Antidepressant interactions with the NMDA NR1-1b subunit

Abstract

The targets for tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), and selective norepinephrine reuptake inhibitors (SNRIs) are known to be the serotonin and norepinephrine transport (reuptake) proteins which are embedded in presynaptic nerve terminals and function to bring these neurotransmitters from the synaptic cleft back into the presynaptic neuron. Using a combination of intrinsic and extrinsic fluorescence quenching, Stern-Volmer analysis, and protease protection assays, we have shown that therapeutics from each of these three classes of antidepressants bind to the extracellular S1S2 domain of the NR1-1b subunit of the NMDA receptor. These results are in agreement with recent work from our lab demonstrating the interaction of antidepressants with the S1S2 domain of the GluR2 subunit of the AMPA receptor, another member of the ionotropic glutamate receptor subfamily, as well as work from other labs, and continue the discussion of the involvement of ionotropic glutamate receptors in depression.

Figure 1

Crystal structure of the NR1-1b S1S2 domain (PDB code 1PB7) [4] with each of the four tryptophans in blue.

Figure 2

The TCAs, SSRIs, and SNRIs above, with the exception of citalopram, were found to bind to the NMDA NR1-1b S1S2 domain.

Figure 3

Intrinsic fluorescence quenching of the NR1-1b S1S2 domain with (a) TCAs and (b) SSRIs. To 5.29 μM NR1-1b S1S2 in buffer 3 was added quenchers in the following final concentrations: (a) TCAs-desipramine, trimipramine, or imipramine: 137 μM, maprotiline: 3.18 mM, nortriptyline: 2.08 mM; (b) SSRIs-paroxetine: 3.75 mM, sertraline: 1.88 mM, fluvoxamine: 2.81 mM.

Figure 4

ANS binding. To 5.29 μM NR1-1b S1S2 in buffer 3 was added ANS in a 1 : 1 molar ratio along with a TCA, SSRI, or SNRI in the following final concentrations: (a) TCAs-desipramine (- - -): 137 μM, trimipramine (⋯): 137 μM, maprotiline (▪): 3.18 mM; (b) TCAs-nortriptyline (⋯): 2.08 mM; (c) SNRI-venlafaxine (- - -): 2.76 mM; (d) SSRI-fluvoxamine (- - -): 2.81 mM, paroxetine (⋯): 3.75 mM, sertraline (▪): 1.88 mM; (e) SSRI-fluoxetine (- - -): 862 μM. In all figures the solid line is the extrinsic fluorescence of ANS:NR1-1b S1S2. In all figures, intensities were corrected by subtracting the drug and ANS blank.

Figure 5

Protease protection assay of NR1-1b S1S2:TCA complexes. To 5.29 μM NR1-1b S1S2 in buffer 3 was added the final quenching concentrations of TCAs prior to addition of a 1 : 1 molar ratio of trypsin. The results of the trypsin cuts were visualized by 12% SDS PAGE. Lanes were loaded as follows, (1): desipramine-bound, (2): imipramine-bound, (3): apo NR1-1b S1S2, (4): nortriptyline-bound, (5): maprotiline-bound, (6): trimipramine-bound, (7): trypsin, (8): uncut NR1-1b S1S2.