Journal
CELL CALCIUM
Volume 45, Issue 2, Pages 198-207Publisher
CHURCHILL LIVINGSTONE
DOI: 10.1016/j.ceca.2008.10.003
Keywords
Calcium; Nicotine; Confocal microscopy; Optical single-channel recordings; Calcium microdomains
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Funding
- Swiss National Science Foundation [Ndegree 3100a0-101787, Ndegree 31-068317.02]
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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 alpha 7-nAChR implicated in schizophrenia. The activity of alpha 7-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 alpha 7-nAChR in neuroblastoma cells stably expressing human alpha 7-nAChRs. Application of nicotine (50 mu M) consistently evoked transient (30 s), stereotyped Ca(2+) responses that were inhibited by the selective alpha 7-nAChRs antagonists methyllycaconitine (MLA) and alpha-bungarotoxin, and greatly increased and prolonged by the allosteric modulator PNU-120596 (1 mu M). Unexpectedly, brief (1-5s), repetitive Ca(2+) transients of sub-micrometric dimension were observed in filopodia of cells expressing alpha 7-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 alpha 7-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 alpha 7-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 alpha 7-nAChRs will reveal the physiological role of these receptor-channels. (C) 2008 Elsevier Ltd. All rights reserved.
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