Functional properties of alpha7 nicotinic acetylcholine receptors co-expressed with RIC-3 in a stable recombinant CHO-K1 cell line.
Heterologous functional expression of alpha7 nicotinic acetylcholine receptors (nAChRs) is difficult to achieve in mammalian cell lines, and the reasons have been associated with a lack of expression of the putative chaperone factor RIC-3. Here, we describe the generation and functional and pharmacological characterization of a Chinese hamster ovary (CHO)-K1 cell line co-expressing the human alpha7 nAChR and RIC-3. Stable recombinant cells expressing alpha7 nAChR on the plasma membrane were selected by binding of fluorochrome-conjugated alpha-bungarotoxin and fluorescence-activated cell sorting. The presence of functional alpha7 channels was demonstrated by whole cell patch clamp recordings. Nicotine and acetylcholine induced rapid desensitizing currents with 50% effective concentration values of 14 and 37 microM, respectively, with agonist-evoked currents detected in approximately 75% of the cell population. Surprisingly, when tested in a FLIPR (Molecular Devices, Sunnyvale, CA) Ca(2+) assay, activation of alpha7 nAChRs was measured only when nicotinic agonists were applied either in the presence of the positive allosteric modulator (PAM) PNU-120596 or after pretreatment of cells with the tyrosine kinase inhibitor genistein. No Ca(2+) influx was measured upon addition of agonists alone or together with allosteric potentiators such as 5-hydroxyindole that predominantly increase the apparent peak amplitude without robustly affecting the current desensitization rate, as exemplified by PNU-120596. These results show that functional alpha7 nAChRs can stably be expressed in the non-neuronal CHO-K1 cell line. This recombinant cell system is useful for characterization of alpha7 nAChRs and to study the mechanism of action of chemical modulators, in particular the detection of PAMs capable of slowing receptor desensitization kinetics.
Roncarati
Renza
R
Discovery Research, Siena Biotech S.p.A., Siena, Italy. rroncarati@sienabiotech.it
Seredenina
Tamara
T
Jow
Brian
B
Jow
Flora
F
Papini
Silvia
S
Kramer
Angela
A
Bothmann
Hendrick
H
Dunlop
John
J
Terstappen
Georg C
GC
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D007555:Isoxazoles / Q000494:pharmacology; D007700:Kinetics; D008856:Microscopy, Fluorescence; D018722:Nicotinic Agonists / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000494:pharmacology; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D064569:alpha7 Nicotinic Acetylcholine Receptor
C508388:1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea; C579167:Chrna7 protein, human; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D007555:Isoxazoles; D018722:Nicotinic Agonists; D010671:Phenylurea Compounds; C496701:RIC3 protein, human; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor; D012313:RNA; D019833:Genistein; D002118:Calcium