实时 cAMP 检测气味受体激活的活细胞测量
Summary
表征气味受体的功能在 deorphanization 过程中起着不可缺少的作用。我们描述了一种方法来测量气味受体的激活, 在实时使用阵营分析。
Abstract
哺乳动物气味受体 (或) 家庭的巨大尺寸在许多挥发性化学物质中发现其同源配体存在困难。为了有效和准确地 deorphanize 口服补液盐, 我们结合使用异种细胞线表达哺乳动物口服补液盐和基因修饰的生物传感器质粒来测量营地生产的下游或实时激活。这种方法可以用于筛选气味对口服补液盐和反之亦然. 通过对各种气味浓度的测试, 产生浓度-反应曲线, 可以证实从屏幕上的正气味受体相互作用。在这里, 我们使用这种方法进行高通量筛选的气味化合物对人类或图书馆表达的 Hana3A 细胞, 并证实, 积极反应受体是同源受体的化合物的兴趣。我们发现这种高通量的检测方法在评估或激活时是有效和可靠的, 我们的数据提供了它的潜在用途或功能研究的一个例子。
Introduction
嗅觉在动物的生存中起着重要作用, 因为它们依赖嗅觉才能获得食物, 避免捕食者和危险, 区分物种, 并选择交配1,2。这些功能的实现取决于气味受体 (口服补液盐), 这是单独表达在纤毛表面的嗅觉感觉神经元 (OSNs) 位于嗅觉上皮 (OE)。口服补液盐组成的 G 蛋白偶联受体 (化学) 超家族的最大的家庭与大约400和1200多样或基因在人和小鼠, 分别3,4,5。气味激活的口服补液盐, 通过嗅 g 蛋白 (g阵线) 和 III 型腺苷 (ACIII) 的顺序激活, 导致细胞内 cAMP 水平增加。细胞内 cAMP 的增加可以作为第二信使, 在细胞表面打开核苷酸门控通道, 触发阳离子的流入, 包括 Ca2 +和动作电位, 并最终启动神经-潜在的传播和嗅觉知觉。检测和鉴别大量气味的过程被认为是嗅觉知觉的第一步6,7。
由于降压和一周半跳8首次成功地克隆了气味受体, 阐明了由口服补液盐引发的嗅觉知觉机制, deorphanization 或家族成为该领域的热点之一。各种体内, ex 体内和体外测量或激活方法已被报告9,10,11,12。传统的方法, 使用 Ca2 +成像后, 单细胞 RT PCR 在 OSNs 启用了识别不同的口服脂肪族气味13,14,15。最近, large-scale 转录分析的问世促进了更高通量的在体内方法的发展。肯塔基试验鉴定了丁香酚和麝香反应的老鼠, 使用了 S100a5-tauGFP 报告鼠株和微阵列分析9。根据气味暴露后的下降或 mRNA 水平, 梦想技术采用了一种组的方法来确定或激活脊椎动物和无脊椎动物物种的剖面图16。同样, 考虑到 S6 在神经元激活中的磷酸化, Matsunami 组从磷酸酯核糖体 immunoprecipitations 测序基因, 以确定反应灵敏的口服补液盐12。最后, 该组报告了超级嗅探小鼠, 可以作为一个平台, 研究气味编码在体内, 称为 MouSensor 技术17。
在体外领域, 培养 OSNs 的挑战使一个异源表达系统模仿或功能表达在体内一个理想的解决方案, 进行 large-scale 筛选气味化学品的口服补液盐。然而, 因为培养的细胞系 non-olfactory 起源不同于当地的 OSNs, 或蛋白质被保留在内质网并且不能交通对血浆膜, 导致或退化和损失受体作用18,19. 为了解决这一问题, 在外源细胞系中对细胞膜的复制或功能表达做了大量的工作。Krautwurst et al.首先将前20氨基酸的视 (ρ标签) 附加到 N 终端或蛋白质, 这促进了一些口服补液盐在人类胚胎肾脏 (HEK) 细胞的细胞表面表达,20。通过对单 OSNs、斋藤et al的基因表达 (SAGE) 库分析进行序列分析。首先克隆受体转运蛋白 (RTP) 家族成员, RTP1 和 RTP2, 和受体表达增强蛋白 1 (REEP1), 促进或贩运到细胞膜和增强气味介导的口服补液盐在 HEK293T 细胞的反应21. 根据这些发现, Matsunami 集团成功地建立了 Hana3A 细胞系, 稳定地转染了 RTP1、RTP2、REEP1 和 Gα阵线HEK293T, 并瞬时转染了ρ标记的口服补液盐, 用于高效或功能表达.随后的研究揭示了 1) 一种较短的形式的 RTP1, RTP1S, 可以更有力地促进或功能比原 RTP1 蛋白和 2) 类型3毒蕈乙酰胆碱受体 (M3R), 可以加强或活动通过抑制β-arrestin-2招聘, 这两个被引入到外源表达系统优化实验输出22,23。
几种检测方法已被用来量化受体激活在异源系统。分泌的胎盘碱性磷酸酶 (SEAP) 检测工作与一个记者酶转录调节的阵营反应元素 (CREs), 使它成为一个有吸引力的选择评估或激活。荧光在培养基样品中容易地被发现在培养媒介以后与 SEAP 检测试剂24。使用这种方法, 口服补液盐的功能以及第二类化学受体表达的 OE-微量胺相关受体 (TAARs) 已被描绘成25,26,27。另一种常见的方法, 荧光检测, 使用了萤火虫荧光报告基因的控制下的阵营反应元件 (综合考试)。测量荧光生成的发光提供了一种有效且健壮的方法来量化或激活10、11、28。
实时营区检测也被广泛应用于动态监测外源或内受体的功能。这种先进的分析的一个例子是利用基因编码的生物传感器变体, 它拥有一个阵营结合的领域融合到一个突变形式的荧光。当阵营束缚, 构象变动导致激活荧光, 发光从然后可以被测量用化学发光的读者29,30。据报道, real-time 营技术适用于 HEK293 和 NxG 108CC15 细胞中人类气味受体的 de-orphaning驴子 = “xref” >> 31,32,33, 以及在 HEK293T-derived Hana3A 单元格34,35。Krautwurst 组还详细描述了 real-time 阵营技术适合双向 large-scale 或筛选方法32,33。
在这里, 我们描述了一个用于测量或激活使用 real-time cAMP 测定 Hana3A 细胞的协议。在本协议中, pre-equilibrated 活细胞的发光是动力学测量的30分钟后, 特定的挥发性化合物的治疗, 代表一个更有效和准确的分析或活化, 不太容易在细胞环境中发生的工件, 具有长时间和气味诱导的细胞毒性。这种 real-time 的测量允许 large-scale 筛选的口服补液盐和配体, 以及表征特定或配基对的兴趣。使用这种方法, 我们成功地确定了 OR5AN1 作为麝香化合物麝香受体, 通过对379人体口服补液盐进行筛选, 并随后确认阳性筛查结果。
Protocol
Representative Results
Discussion
在接触到某气味时, 准确地测量或激活是破译嗅觉信息编码的第一步。本研究中所示的实验表明了一个例子, 说明如何使用一个体外或表达系统来识别人类或有气味的化学物质的反应性口服补液盐, 并随后表征该受体药理学使用各种浓度的化学物质。我们的结果证实 OR5AN1 作为一个善意受体的麝香。这与以前的报告39一致, 并提供了进一步的证据, 推测只有少数的受体?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家科学基金会 (31070972)、上海市科学技术委员会 (16ZR1418300)、上海市教育委员会创新研究小组项目、上海东部学者计划 (J50201) 和中国国家基础研究计划 (2012CB910401)。
Materials
| Amphotericin B | Sigma | A2942 | |
| DMSO | Sigma | D2650 | |
| FBS | Gibco | 10099-141 | |
| GloSensor cAMP reagent | Promega | E1290 | |
| pGloSensor-20F cAMP plasmid | Promega | E1171 | |
| Hana3A cells | available from authors upon request | ||
| HBSS, w/o calcium chloride and magnesium chloride | GIBCO | 14175095 | |
| HEPES | Hyclone | SH30237 | |
| Lipofectamine2000 | Invitrogen | 11668-019 | |
| M3R plasmid | cloned into a mammalian expression vector such as pCI | ||
| MEM, w/EBSS and w/L-glutamine | Hyclone | SH30024 | |
| Muscone | Santa Cruz | sc-200528 | |
| Musk tibetene | Sigma-Aldrich | S359165 | |
| OR plasmids | cloned with a Rho-tag into a mammalian expression vector such as pCI | ||
| PBS, w/o calcium or magnesium | Cellgro | 21-040-CV | |
| Penicillin-streptomycin | Hyclone | SV30010 | |
| Plasmid miniprep kit | Tiangen | DP103-03 | |
| Puromycin | Sigma | P8833 | |
| RTP1S plasmid | cloned into a mammalian expression vector such as pCI | ||
| Trypsin-EDTA | Hyclone | SH30236 | |
| 0.2-mL PCR tube | Axygen | PCR-02-C | |
| 1.5-mL Eppendorf tube | Eppendorf | ||
| 15-mL 17 mm x 120 mm conical tube | BD Falcon | 352096 | |
| 8-well and/or 12-well multichannel pipetman | Eppendorf | ||
| 96-well flat-bottomed white cell culture plate | Greiner | 655098 | |
| 100 mm x 20 mm cell culture dish | BD Falcon | 353003 | |
| Class II biological safety cabinet with laminar flow | |||
| Cell culture incubator, w/ 5% CO2 | |||
| Centrifuge, with swinging bucket rotor for 15-ml conical tubes | |||
| Infinite F200 plate reader | Tecan | ||
| Phase-contrast microscope with x10 and x20 objectives | |||
| Spectrophotometer | |||
| Sterile reagent reservoirs for multichannel distribution | |||
| Sterile paper towel |
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