Effects of ethanol on phosphorylation site mutants of recombinant N-methyl-D-aspartate receptors

Abstract

N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by the neurotransmitter glutamate. These channels are highly expressed by brain neurons and are critically involved in excitatory synaptic transmission. Results from previous studies show that both native and recombinant NMDA receptors are inhibited by ethanol at concentrations associated with signs of behavioral impairment and intoxication. Given the important role that NMDA receptors play in synaptic transmission and brain function, it is important to understand the factors that regulate the ethanol inhibition of these receptors. One dynamic mechanism for regulating ethanol action may be via phosphorylation of NMDA subunits by serine-threonine and tyrosine kinases. Both NR1 and NR2 subunits contain multiple sites of phosphorylation; and in the NR1 subunit, most of these are contained within the C1 domain, a carboxy-terminal cassette that is subject to alternative splicing. Although results from our previous studies suggest that single phosphorylation sites do not greatly affect ethanol sensitivity of NMDA receptors, it is likely that in vivo, these subunits are phosphorylated at multiple sites by different kinases. In the present study, we constructed a series of NMDA receptor mutants at serine (S) or threonine (T) residues proposed to be sites of phosphorylation by protein kinase A and various isoforms of protein kinase C. Ethanol (100mM) inhibited currents from wild-type NR1/2A and NR1/2B receptors expressed in human embryonic kidney 293 cells by approximately 25 and 30%, respectively. This inhibition was not different in single-site mutants expressing alanine (A) or aspartate/glutamate (D/E) at positions T879, S896, or T900. The mutant NR1(S890D) showed greater ethanol inhibition than NR1(890A) containing receptors, although this was only observed when it was combined with the NR2A subunit. Ethanol inhibition was not altered by aspartate substitution at four serines (positions 889, 890, 896, and 897) or when T879D was added to the four serine-substituted mutant. Ethanol inhibition was increased when T900E was added to the five serine-/threonine-substituted mutants, but again this was selective for NR2A containing receptors. Together with previously published data, these findings suggest that modification of putative phosphorylation sites could contribute to the overall acute ethanol sensitivity of recombinant NMDA receptors. Supported by R37AA009986.

Figure 1

NR1 Subunit Phosphorylation Sites. The schematic diagram shows the organization of the NR1 subunit. Shaded boxes denote the transmembrane domains (note that the second transmembrane domain does not fully traverse the membrane) and open boxes show location of three discrete C-terminal domains. The amino acids shown below the diagram represent the sequence of a portion of the C1 domain with residues in boxes identified as potential phosphorylation sites for PKC or PKA. The table lists the various isoforms of PKC and PKA and their potential targets for phosphorylation within the C1 domain sequence.

Figure 2

Effects of ethanol on wild-type and single-site mutant NMDA receptors. Graphs show the inhibition of glutamate-evoked currents (10 μM plus 10 μM glycine) by 100 mM ethanol for each of the mutant receptors tested. Results for alanine-substituted mutants are shown in red while those for aspartate/glutamate mutants are in green. Values represent the mean (± S.E.M.) percent inhibition by ethanol. Sample sizes for each mutant tested are shown in Table 1. Note that values for the controls for wild-type NR1/2A and NR1/2B receptors were pooled and are shown in each graph for comparison. Symbol(*): value significantly different from S890A; p<0.05, 1-way Anova with Bonferroni’s multiple comparison test.

Figure 3

Effects of ethanol on wild-type and multiple-site mutant NMDA receptors. Graphs show the inhibition of glutamate-evoked currents (10 μM plus 10 μM glycine) by 100 mM ethanol for each of the mutant receptors tested. Values represent the mean (± S.E.M.) percent inhibition by ethanol. Sample sizes for each mutant are shown in Table 1. Note that values for the controls for NR1/2A and NR1/2B receptors were pooled and are shown in each graph for comparison. Symbol(*): value significantly different from control; p<0.05, unpaired t-test. Abbreviations: SSDD (S889D/S890D/S896D/S897D), ATD (T879D/S889D/S890D/S896D/S897D), ALLDE (T879D/S889D/S890D/S896D/S897D/T900E).

Figure 4

Concentration-response relationship for ethanol inhibition of NR1 S890A, S890D and ALLDE mutant receptors. Graphs show the percent inhibition of glutamate-evoked currents (10 μM plus 10 μM glycine; N=6–10 cells for each concentration) by various concentrations of ethanol (10–500 mM). A) Ethanol inhibition of NR1(S890A) and NR1(S890D) mutants. The dashed line represents data for wild-type NR1/NR2A receptors (see next panel). B) Ethanol inhibition of NR1/NR2A and the NR1(ALLDE) mutant. Non-linear regression analysis resulted in calculated IC₅₀ values for ethanol inhibition of 193.5 mM (NR1/NR2A; 95% confidence intervals 153.2–244.4), 225.5 mM (S890A; 95% confidence intervals 183.9–269.1), 148.0 mM (S890D; 95% confidence intervals 132.2–165.7), and 162.9 mM (ALLDE; 95% confidence intervals 130.8–202.8). Traces adjacent to each figure are representative examples of currents in the absence (bottom trace of each pair) and presence (top trace of each pair) of 100 mM ethanol. Currents after washout of the ethanol solution have been removed for clarity. Scale bars: X-axis (2 sec), Y-axis (S890A, 50 pA, S890D, 200 pA, NR1, 100 pA, ALLDE, 200 pA).