Functional Deactivation of the Major Neuronal Nicotinic Receptor Caused by Nicotine and a Protein Kinase C- Dependent Mechanism

SUMMARY The effect of nicotine on the major human neuronal nicotinic receptor (␣4␤2 subtype) was studied in permanently transfected HEK 293 cells. Prolonged exposure to low concentrations of nicotine (1 M) increased epibatidine binding but functionally deactivated the nicotinic receptor, abolishing Ca 2ϩ influx in response to an acute nicotine challenge. Deactivation could also be caused by down-regulating protein kinase C (PKC) activity with 0.5 M phorbol-12,13-dibutyrate or briefly incubating cells with the PKC inhibitor NPC-15437. Recovery from receptor deactivation caused by either nicotine treatment or PKC inhibition occurred slowly (4 -6 hr). Reversal of nicotineinduced deactivation was accelerated by the addition of inhibitors of protein phosphatases 2A and 2B. These data suggest a hypothetical mechanism of nicotine-induced deactivation that involves dephosphorylation of nicotinic receptors at PKC phosphorylation sites. The action of nicotine in the brain is mediated by a family of oligomeric ion channels whose opening is regulated by the binding of the neurotransmitter acetylcholine and drugs such as nicotine. Eleven different mammalian nAChR subunits (␣2-9 and ␤2-4) have been cloned (for reviews, see Refs. 1 and 2), and all the neuronal nicotinic receptors display a pronounced selectivity for Ca 2ϩ relative to Na ϩ (3-5). The most abundant receptor is composed of the ␣4 and ␤2 subunits with a stoichiometry of 2␣:3␤ (6) and is responsible for ϳ85% of the high affinity nicotine binding in the brain (6 -8). When nicotine is administered chronically to rats, a ϳ2-fold up-regulation of high affinity brain nicotinic receptors has been observed (9). This seems to be due to a dramatic decrease in the rate of degradation of the receptor in the cell membrane after nicotine treatment (10). The basis for this change in turnover is obscure. It has also been shown that chronic nicotine exposure leads to what has been termed "functional deactivation" of receptors to distinguish it from short term desensitization (11). Deactivation of neuronal nAChRs was first described and distinguished from receptor desensitization by Simasko et al. (12). The fact that chronic nicotine administration results in an increase in receptor number, coupled with a functional deactivation, suggests a mechanism for the addictive effects of nicotine (13). In this model, withdrawal of nicotine from an individual chronically exposed to the drug would result in reactivation of excess receptors, leading to craving, and prompting a further deactivating dose of the drug (13). Therefore, the relationship between nicotinic receptor number and intrinsic activity is a critical issue. In this report, we show that HEK 293 cells stably expressing the human ␣4␤2 nicotinic receptor subtype, after prolonged exposure to nicotine, display both a dramatic up-regulation of the receptor, together with a functional deactivation that is easily distinguished from simple short term desensitization. This functional deactivation can also be achieved by inhibition of PKC activity in the cells. Furthermore, phosphatase inhibitors increase the rate of recovery from nicotine-induced deactivation of the ␣4␤2 nicotinic receptor. Our data also indicate that the open state conformation per se, rather than calcium ion influx, directs formation of a deactivated receptor structure. Experimental Procedures Cloning human nicotinic receptor subunits. A human ␣4 subunit cDNA probe was generated by RT-PCR of total human brain RNA, with primers based on the rat ␣4 sequence. The resulting PCR fragment, which contained part of the human ␣4 sequence spanning nucleotides 755-985 (all numbering is from the ATG initiatio