Dephosphorylation of GluN2B C-terminal tyrosine residues does not contribute to acute ethanol inhibition of recombinant NMDA receptors

Abstract

N-methyl-d-aspartate (NMDA) receptors are ion channels activated by the neurotransmitter glutamate and are highly expressed by neurons. These receptors are critical for excitatory synaptic signaling and inhibition of NMDA receptors leads to impaired cognition and learning. Ethanol inhibits NMDA currents at concentrations associated with intoxication and this action may underlie some of the behavioral effects of ethanol. Although numerous sites and mechanisms of action have been tested, the manner in which ethanol inhibits NMDA receptors remains unclear. Recent findings in the literature suggest that ethanol, via facilitation of tyrosine phosphatase activity, may dephosphorylate key tyrosine residues in the C-terminus of GluN2B subunits resulting in diminished channel function. To directly test this hypothesis, we engineered GluN2B mutants that contained phenylalanine in place of tyrosine at three different sites and transiently expressed them with the GluN1 subunit in human embryonic kidney (HEK) cells. Whole-cell patch clamp electrophysiology was used to record glutamate-activated currents in the absence and presence of ethanol (10-600 mM). All mutants were functional and did not differ from one another with respect to current amplitude, steady-state to peak ratio, or magnesium block. Analysis of ethanol dose-response curves showed no significant difference in IC50 values between wild-type receptors and Y1252F, Y1336F, Y1472F or triple Y-F mutants. These findings suggest that dephosphorylation of C-terminal tyrosine residues does not account for ethanol inhibition of GluN2B receptors.

Figure 1

Schematic cartoon of the NMDA GluN2B subunit. The extracellular N-terminus, four transmembrane domains denoted by the four blue boxes, and the intracellular C-terminus are depicted in the diagram. Red circles on the intracellular C-terminus indicate the approximate location of tyrosine residues 1252, 1336, and 1472. Also depicted are comparisons of wild-type (top) and mutant (bottom) amino acid sequences for each mutation studied.

Figure 2

Concentration-response relationship for glutamate activation of mutant and wild-type GluN2B-containing NMDARs. (A) Example traces showing currents from wild-type NMDAR transfected cell during exposure to to 0.03 μM (black), 1 μM (light blue), and 10 μM glutamate (dark blue). (N = 6 – 8 cells). All recordings performed in the presence of 10 μM glycine. (B) Currents are expressed as percent of maximal response to various concentrations of glutamate (0.03–10 μM). Non-linear regression yielded EC₅₀ values of 0.92 μM (wild-type), 0.88 μM (Y1472F), 0.95 μM (Y1336F), 0.78 μM (Y1252F), and 0.69 μM (triple mutant). Inset bar graph shows a comparison of ln EC₅₀ values expressed as mean ± SEM.

Figure 3

Current-voltage relationship of wild-type and phospho-mutant NMDARs. (A) Cells were voltage clamped at 0 mV and then stepped to −80 mV for 200 ms followed by a 1.3s ramp to +80 mV for another 200 ms before returning to 0 mV. All recordings were performed in the presence of 2 mM Mg⁺⁺. (N = 7 – 9 cells) (B) Current-voltage relationship of wild-type and mutant receptors after normalizing current to that obtained at +80 mV.

Figure 4

Concentration-response effect of ethanol on wild-type and mutant GluN2B-containing NMDARs. (A) Example traces from a cell expressing wild-type and mutant GluN2B receptors. Agonist-only (blue) current; agonist + 100 mM EtOH (black). (B) Data are expressed as percent inhibition by ethanol (10–600 mM) of glutamate-evoked currents. (N = 6–8 cells per concentration). Nonlinear regression yielded estimated IC₅₀ values for ethanol of 136 mM (wild-type), 135 mM (Y1472F), 118 mM (Y1336F), 104 mM (Y1252F), and 156 mM (triple mutant). Inset bar graph shows a comparison of ln IC₅₀ values expressed as mean ± SEM.