A Systematic Evaluation of High-Affinity α4β2 Nicotinic Acetylcholine Receptor Phosphorylation

Addiction to nicotine is mediated through nicotinic acetylcholine receptors containing the α4 and β2 subunits (α4β2* nAChRs). Upon nicotine exposure, α4β2* nAChRs are activated, then rapidly desensitized. Chronic nicotine exposure, as would be observed in the brain of a smoker, is also associated with a robust upregulation of α4β2* nAChR expression, which is post-translationally mediated. As such, the molecular mechanisms of α4β2* nAChR regulation are paramount to our understanding of the physiological and behavioral effects of nicotine. In vitro and cell biological studies have demonstrated that the assembly, trafficking and desensitization of nicotinic receptors are all regulated by phosphorylation of the receptor by PKA and PKC. However, phosphorylation of α4β2* nAChRs has not been demonstrated in vivo and other important brain kinases, like CaMKII, have not been evaluated. In this study, we will utilize the cutting edge mass spectrometry techniques and expertise at the Yale/NIDA Neuroproteomics Center to directly assess phosphorylation of α4β2* nAChRs with two major aims: (1) To evaluate phosphorylation of α4β2* nAChRs in the mouse brain at baseline and following acute and chronic nicotine exposure, and (2) To assess the ability of CaMKIIα to phosphorylate α4 and β2 nAChR subunits. The results from this study will provide a necessary extension of previous studies of α4β2 nAChR regulation by characterizing receptor subunit phosphorylation in vivo. Additionally, we will, for the first time, evaluate the effects of nicotine on nAChR phosphorylation and explore the role of a novel interacting kinase, CaMKIIα. Results from these studies will improve our understanding of the cell signaling mechanisms underlying nicotine addiction and may also inform studies of other disorders linked to nAChR dysfunction, including schizophrenia, depression and other addictions.