Abstract
Activation of nicotinic acetylcholine receptors containing α4 and β2 subunits (α4/β2* nAChRs) in the mammalian brain is necessary for nicotine reinforcement and addiction. We previously identified interactions between α4/β2* nAChRs and calcium/calmodulin-dependent protein kinase II (CaMKII) in mouse and human brain tissue. Following co-expression of α4/β2 nAChR subunits with CaMKII in HEK cells, mass spectrometry identified 8 phosphorylation sites in the α4 subunit. One of these sites and an additional site were identified when isolated α4/β2* nAChRs were dephosphorylated and subsequently incubated with CaMKII in vitro, while 3 phosphorylation sites were identified following incubation with protein kinase A (PKA) in vitro. We then isolated native α4/β2* nAChRs from mouse brain following acute or chronic exposure to nicotine. Two CaMKII sites identified in HEK cells were phosphorylated, and 1 PKA site was dephosphorylated following acute nicotine administration in vivo, whereas phosphorylation of the PKA site was increased back to baseline levels following repeated nicotine exposure. Significant changes in β2 nAChR subunit phosphorylation were not observed under these conditions, but 2 novel sites were identified on this subunit, 1 in HEK cells and 1 in vitro. These experiments identified putative CaMKII and PKA sites on α4/β2* nAChRs and novel nicotine-induced phosphorylation sites in mouse brain that can be explored for their consequences on receptor function.
Highlights
High-affinity nicotinic acetylcholine receptors containing the α4 and β2 subunits (α4/β2* nAChRs, where * denotes other, potentially unidentified, subunits) are essential for the rewarding and reinforcing properties of nicotine in the mouse [1,2,3,4]. α4/β2* nAChRs are intrinsic ion channel-containing proteins that flux positive ions, including calcium, in response to nicotine or the endogenous neurotransmitter acetylcholine
We identified 8 serine residues on the α4 subunit with significantly increased phosphorylation following co-expression with CaMKIIα-mRuby, compared to those co-expressing mRuby alone (Figure 2, see Supplementary Materials for representative spectra)
These findings identify distinct phosphorylation sites on the α4 subunit for distinct phosphorylation sites on the α4 subunit for protein kinase A (PKA) and CaMKIIα, as well as sites that mayand be CaMKIIα, as well sites that may be phosphorylated at as baseline by these orphosphorylated other kinases. at baseline by these or other kinases
Summary
High-affinity nicotinic acetylcholine receptors containing the α4 and β2 subunits (α4/β2* nAChRs, where * denotes other, potentially unidentified, subunits) are essential for the rewarding and reinforcing properties of nicotine in the mouse [1,2,3,4]. α4/β2* nAChRs are intrinsic ion channel-containing proteins that flux positive ions, including calcium, in response to nicotine or the endogenous neurotransmitter acetylcholine. In addition to initiating calcium signaling, nicotine increases the number of nAChRs and can alter the associated proteome of α4/β2* nAChRs in mouse and human brain [6]. Biochemical studies have identified a number of interacting proteins that regulate assembly, trafficking, and function of α4/β2* nAChRs. For example, the chaperone 14-3-3 has been identified as an α4 nAChR subunit interactor in multiple studies [6,7,8], and this interaction can alter the physiological properties of α4/β2* nAChRs [7,9,10]. Phosphorylation of the α4 nAChR subunit by the calcium-dependent protein kinase PKC, as well as dephosphorylation by the calcium-dependent phosphatase calcineurin, regulate desensitization of α4/β2* nAChRs in response to prolonged nicotine exposure [13,14,15]. Biochemical studies have established an important role for nAChR phosphorylation in regulation of nicotine signaling through its receptors
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