先导α-Ctx 通过筛选现有的一组>400种新型肽，并开发具有α3β2*-nAChR选择性的配体， 以增强选择性。我们已经确定了2个α-Ctx先导物，其α3β2*-nAChR选择性是 其他亚型。最具选择性的电极导线将使用一种新的简化方法开发，以改善 α-Ctx选择性。2)阐明MH和IPN α3β2*-nAChR亚基组成。我们要做无线电-还有 目标1中开发的高选择性肽的荧光标记衍生物。使用这些标记 肽，详细的α3β2*-nAChR组成将首次使用nAChR亚基-null确认 突变小鼠3)确定MH和IPN α3β2*-和α6β2*-nAChR在尼古丁强化中的重要性 和撤退。我们将在大鼠中测试是否局部输注选择性拮抗剂α-CtxMII（α3β2* & α 6 β 2 *）、α-CtxMII（α3β2* & α6β2*）、α-CtxMII（α3β2* & α6β2*）、α-CtxMII（α 3 β 2 *& α6 β 2 *）和α-CtxMII（α 6 β 2 *& α 6 β2*）。 CtxPIA（仅α6β2*-）或新的仅α3β2*-α-Ctx进入MH、IPN或2个以上控制区a）影响动机 对尼古丁起作用（在渐进比例计划下）和B）影响自发的身体和行为 戒断症状对于目标2和3，我们描述了如何在极端条件下使用现有化合物进行 不太可能的情况是，Aim 1不能产生适当的α3β2*-nAChR-选择性α-Ctx。这个提案的新筛选 和肽开发功能将从根本上推动未来利用宝贵的α-Ctx资源。的 在这项建议中开发的资源将是至关重要的，使未来的研究探索nAChR的功能， 成瘾相关网络，MH和IPN广泛连接。用我们新颖有效的方法 用于设计α-Ctx选择性，并结合男性和女性受试者的精细行为测试 大大增强了翻译的影响力。该提案有望将α3β2*-nAChR鉴定和表征为 新的、可药物化的吸烟治疗靶点，并证实α6β2*-nAChR是可行的戒烟靶点。 这些进步可能通过促进戒烟对公共健康产生重大影响。

SUMMARY / ABSTRACT α-Conotoxin MII (α-CtxMII) selectively antagonizes α3β2*- and α6β2*-nAChR. We have previously developed α6β2*-nAChR-selective α-Ctxs to define mesolimbic α6β2*-nAChR contributions to nicotine and other drug abuse phenotypes. A lack of selective compounds, and lethality in α3 nAChR null mutant mice means virtually no α3β2*-nAChR studies have been performed. Nor have extra-limbic α6β2*-nAChR contributions to addiction- relevant behaviors been investigated extensively. Therefore, we will develop α-Ctx ligands to discriminate between, characterize, and define the roles of α3β2*- and α6β2*-nAChR. The medial habenula (MH) and interpeduncular nucleus (IPN) contain the densest CNS α3β2*-nAChR populations (which outnumber MH and IPN α6β2*-nAChR). α3β4*- and α5*-nAChR in MH and IPN support nicotine dependence but ganglionic expression of α3β4*-nAChR and lack of α5*-nAChR in orthosteric binding sites may make these problematic smoking cessation targets. To establish and differentiate MH and IPN α3β2*- and α6β2*-nAChR contributions to nicotine abuse and addiction phenotype, three Specific Aims are proposed: 1) To discover further lead α-Ctx ligands with α3β2*-nAChR selectivity by screening an existing panel of >400 novel peptides, and develop them for enhanced selectivity. We have already identified 2 α-Ctx leads with >10-fold α3β2*-nAChR selectivity over other subtypes. The most-selective leads will be developed using a novel and streamlined approach to improve α-Ctx selectivity. 2) To elucidate MH and IPN α3β2*-nAChR subunit composition. We will make radio- and fluorescence-labeled derivatives of the highly-selective peptides developed in Aim 1. Using these labeled peptides, detailed α3β2*-nAChR composition will be confirmed for the first time using nAChR subunit-null mutant mice. 3) To define the importance of MH and IPN α3β2*- and α6β2*-nAChR in nicotine reinforcement and withdrawal. We will test in rats if local infusion of the selective antagonists α-CtxMII (α3β2* & α6β2*), α- CtxPIA (α6β2*-only), or a novel α3β2*-only α-Ctx into MH, IPN or 2 more control regions a) affects motivation to work for nicotine (under a progressive ratio schedule) and b) affects spontaneous somatic and behavioral withdrawal symptoms. For Aims 2 & 3, we describe how to proceed using existing compounds in the extremely unlikely case that Aim 1 does not yield suitably α3β2*-nAChR-selective α-Ctxs. This proposal's new screening and peptide-development features will radically advance future utilization of the invaluable α-Ctx resource. The resources developed in this proposal will be vital to enable future studies probing nAChR function within the addiction-related network to which MH and IPN are extensively connected. Using our novel and potent method for engineering α-Ctx selectivity in combination with refined behavioral testing across male and female subjects greatly enhances translational impact. This proposal promises to identify and characterize α3β2*-nAChR as novel, druggable, smoking therapeutic targets, and confirm α6β2*-nAChR as a viable tobacco cessation target. These advances may produce a major impact on public health by promoting smoking cessation.