Gilbert Dorothée, Lecchi Marzia, Arnaudeau Serge, Bertrand Daniel, Demaurex Nicolas
Bioimaging Core Facility, University of Geneva, Switzerland.
Cell Calcium. 2009 Feb;45(2):198-207. doi: 10.1016/j.ceca.2008.10.003. Epub 2008 Nov 26.
Neuronal nicotinic acetylcholine receptors (nAChRs) are Ca(2+)-permeable ligand-gated channels widely expressed in the central and peripheral nervous system. One of the most Ca(2+) selective isoform is the homopentameric alpha7-nAChR implicated in schizophrenia. The activity of alpha7-nAChRs is usually recorded electrophysiologically, which limits the amount of information obtained. Here, we used fluorescence imaging to record Ca(2+) transients associated with activation of the alpha7-nAChR in neuroblastoma cells stably expressing human alpha7-nAChRs. Application of nicotine (50 microM) consistently evoked transient (30s), stereotyped Ca(2+) responses that were inhibited by the selective alpha7-nAChRs antagonists methyllycaconitine (MLA) and alpha-bungarotoxin, and greatly increased and prolonged by the allosteric modulator PNU-120596 (1 microM). Unexpectedly, brief (1-5s), repetitive Ca(2+) transients of sub-micrometric dimension were observed in filopodia of cells expressing alpha7-nAChR. PNU-120596 increased the frequency and slowed the decay kinetics of these miniature Ca(2+) elevations, which were insensitive to ryanodine, preserved during hyperpolarisation, and prevented by MLA, alpha-bungarotoxin, or Ca(2+) removal. Global Ca(2+) responses were also recorded in ganglion cells of embryo chicken retina during co-application of PNU-120596 and nicotine, together with whole-cell currents and brief current bursts. These data demonstrate that Ca(2+) signals generated by alpha7-nAChRs can be recorded optically both in cell lines and in intact tissues. The possibility to image miniature Ca(2+) signals enables to map the location of functional alpha7-nAChR channel clusters within cells and to analyze their single channel properties optically. Deciphering the rich pattern of intracellular Ca(2+) signals generated by the activity of the alpha7-nAChRs will reveal the physiological role of these receptor-channels.
神经元烟碱型乙酰胆碱受体（nAChRs）是一种Ca(2+)可通透的配体门控通道，在中枢和外周神经系统中广泛表达。其中Ca(2+)选择性最强的亚型之一是与精神分裂症有关的同五聚体α7-nAChR。α7-nAChRs的活性通常通过电生理学方法记录，这限制了所获得的信息量。在此，我们利用荧光成像记录稳定表达人α7-nAChRs的神经母细胞瘤细胞中与α7-nAChR激活相关的Ca(2+)瞬变。施加尼古丁（50 microM）持续诱发短暂（30秒）、刻板的Ca(2+)反应，该反应被选择性α7-nAChRs拮抗剂甲基lycaconitine（MLA）和α-银环蛇毒素抑制，并被变构调节剂PNU-120596（1 microM）极大增强和延长。出乎意料的是，在表达α7-nAChR的细胞丝状伪足中观察到短暂（1-5秒）、重复的亚微米级Ca(2+)瞬变。PNU-120596增加了这些微小Ca(2+)升高的频率并减缓了其衰减动力学；这些微小Ca(2+)升高对ryanodine不敏感，在超极化期间保持，并被MLA、α-银环蛇毒素或去除Ca(2+)所阻止。在共同施加PNU-120596和尼古丁期间，还在胚胎鸡视网膜神经节细胞中记录了整体Ca(2+)反应，同时记录了全细胞电流和短暂电流爆发。这些数据表明，α7-nAChRs产生的Ca(2+)信号可以在细胞系和完整组织中通过光学方法记录。对微小Ca(2+)信号进行成像的可能性使得能够绘制细胞内功能性α7-nAChR通道簇的位置，并通过光学方法分析其单通道特性。解读由α7-nAChRs活性产生的丰富的细胞内Ca(2+)信号模式将揭示这些受体通道的生理作用。
