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

摘要/摘要 α-芋螺毒素MII(α-CtxMII)选择性拮抗α3β2*-和α6β2*-nAchR。我们之前已经开发出 α6β2*-nAChR选择性α-Ctxs用于确定中脑边缘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，对α3nAChR缺失突变小鼠的致命性几乎意味着 尚未进行α3β2*-nAChR研究。边缘外的α6β2*-nAChR对成瘾也没有贡献- 对相关行为进行了广泛调查。因此，我们将开发α-ctx配体来区分 在α3β2*-和α6β2*-nAChR之间，表征和定义角色。内侧缰核(MH)和 脚间核(IPN)含有密度最高的中枢神经系统α3β2*-nAChR群体(数量超过MH和 IPNα6β2*-nAChR)。MH和IPN中的α3β4*-和α5*-nAChR支持尼古丁依赖，但神经节细胞 α3β4*-nAChR的表达和α5*-nAChR在正构体结合部位的缺失可能会使这些问题成为问题 戒烟目标。建立和鉴别MH和IPNα3β2*-和α6β2*-nAChR的贡献 对于尼古丁滥用和成瘾的表型，我们提出了三个具体的目标：1)进一步发现α-CTX 具有α3β2*-nAChR选择性的配体通过筛选现有的一组&gt；400新肽并开发它们 以提高选择性。我们已经确定了2条α-ctx引线，其α3β2*-nAChR选择性高于 其他子类型。将使用一种新颖和简化的方法开发最具选择性的线索，以改进 α-环磷酰胺选择性。2)阐明MH和IPNα3β2*-nAChR亚基组成。我们会制作收音机--还有 目标1中开发的高选择性多肽的荧光标记的衍生物。使用这些标记的 多肽，详细的α3β2*-nAChR组成将首次使用nAChR亚单位确认-空 突变的小鼠。3)明确MH和IPNα3β2*-和α6β2*-nAChR在尼古丁强化中的重要性 和戒烟。我们将在大鼠身上测试局部输注选择性拮抗剂α-CtxMII(α3β2*&α6β2*)，α- CtxPIA(α6β2*-Only)，或一种新的α3β2*-Onlyα-CTX进入MH、IPN或2个以上控制区a)影响动机 为尼古丁工作(在累进比率时间表下)和b)影响自发的躯体和行为 戒断症状。对于目标2和目标3，我们描述了如何继续使用现有的化合物 不太可能的情况是，Aim 1没有产生适当的α3β2*-nAChR选择性α-CTX。这项提案的新放映 而多肽开发功能将从根本上促进未来对宝贵的α-CTX资源的利用。这个 本提案中开发的资源对于使未来的研究能够探索nAChR在 MH和IPN广泛连接的成瘾相关网络。使用我们新颖而有效的方法 用于设计α-CTX的选择性，并结合对男性和女性受试者的精细行为测试 极大地增强了翻译效果。该提案承诺将α3β2*-nAChR确定和表征为 新型、可药物的吸烟治疗靶点，并确认α6β2*-nAChR是可行的戒烟靶点。 这些进展可能会通过促进戒烟对公众健康产生重大影响。

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.