In this study, we used site-directed mutagenesis to either block or mimic phosphorylation of residues within the C1 domain of the NR1 subunit that are thought to be targets for various serine/threonine kinases. Expression of these subunits in HEK293 cells in combination with either the NR2A or NR2B subunit generated glutamate-activated receptors that were all inhibited to some degree by ethanol. Of the mutants tested, both a single site mutant (S890A/D) and a multi-site mutant (ALLDE) showed changes in ethanol inhibition when expressed with the NR2A subunit. These results suggest that phosphorylation of specific residues contained within the C1 cassette of the NR1 subunit may contribute to the overall sensitivity of NMDA receptors to ethanol.
Previous studies have investigated the phosphorylation status of NR1 subunits and report that multiple residues within the C1 domain are targets of serine/threonine kinases including PKC and PKA. For example, phosphorylation of NR1 fusion proteins by purified PKC is dramatically reduced (but not eliminated) when serines at positions 889, 890, 896, and 897 are mutated to alanine (Tingley et al., 1997
). Using a similar approach, these authors reported that PKA mediated phosphorylation is largely restricted to serines 896 and 897. Antibodies generated against specific phosphorylated residues of NR1 largely confirmed these findings with PKC activators increasing the intensity of the anti-phosphoserine 890 and 896 antibody signal while treatment with the PKA activator forskolin enhanced anti-phosphoserine 897 immunoreactivity (Tingley et al., 1997
). Cellular imaging studies revealed that some of these sites also affect receptor clustering and surface expression. In particular, mutation of serine 890 to alanine was sufficient to prevent the re-distribution of NR1 clusters during treatment of transfected fibroblasts with PKC activators (Ehlers et al., 1995
; Tingley et al., 1997
). This observation is particularly interesting with respect to the present study as among the single phosphorylation sites tested, only the S890 pair of mutants showed a statistically significant difference in ethanol inhibition. It is not clear whether this difference reflects changes in receptor clustering or whether some other biophysical property that affects ethanol inhibition is involved. Clearly, this effect does not involve differences in macroscopic receptor kinetics as although both S890 mutants showed a similar reduction in SS/Pk ratios, the ethanol sensitivity of these two mutants was different. The effects of PKC activation on NMDA receptor currents are complex and appear to involve other kinases including Src family tyrosine kinases (Lu et al., 1999
). In addition, PKC activators can potentiate NMDA receptor currents even in mutants lacking all known phosphorylation sites suggesting that other protein targets are involved (Liao et al., 2001
; Zheng et al., 1999
). Nonetheless, these results suggest that the phosphorylation status of S890 could influence the degree of ethanol inhibition of neuronal NMDA receptors.
Interestingly, the anti-phosphoserine 890 antibody has also been used to examine NMDA receptor expression in a subset of brain regions. This antibody gave strong signals on western blots from cultured cerebellar granule neurons (Sanchez-Perez and Felipo, 2005
) and variable but discrete labeling of neurons within cortical and striatal regions of rat brain slices (Liu et al., 2004
). In cerebellar granule neurons, the intensity of the anti-phosphoserine 890 antibody signal was reduced by inhibitors of PKC-γ or δ/θ while blockers of PKC-α/β isoforms had less effect (Sanchez-Perez and Felipo, 2005
). In cortex and striata, the expression of phosphorylated S890 immunofluorescence was almost completely restricted to neurons that express the calcium binding protein parvalbumin (Liu et al., 2004
). Parvalbumin positive neurons are typically GABAergic interneurons that show high-frequency patterns of firing during depolarization (Markram et al., 2004
). Together with the results of the present study, these results suggest the possibility that modulation of ethanol inhibition of NMDA receptors by serine phosphorylation may be highly restricted to neurons that show strong expression of both PKC γ/δ/θ and phosphorylated NR1 serine 890.
The other receptor that displayed a change in ethanol inhibition, albeit modest, was the ALLDE mutant that contains aspartate or glutamate at 6 different positions including serines at positions 890 and 897. In a previous study, we showed that NR1 S897D mutants were also inhibited to a greater degree by ethanol than S897A mutants suggesting that in the ALLDE mutant, either S890D or S897D may confer additional ethanol inhibition. However, these aspartate substitutions were also contained in the SSDD and ATD mutants that in the present study showed no significant change in ethanol inhibition. These findings indicate that the ability of specific serine/threonine phosphorylation site mutants to enhance ethanol inhibition may be influenced by substitutions at neighboring sites.
An important caveat regarding the conclusions of the present study is that all experiments were conducted with mutants designed to block or mimic phosphorylation. While alanine substitution clearly precludes phosphorylation of that site by endogenous kinases, replacing serine/threonine residues with aspartate or glutamate can only approximate the additional negative charge associated with the phosphorylated residue. Despite this limitation, there is ample evidence from a variety of studies that phospho-site mutants produce effects that are consistent with those produced by a kinase. For example, introducing aspartate residues at S896 and S897 of the NR1 subunit enhances surface expression of this protein when it is expressed alone in HEK293 cells or neurons (Scott et al., 2003
; Xia et al., 2001
). This is presumably due to masking of an ER retention domain that serves as a quality control check for mis-folded or mismatched subunits. Adding the NR2 subunit also overcomes this retention signal even in non-phosphorylated (eg. alanine containing) NR1 mutants that would be retained intracellularly if expressed alone (see in this study and Figure 1 in Xu et al, 2006
). Similarly, in studies with the serotonin transporter (SERT), replacing threonine 276 with alanine had no effect of transporter function but substitution with aspartate to mimic phosphorylation enhanced 5-HT uptake similar to that observed after activation of PKG (Ramamoorthy et al., 2007
). Finally, substitution of glutamate for threonine 107 in the BK potassium channel alpha subunit mimicked the effects of CaMKII phosphorylation on channel modulation and alcohol sensitivity (Liu et al., 2006
). Together, the results from these studies suggest that amino acid substitution is a reasonable first step in testing the role that phosphorylation plays in regulating receptor function and ethanol sensitivity.
In summary, the findings of the present study complement those from previous reports and suggest that certain phosphorylation sites within the C1 domain of the NR1 subunit can affect the degree of ethanol inhibition of NR1/NR2 receptors. While these sites do not appear to define the major site of action for ethanol, the phosphorylation status of serines 890 and 897 in the NR1 subunit may be particularly important in determining the overall ethanol sensitivity of NMDA receptors.