Local and global calcium signals associated with the opening of neuronal alpha7 nicotinic acetylcholine receptors.
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.
Gilbert
Dorothée
D
Bioimaging Core Facility, University of Geneva, Switzerland.
Lecchi
Marzia
M
Arnaudeau
Serge
S
Bertrand
Daniel
D
Demaurex
Nicolas
N
D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D045744:Cell Line, Tumor; D002645:Chickens; D055724:Electrophysiological Phenomena / Q000187:drug effects; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009538:Nicotine / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D064569:alpha7 Nicotinic Acetylcholine Receptor
C508388:1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea; C579167:Chrna7 protein, human; D007555:Isoxazoles; D010671:Phenylurea Compounds; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D009538:Nicotine