Name: a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate
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作者要感谢毕业后的学者计划办公室和诺华医学研究所作为一个整体为这项研究提供资金。

1. 细胞的制备
注意: 这个协议, 包括数据生成, 使用 HEK293 细胞转基因与杆状病毒编码 NR1 和 NR2A 细胞。
<ol><li>种子适当数量的 HEK293 细胞, 并添加 NR1 和/或 NR2A 病毒在适当的最终浓度 (1.00 µL 每个)。对于一个384井板, 使用1万细胞/以及在最后的体积30µL。</li>
	<li>或者, 种子 HEK293-NR1-NR2A 细胞在适当的细胞数量和容量 (为384井板材, 使用1万个细胞/井在最后容量30µL)。添加四环素在2...

<ol>
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M., Rodriguez-Pena, A., Diaz-Guerra, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxicity+and+focal+cerebral+ischemia+induce+truncation+of+the+NR2A+and+NR2B+subunits+of+the+NMDA+receptor+and+cleavage+of+the+scaffolding+protein+PSD-95.">Excitotoxicity and focal cerebral ischemia induce truncation of the NR2A and NR2B subunits of the NMDA receptor and cleavage of the scaffolding protein PSD-95.</a> Molecular Psychiatry. 13 (1), 99-114 (2008).</li><li>Uttara, B., Singh, A. V., Zamboni, P., Mahajan, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+neurodegenerative+diseases:+a+review+of+upstream+and+downstream+antioxidant+therapeutic+options.">Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options.</a> Current Neuropharmacology. 7 (1), 65-74 (2009).</li><li>Hansen, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+a+fluorescence-based+screening+assay+identifies+histamine+H3+receptor+antagonists+clobenpropit+and+iodophenpropit+as+subunit-selective+N-methyl-D-aspartate+receptor+antagonists.">Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.</a> Journal of Pharmacology and Experimental Therapeutics. 333 (3), 650-662 (2010).</li><li>Bettini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+novel+NMDA+receptor+antagonists+selective+for+NR2A-+over+NR2B-containing+receptors.">Identification and characterization of novel NMDA receptor antagonists selective for NR2A- over NR2B-containing receptors.</a> Journal of Pharmacology and Experimental Therapeutics. 335 (3), 636-644 (2010).</li><li>Feuerbach, D., Loetscher, E., Neurdin, S., Koller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+pharmacology+of+the+human+NMDA-receptor+subtypes+R1-2A,+R1-2B,+R1-2C+and+R1-2D+using+an+inducible+expression+system.">Comparative pharmacology of the human NMDA-receptor subtypes R1-2A, R1-2B, R1-2C and R1-2D using an inducible expression system.</a> European Journal of Pharmacology. 637 (1-3), 46-54 (2010).</li><li>Hansen, K. 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这种化验的成功主要取决于使用的 HEK 细胞的健康状况。需要使用指数增长和低通道数的细胞。这种分析涉及许多转移和补充的解决方案, 所以使用谨慎将确保更高的准确性, 其结果。化合物和所有其他试剂的浓度也应进行交叉检查, 以尽量减少错误。当用测定缓冲液取代细胞培养基进行钙通量测定时, 应轻轻倾斜板, 使细胞不分离。最后, 在本协议中使用聚 d-赖氨酸涂层板增加了细胞对板的附着。</...

随着目前医学的进步, 预期寿命显著增加;然而, 与年龄相关的疾病的流行率也是如此。中枢神经系统疾病 (CNS), 如精神分裂症, 肌萎缩侧索硬化症, 阿尔茨海默氏病, 帕金森病等, 也不例外, 并预计将增加在未来十年1,2,3. 被称为 n-甲基-d-天门冬氨酸受体 (NMDAR) 的离子型谷氨酸受体的故障与阿尔茨海默病、精神分裂症、外伤性脑损伤、中风、糖尿病和青光眼等有联系, 这就保证了需要研究他们的生物学为发展有效, 疾病修改疗法4,5,6,7。
NMDARs 由四单体或亚基4,8,9组成。NMDAR 的结构构成在脑子7,10显示发展和区域可变性。NMDARs 参与突触可塑性, 认知和节奏的世代为呼吸和运动11,12,13。作为电压门控通道, 它主要是在静止膜电位 (-70 mV) 不导电, 并被镁堵塞, 以防止离子进一步渗透。该通道是由两个配体, 谷氨酸和甘氨酸/d-丝的结合激活, 并在突触膜 AMPA 受体介导的, 另一子类离子型谷氨酸受体的同时去极化。退极化去除 NMDAR 镁堵塞, 使阳离子的汇集, 特别是钙14,15,16。尽管激活 NMDAR 是细胞存活的必要条件, 但过度活化可能导致细胞死亡17,18,19通过兴奋。这, 除了受体的复杂性质, 使其具有挑战性的研究开发有效的治疗所必需的。
对 NMDAR 的研究开发了不同的方法。然而, 每一个都有相应的警告。例如, 一种广泛使用的技术是一种基于荧光的检测方法, 用于测量在四环素诱导启动子 (NMDAR) 控制下稳定细胞系内细胞内钙的变化.然而, 在这个系统中, 所需配体的 supramaximal 浓度和 NMDAR 抑制剂在测量过程中存在的要求, 几乎不可能检测到竞争对手的活动。在其他类似的系统中, 功能受体的表达导致毒性, 要求通道阻滞剂, 如氯胺酮21,22保存细胞培养。这些通道阻滞剂坐在受体的核心, 很难洗出来, 尤其是以平板为基础的格式, 所以他们干扰的功能研究的受体。最后, 在电生理测量, 如补丁夹紧, 有有限的吞吐量, 和大规模的研究是非常昂贵的23。尽管如此, 上述系统对甘氨酸刺激不敏感;因此, 研究 NMDAR 的甘氨酸依赖性活动成为一项挑战。
在这里, 我们描述了一个新的方法来研究 NMDAR, 克服了讨论的局限性。我们的技术是利用杆状病毒表达系统来表达受体在功能水平上的最佳比例的亚基在16小时。此外, 杆状病毒的使用允许一种简单的组合方法, 它提供了一个广泛的描述不同的重组 NMDAR 亚型。与其他化验不同, 该协议不需要通道阻滞剂, 因为使用弱拮抗剂。该方法的最大优势是, 在弱拮抗剂的冲刷后, 受体对个体甘氨酸和谷氨酸结合部位的调制敏感, 除了甘氨酸/d-丝氨酰胺和谷氨酸配体结合的双重调制网站。该检测概括已知的 NMDAR 受体药理作用及其已知的阳性和阴性调节剂的影响。最后, 这种体外细胞检测的产生克服了过量钙流入引起的细胞毒性, 允许高通量的受体功能研究, 从而加速 NMDAR 调制器的发现。在疾病状态。

<table>
	<tbody>
		<tr>
			<td>HEK-293</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Human NMDA (NR1/NR2A) Receptor Cell Line</td>
			<td>ChanTest Corporation</td>
			<td>CT6120</td>
			<td></td>
		</tr>
		<tr>
			<td>pFastBac Dual Expression Vector</td>
			<td>ThermoFisher Scientific</td>
			<td>10712-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 384-well Clear Flat Bottom Microplate</td>
			<td>Corning Life Sciences</td>
			<td>3844</td>
			<td></td>
		</tr>
		<tr>
			<td>FLIPR Calcium 6-QF Assay Kit</td>
			<td>Molecular Devices</td>
			<td>R8192</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>49621</td>
			<td></td>
		</tr>
		<tr>
			<td>D-serine</td>
			<td>Sigma-Aldrich</td>
			<td>S4250</td>
			<td></td>
		</tr>
		<tr>
			<td>L701,324</td>
			<td>Tocris</td>
			<td>907</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Boston Bio Product</td>
			<td>BB-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>63069</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>VWR</td>
			<td>E506</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher Scientific</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>ThermoFisher Scientific</td>
			<td>P36400</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX media</td>
			<td>ThermoFisher Scientific</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>MDL105,519</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
		<tr>
			<td>NVP-AAM077</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
		<tr>
			<td>CGP070667</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate

n-甲基-d-天门冬氨酸 (NMDA) 受体 (NMDAR) 被归类为离子型谷氨酸受体, 在学习和记忆中具有重要作用。NMDAR 的故障, 表现为过度或活动不足造成的突变, 改变表达, 贩运, 或本地化, 可能导致许多疾病, 特别是在中枢神经系统。因此, 了解受体的生物学以及促进发现化合物和小分子, 对于目前防治神经系统疾病的努力至关重要。目前研究该受体的方法有限制, 包括低吞吐量, 高成本, 并无法研究其功能能力, 由于必要存在的渠道阻滞剂, 以防止 NMDAR 介导的兴奋。此外, 现有的检测系统只对谷氨酸的刺激很敏感, 对 NMDAR 的另一种共配体甘氨酸的刺激缺乏敏感性。在这里, 我们提出了第一个以板块为基础的高通量的检测方法, 研究一种对同配体、谷氨酸和 d-丝氨酸/甘氨酸有敏感性的 NMDA 受体。这种方法允许研究不同的 NMDAR 亚基组成, 并允许功能研究的受体在甘氨酸和/或谷氨酸敏感模式。此外, 该方法不需要在测量过程中存在抑制剂。通过本试验可以检测到正、负构调节剂的作用, 并在本系统中复制了 NMDAR 的已知药理学。该技术克服了现有方法的局限性, 具有很高的成本效益。我们相信, 这项新的技术将加快发现治疗 NMDAR 介导的病理。

在测试小分子的影响之前, 你必须确定 NMDARs 的最佳表达水平以及最佳配体浓度。如所述, 在384井板中, HEK293 细胞每井1万个细胞, 在5µM CGP060667, 然后转基因的 NR1 和 NR2A 病毒数量不等。在一夜之间孵化后, 配体诱导的钙通量被测量 (图 1)。这些实验的结果显示了每一个亚基使用的病毒的最佳数量和比率 (图 1, 黄色盒)。
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Watch this Scientific Journal Video about A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate at JoVE.com

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

本议定书的目的是促进对 NMDA 受体 (NMDAR) 的研究, 使之更大规模地进行, 并允许检查小分子的调节作用及其治疗应用。

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Research

JoVE Journal

Neuroscience

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科研

神经科学

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