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 ) [ 14 ] . 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 ) [ 14 ] . 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 , 1113 ] . 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 . 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 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 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 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 . protein samples 
 were at a final concentration of 5.29 m 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 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 136.9 m , 136.9 m , 136.9 m , 2.08 mm , and 3.18 mm , 
 respectively . the final concentrations of each ssri - paroxetine , citalopram , and 
 sertraline were 3.75 mm , 
 1.88 m , and 1.88 mm , respectively . antidepressants were added to final concentrations as follows : nortriptyline : 
 1.243.61 mm , 
 imipramine or desipramine : 8570 m , 
 trimipramine : 130570 m , 
 sertraline : 0.741.84 mm , fluvoxamine : 0.742.37 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/f = 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 ] , f is ( f  fo ) , and kd is the 
 dissociation constant . 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 . acrylamide 
 ( sigma ) ranging in final concentration from 16.3 to 113.9 mm was added to 5.29 m 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 ) . extrinsic fluorescence studies were performed in a cary 
 ec - lipse fluorometer at 25c . 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 blanks were collected both for 
 the antidepressants and the nr1 - 1b s1s2 domain . 8-anilino-1-naphthalenesulfonic 
 acid ( ans , tci america ) , in a 1 : 1 molar ratio , was added to 5.29 m of the 
 nr1 - 1b s1s2 domain premixed with antidepressant ( at final concentrations as 
 above for intrinsic quenching studies in addition to the ssris fluoxetine ( 8.62 m ) and fluvoxamine ( 2.81 mm ) 
 and the snri venlafaxine ( 29.07 m ) ) . as with all intrinsic assays , antidepressants ( at final concentrations as above for the 
 extrinsic assays , with the exception of sertraline which was added to a final 
 concentration of 940 m ) were added to 60 l aliquots of the nr1 - 1b s1s2 domain 
 at a final concentration of 5.29 m . 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 5.29 m , yielding a 1 : 1 molar ratio of 
 protein to trypsin . digestion was stopped by the addition of sample loading dye , dtt 
 ( 50 mm ) , and incubation at 100c for 10 minutes . 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 ( max  ) and fluorescence 
 intensity of a tryptophan residue depends upon the polarity of its environment . 
 by comparing the emission spectrum ( max   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 max   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      150 m ; 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      150 m ) . 
 the data in figure 3(b ) demonstrates the binding of the ssris paroxetine , 
 sertraline ( kd : 240      90 m ) , 
 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 . 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 max   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 . the only other 
 ligand which caused a statisticallysignificant shift in max   of the 
 s1s2 domain is sertraline , which upon complex formation is shifted to 329.07    
 0 nm ( table 1 ) . this increase in max   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 . 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 max   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 max   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 . 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 , 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 , 0 , 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 [ 1517 ] , 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 . to differentiate the binding of desipramine and 
 nortriptyline as well as trimipramine and imipramine to the s1s2 domain of the nr1 - 1b 
 subunit 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 . 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 , 1113 ] .   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 
 0.247.36 mm ) , and typical therapeutic plasma concentrations ( 0.171 m ) , 
 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 [ 13 , 2022 ] .
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 .