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.
1. Introduction
Ionotropic glutamate receptors
(iGluRs) are a family of ligand-gated ion channels located on the postsynaptic
neural membrane which open in response to extracellular binding of the neurotransmitter glutamate. These
receptors play an important role in memory, learning, development, and neu-ral
plasticity. As such, their misregulation has been implicated in a number of
disease states such as the ischaemic stroke cascade, schizophrenia, epilepsy,
and Alzheimer’s, Huntington’s, and Parkinson’s Disease.
The functional unit of each member
of the iGluRfamily (NMDA, AMPA, and kainate receptors) is a tetramer;
homotetramer for AMPA and kainate receptors and heterotetramer, composed of NR1
(one of eight alternatively spliced versions) and NR2 subunits (one of four
versions encoded by four different genes) for NMDA receptors. Subunits from any
of these family members are modular in nature, containing seven domains: an
amino terminal domain (ATD) important for the interaction between subunits, a
nonsequential extracellular S1S2 neurotransmitter binding do main, three
membrane spanning domains, a re-entrant loop which forms the pore of the ion
channel when the tetramer is assembled, and an intracellular C-terminal tail. Soluble extracellular GluR2 (AMPA) and NR1-1b (NMDA) S1S2 domains have
been constructed
by eliminating all three transmembrane
spanning regions plus the re-entrant loop and linking the S1 and S2 domains by two
amino acids (GT) [1–4].
These S1S2 domains have been shown to possess
near native binding affinities for both agonists and antagonists and their
structures have been studied by X-ray crystallography (Figure 1) [1–4].
Crystal structure of the NR1-1b S1S2 domain (PDB code 1PB7) [4] with each of the four tryptophans in blue.
In the
past twenty years, there has been an accumulation of data suggesting that
iGluRs are also involved in the mechanism of action of antidepressants. This is
a de-parture from the idea that antidepressants function solelyby either
inhibiting monoamine oxidase (MAOIs) or the serotonin or norepinephrine
transport proteins (TCAs, SSRIs, or SNRIs). Data linking iGluRs to
antidepressant activities include experiments demonstrating direct binding of tricyclic
antidepressants to the extracellular S1S2 region of AMPA receptors [5, 6], open channel block of NMDA receptors
by antidepressants [7, 8], and altered expression and phosphorylation of AMPA
receptors by antidepressants [9, 10]. In
addition, AMPA receptor potentiators and NMDA receptor antagonists have been
shown to have antidepressant-like effects [9, 11–13]. This data has led to the suggestion that
increased activity of AMPA receptors leads to an increase in the expression of
brain derived neurotrophic factor (BDNF), which in turn promotes
neurogenesis in the hippocampus, leading to antidepressant activities [9].
The intrinsic and
extrinsic fluorescence studies presented here add to the body of knowledge suggesting
a role for NMDA receptors in antidepressant activities. Specifically, they demonstrate
that five tricyclic antidepressants (TCAs) (desipramine, trimipramine,
maprotiline, nortriptyline, and imipramine), four selective serotonin reuptake
inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, and fluoxetine), and
one selective norepinephrine reuptake inhibitor (SNRI) (venlafaxine), see
Figure 2, bind to the S1S2 domain of the NR1-1b subunit of the NMDA receptor.
The only antidepressant that showed no binding in any of our studies, at
concentrations as high as 5.21 mM, was the SSRI citalopram.
The TCAs, SSRIs, and SNRIs above, with the exception of citalopram, were found to bind to
the NMDA NR1-1b S1S2 domain.
2. Experimental Procedures2.1. Protein Expression and Purification of the NMDA NR1-1b S1S2 Domain
The cloning
plasmid for the NMDA NR1-1b S1S2 domain, provided by Eric Gouaux (Oregon Health
and ScienceUniversity), was chemically transformed
into E. coli Origami B(DE3) cells.
The expression system was designed as follows: (His)8-TSG-LVPRG(thrombin
cut site)-S1(394-544)-GT-S2(663-800). Expression and purification of NMDA
NR1-1b S1S2 protein were performed as in Furukawa and Gouaux [4].
2.2. Intrinsic Fluorescence
Intrinsic fluorescence studies were performed in a Cary Eclipse fluorometer at 25°C. All
emission scans were acquired from 300 to 400 nm upon excitation at 290 nm, with
10 nm excitation and emission slit widths in a 10 mm-path-length cell. Excitation
was performed at 290 nm to minimize interference from both tyrosine residues in
the NMDA NR1-1b S1S2 domain as well as the aromatic antidepressants being
studied. Before data acquisition, samples were incubated for thirty seconds in
the sample holder at 25°C. Analysis of the buffer emission signal showed that
it contributed less than half a percent to the fluorescence intensity of the
NR1-1b S1S2 domain. Thus, data was reported without a buffer subtraction.
Protein samples
were at a final concentration of 5.29μM in buffer 3 (10 mM MES, pH 6.5, 25 mM
NaCl, 1 mM glycine). All antidepressants were solubilized in 100% methanol to
the highest attainable concentrations. To account for any effects methanol had
on fluorescence intensity, protein spectra with methanol were collected,
allowing direct comparison between samples with and without antidepressant.
Each data set consisted of at least three independent emission spectra of
identically prepared samples. The data is reported with error determined at the
95% confidence level.
2.3. Intrinsic Quenching Studies
The fluorescence intensity of the NR1-1b S1S2 domain was determined in the presence
and absence of eachantidepressant with the appropriate blank subtraction. The
final concentrations of each TCA-desipramine, imipramine, trimipramine,
nortriptyline, and maprotiline were 136.9μM, 136.9μM, 136.9μM, 2.08 mM, and 3.18 mM,
respectively. The final concentrations of each SSRI-paroxetine, citalopram, and
sertraline were 3.75 mM,
1.88μM, and 1.88 mM, respectively. For all samples, the final concentration of
methanol was kept at 1.8%.
2.4. Kd Determination
Antidepressants were added to final concentrations as follows: nortriptyline:
1.24–3.61 mM,
imipramine or desipramine: 8–570μM,
trimipramine: 130–570μM,
sertraline: 0.74–1.84 mM, fluvoxamine: 0.74–2.37 mM. A double reciprocal plot was used to determine Kd,
which derives from the Benesi-Hildebrand relationship, provided there is a 1 : 1
binding stoichiometry. The equation is: 1/ΔF=1/((F∞−Fo)⋅Kd[L])+1/(F∞−Fo), where Fo is the fluorescence intensity in the
absence of ligand, F∞ is
the fluorescence intensity with saturating concentration of ligand, F is thefluorescence intensity at some particular [ligand], ΔF is (F−Fo), and Kd is the
dissociation constant. When 1/ΔF is plotted against 1/[L], a linear relationship exists.
The intercept on the x-axis is −Kd. [14].
Note, this method to determine Kd is based on the formation of
ground state NMDA NR1-1b S1S2 domain: antidepressant complexes, and could be
altered by dynamic quenching.
2.5. Stern-Volmer Analysis
Acrylamide
(Sigma) ranging in final concentration from 16.3 to 113.9 mM was added to 5.29μM NR1-1b S1S2 premixed with antidepressant (at final concentrations as above
for intrinsic quenching studies, with the exception of sertraline which was at
0.94 mM). Plots of (F0/F)−1 versus
acrylamide concentration were constructed.
2.6. Extrinsic Fluorescence
Extrinsic fluorescence studies were performed in a Cary
Ec-lipse fluorometer at 25°C. All emission scans were acquired from 400 to 600 nm after excitation at 380 nm with 20 nm slit widths for emission and
excitation in a 10 mm-path-length cell. Before data acquisition, samples were incubated
for thirty seconds in the sample holder at 25°C. Blanks were collected both for
the antidepressants and the NR1-1b S1S2 domain. Three emission spectra of
identicallyprepared samples were averaged for each plot. 8-Anilino-1-naphthalenesulfonic
acid (ANS, TCI America), in a 1 : 1 molar ratio, was added to 5.29μM of the
NR1-1b S1S2 domain premixed with antidepressant (at final concentrations as
above for intrinsic quenching studies in addition to the SSRIs fluoxetine (8.62μM) and fluvoxamine (2.81 mM)
and the SNRI venlafaxine (29.07μM)). As with all intrinsic assays, total methanol
concentration was kept at 1.8%. Plots of intensity versus wavelength were
constructed from averaged emission spectra.
2.7. Protease Protection
Antidepressants (at final concentrations as above for the
extrinsic assays, with the exception of sertraline which was added to a final
concentration of 940μM) were added to 60μL aliquots of the NR1-1b S1S2 domain
at a final concentration of 5.29μM. The NR1-1b S1S2 domain/antidepressant complexes
were allowed to incubate on ice for 5 minutes before trypsin (Sigma: 13500 U/mg)
was added to a final concentration of 5.29μM, yielding a 1 : 1 molar ratio of
protein to trypsin. The reactions were then incubated at room temperature for
45 minutes. Digestion was stopped by the addition of sample loading dye, DTT
(50 mM), and incubation at 100°C for 10 minutes. Each reaction was visualized
by 12% SDS-PAGE.
3. Results and Discussion3.1. Intrinsic Tryptophan Fluorescence Demonstrates Binding of 5 TCAs and 3 SSRIs to the NMDA NR1-1b S1S2 Domain
The environment of the four tryptophan residues in the NMDA NR1-1b S1S2 domain,
W498, W731, W768, W792, can be monitored by fluorescence spectroscopy. Uponexcitation, the emission maximum (λmax) and fluorescence
intensity of a tryptophan residue depends upon the polarity of its environment.
By comparing the emission spectrum (λmax and fluorescence
intensity) of the apo NMDA NR1-1b S1S2 domain to antidepressant bound
complexes, it is possible to determine if the antidepressant has bound to the
S1S2 domain and caused a change in the environment of the NR1-1b S1S2
tryptophan residues. An increase in the polarity of the environment surrounding
a tryptophan causes an increase in the λmax of emission and a
decrease in the fluorescence intensity. The data in Figure 3(a), which monitors
the intensity of the tryptophan emission from the NMDA NR1-1b S1S2 domain shows
binding of the TCAs desipramine (Kd: 730 ± 150μM; for Kd data see supplemental
Figures 1S and 2S), trimipramine (Kd: 1.09±0.24 mM),
maprotiline, nortriptyline (Kd: 7.36±2.06 mM), and
imipramine (Kd: 830 ± 150μM).
The data in Figure 3(b) demonstrates the binding of the SSRIs paroxetine,
sertraline (Kd: 240 ± 90μM),
and fluvoxamine (Kd: 6.54±3.48 mM). These intrinsic fluorescence studies are
complicated by the fact that paroxetine, sertraline, citalopram, and
venlafaxine, the former two that showed evidence of binding to the NR1-1b S1S2
domain, and the latter two that did not, all had relatively high intrinsic fluorescence
signals in the emission range of tryptophan. To compensate for this, lower drug
concentrations had to be used which reduced the range of workable
concentrations, proving problematic for Kd determination. Low drug solubility in methanol (one of the only organics
compatible with a folded S1S2 domain) further complicated Kd determination for maprotiline and sertraline.
Intrinsic fluorescence quenching of the NR1-1b S1S2 domain with (a) TCAs and (b) SSRIs. To 5.29μM NR1-1b S1S2 in buffer 3 was added quenchers in the following final concentrations: (a) TCAs-desipramine, trimipramine, or imipramine: 137μM, maprotiline: 3.18 mM,
nortriptyline: 2.08 mM; (b) SSRIs-paroxetine: 3.75 mM, sertraline: 1.88 mM, fluvoxamine: 2.81 mM.
Based on this intrinsic
fluorescence quenching data, the only antidepressant which either binds in a unique
location on the S1S2 domain or causes a unique conformational change upon
binding is maprotiline, the binding of which increases, rather than quenches,
the tryptophan fluorescence emission. The unique mode of binding of maprotiline
is further supported by the shift in the maximum wavelength of emission of the maprotiline
bound S1S2 complex, from 326.24±1.54 nm (apo) to 322.04±1.83 nm (bound),
Table 1. This decrease in λmax and increase in fluorescence emission
upon binding of maprotiline are consistent with the tryptophan residues being
in a more nonpolar environment upon complex formation, either due to direct
contact with maprotiline or to a conformational change upon maprotiline binding
that places them in a more nonpolar portion of the protein.
λmax of the NR1-1b S1S2 apo
domain and of the NR1-1b drug bound complexes.
Antidepressant
λmax (nm)
Standard deviation (nm)
Desipramine
326.77
1.90
Nortriptyline
327.8
0.950
Imipramine
328.04
1.38
Trimipramine
327.27
2.19
Maprotiline
322.04
1.83
Fluvoxamine
328.57
1.91
Paroxetine
328.3
0.960
Sertraline
329.07
0
Apo
326.24
1.54
The only other
ligand which caused a statisticallysignificant shift in λmax of the
S1S2 domain is sertraline, which upon complex formation is shifted to 329.07 ±
0 nm (Table 1). This increase in λmax and decrease in fluorescence emission upon binding of sertraline, the tightest
binding antidepressant used in these studies, are consistent with the
tryptophan residues being in a more polar environment upon complex formation,
either due to direct contact with sertraline or to a conformational change upon
sertraline binding that places them in a more polar or solvent exposed portion
of the protein.
3.2. Extrinsic ANS Fluorescence Confirms the Binding of 4 TCAs and 3 SSRIs and Demonstrates Binding of Fluoxetine (SSRI) and Venlafaxine (SNRI) to the NMDA NR1-1b S2S2 Domain
If an antidepressant
bound to the S1S2 domain of the NR1-1b subunit of the NMDA receptor, but did
not change the polarity surrounding the tryptophans or changed them in such a
way that the results canceled each other out, it would not be possible to
determine binding from the intrinsic fluorescence studies described above. To
address this, the extrinsic fluor ANS was used to probe antidepressant
binding. Upon binding to hydrophobic
patches on the surface of proteins, the λmax of ANS decreases and its
emission intensity increases. As can be
seen in Figures 4(a) and 4(b), the emission spectrum of ANS bound to NR1-1b S1S2
complexes of desipramine, trimipramine, maprotiline, or nortriptyline was
significantly different from ANS bound to the apo S1S2 domain, confirming the
binding of these antidepressants. Again, the uniqueness of maprotiline binding
is confirmed by the shift in λmax of the complex. No data is
presented for the ANS bound NR1-1b S1S2-imipramine complex. In this case, a
strong, shifted fluorescence signal from the ANS, imipramine, buffer blank
suggested a direct binding of ANS to imipramine.
ANS binding. To 5.29μM 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 (- - -): 137μM, trimipramine (⋯): 137μM, 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 (- - -): 862μM. 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.
The three SSRIs
that showed binding by intrinsic fluorescence quenching, sertraline,
paroxetine, and fluvoxamine,all had spectra that were significantly different
from the apo spectrum, confirming their binding (Figure 4(d)). In addition, ANS emission spectra confirmed
the binding of both the SNRI venlafaxine (Figure 4(c)) and the SSRI fluoxetine
(Figure 4(e)), neither of which showed binding in the intrinsic fluorescence
assays. Citalopram is the only antidepressant studied which showed no evidence
of bindingin either the intrinsic or extrinsic assays at concentrations as
high as 5.21 mM.
To probe the
conformational change elicited upon binding of each of the antidepressants, a
Stern-Volmer analysis was performed. In
these experiments, the ability of an external molecule, in this case
acrylamide, to quench tryptophan fluorescence is monitored in both the apo
NR1-1b S1S2 and in the antidepressant bound NR1-1b S1S2 domains. The
Stern-Volmer constant, Ksv,
defined as the product of the bimolecular collisional constant, kq, and the lifetime of the
tryptophan residues in the absence of quencher, τ0, is measured. The bimolecular collisional constant is
a measure of the accessibility of acrylamide to the tryptophans with an
increase in kq equating to an increase in
accessibility to the fluorophore. If it is assumed that the lifetime of the
tryptophans do not change upon antidepressant binding [15–17], then changes in Ksv are due to changes in kq and therefore reflect the
change in accessibility of the tryptophan residues to acrylamide upon antidepressant
binding. The data in Table 2 shows that upon binding to any of the
antidepressants, the accessibility of the tryptophan residues in the S1S2
domain to acrylamide increased when compared to the apo protein. Within the TCAclass of antidepressants, the conformational change upon
imipramine/trimipramine bindingappears distinct from that of
desipramine/nortriptyline binding, whichappears distinct from maprotiline
binding. In these assays, the binding of
imipramine could not be distinguished from trimipramine, and the binding of
desipramine could not be distinguished from nortriptyline. Within the SSRI
class of antidepressants, all conformational changes elicited upon binding to
the NR1-1b S1S2 domain were found to alter the accessibility of the tryptophan
residues in statistically significant ways.
Ksv values for the NMDA NR1-1b S1S2 apo domain and for the NR1-1b antidepressant
complexes.
Antidepressant (family)
Ksv (μM−1)
Error in Ksv(μM−1)
Desipramine (TCA)
2.67
0.10
Nortriptyline (TCA)
2.56
0.17
Imipramine (TCA)
3.15
0.06
Trimipramine (TCA)
3.02
0.21
Maprotiline (TCA)
5.92
0.47
Sertraline (SSRI)
2.23
0.10
Paroxetine (SSRI)
3.06
0.13
Fluvoxamine (SSRI)
3.77
0.07
Apo
1.61
0.11
3.4. Protease Protection Assays Differentiate
Conformational Changes of Nortriptyline and Desipramine Binding to the NMDA
NR1-1b Domain
To differentiate the binding of desipramine and
nortriptyline as well as trimipramine and imipramine to the S1S2 domain of the NR1-1b
subunit, a limited trypsin digest was performed on the antidepressant bound
complexes. Figure 5 shows that this method was unsuccessful in differentiating
the conformational changes associated with imipramine (lane 2) and trimipramine
(lane 6) binding. However, it was successful in differentiating the
conformational changes associated with nortriptyline (lane 4) and desipramine
(lane 1) binding with the conformational change associated with nortriptyline
affording much less protection to proteolysis than the desipramine bound
complex or the apo S1S2 do-main.
Protease protection assay of NR1-1b S1S2:TCA
complexes. To 5.29μM 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.
3.5. Summary
A combination of intrinsic and
extrinsic fluorescence binding studies, Stern-Volmer analysis, and protease
protection assays provides evidence for the binding of five TCAs, four SSRIs,
and one SNRI to the NMDA NR1-1b S1S2 domain. Data from the SSRI class of
antidepressants supports the hypothesis that the conformational changes
elicited upon binding to the S1S2 domain are unique. Within the TCA class of antidepressants, the
conformational changes of the S1S2 domain upon binding to each drug also appear
to be unique, with the exception of binding to imipramine and trimipramine. Structurally,
the difference between imipramine and trimipramine (Figure 2) is the branched
aliphatic side chain present in trimipramine. While it is possible that this
additional steric hindrance in trimipramine causes it either to bind to a
unique site or in a unique way to the same site within the S1S2 domain, it is
also possible that these two drugs have very similar binding to the S1S2 domain
of the NR1-1b subunit. None of the studies performed provide evidence of binding of this iGluR
domain to the SSRI citalopram. Perhaps the presence of the nitrile group,
present in none of the other antidepressants studied, prevents binding to the
NR1-1b S1S2 domain.
Based upon the
ways in which iGluRs have been proposed to interact with antidepressants, this
direct binding to the extracellular S1S2 domain of the NMDA NR1-1b subunit may
fit into the previously observed category of NMDA antagonists having
antidepressant activities [9, 11–13]. As
the TCAs have been shown to produce open channel block of the NMDA receptors,
however, differentiating antagonist effects from external S1S2 binding and open
channel block will be difficult [7, 8]. As
is the case for open channel block, for direct binding of antidepressants to
the NMDA NR1-1b S1S2 domain to play a major role in the mode of action of these
drugs, the differences in their binding affinities (Kd range
0.24–7.36 mM), and typical therapeutic plasma concentrations (0.17–1μM),
will need to be resolved [7, 8, 18, 19]. Part
of this solution may lie in the fact that while these isolated iGluR S1S2
domains have been shown to possess near native binding
affinities for both agonists and antagonists in vitro, their in vivo interactions
with antidepressants could be quite different [1–3, 20, 21, 22].
Acknowledgments
The authors would like to thank Eric Gouaux (Oregon Health and Science University) for the plasmid containing
NMDA NR1-1b S1S2, James Hall, Christie Yankowski and Ben Telsey for preliminary antidepressant binding studies, and the National Science Foundation for support of this work (MCB-0447541 to L.G.).
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