Functional properties of α7 nicotinic acetylcholine receptors co-expressed with RIC-3 in a stable recombinant CHO-K1 cell line

Heterologous functional expression of alpha 7 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 alpha 7 nAChR and RIC-3. Stable recombinant cells expressing alpha 7 nAChR on the plasma membrane were selected by binding of fluorochrome-conjugated alpha-bungarotoxin and fluorescence-activated cell sorting. The presence of functional alpha 7 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 mu M, respectively, with agonist-evoked currents detected in approximately 75% of the cell population. Surprisingly, when tested in a FLIPR (TM) (Molecular Devices, Sunnyvale, CA) Ca2+ assay, activation of alpha 7 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 Ca2+ 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 alpha 7 nAChRs can stably be expressed in the non-neuronal CHO-K1 cell line. This recombinant cell system is useful for characterization of alpha 7 nAChRs and to study the mechanism of action of chemical modulators, in particular the detection of PAMs capable of slowing receptor desensitization kinetics.