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-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors
Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois
Received May 19, 2004; accepted September 23, 2004
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Abstract |
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-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and [3H]fluorowillardiine to rat brain membranes and homomeric GluR1-4 receptors, with IC50 values in the range of 5 to 180 µM. Inhibition often was less than complete at saturating drug concentrations and thus seems to be noncompetitive in nature. Pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) is a potent antagonist of purinoceptors that shares some structural elements with suramin yet is smaller than the latter. PPNDS also had potent effects on AMPA receptors (EC50 value of 4 µM) but of a kind not seen with the other compounds in that it increased binding affinity for radioagonists severalfold. In addition, PPNDS slowed association and dissociation rates more than 10 times. In physiological experiments with GluR2 receptors, PPNDS at 50 µM reduced the peak current by 30 to 50% but had only small effects on other waveform aspects such desensitization and steady-state currents. This pattern of effects differentiates PPNDS from other compounds such as thiocyanate and up-modulators, which increase agonist binding by enhancing desensitization or slowing deactivation, respectively. Receptor model simulations indicate that most effects can be accounted for by assuming that PPNDS slows agonist binding/unbinding and stabilizes the bound-closed state of the receptor. By extension, suramin is proposed to stabilize the unbound state and thereby to reduce affinity for agonists. These drugs thus act through a novel type of noncompetitive antagonism.
). In vitro, suramin affects a bewildering array of molecular and cellular processes. It inhibits numerous signaling proteins, including growth factors and interleukins (Voogd et al., 1993); among its many other targets are G proteins (Freissmuth et al., 1996) and enzymes such as reverse transcriptase (De Clercq, 1987) and ectonucleotidases (Lambrecht, 2000). However, suramin seems particularly potent in blocking purinergic receptors with low micromolar inhibition constants (Charlton et al., 1996; Lambrecht, 2000).
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There are also reports that suramin inhibits glutamatergic synaptic transmission (Motin and Bennett, 1995; Gu et al., 1998) and whole-cell currents mediated by AMPA receptors (Nakazawa et al., 1995; Zona et al., 2000) and NMDA receptors (Ong et al., 1997; Peoples and Li, 1998). Suramin was observed to inhibit binding of radioligands to NMDA receptors (Balcar et al., 1995) but not kainate receptors (Ong et al., 1997), which suggested some selectivity among glutamate receptors. Apart from these studies, there have been few efforts to characterize the molecular actions of suramin and the nature of the impact on glutamate receptor function. Even less is known about possible interactions with more recent P2X receptor antagonists, many of which possess structural elements similar to those in suramin. Figure 1 shows examples of three such compounds with terminal naphthyl-polysulfonate groups (Freissmuth et al., 1996; Lambrecht, 2000; Rettinger et al., 2000). A somewhat distinct structural family of purinoceptor antagonists was established some time ago with the development of PPADS (Lambrecht et al., 1992), which has since been widely used as a highly selective P2X receptor blocker. This compound does not have mirror symmetry, is much smaller than suramin, and was indeed found to have no or weak effects on glutamate receptor responses (Motin and Bennett, 1995; Gu et al., 1998; Zona et al., 2000). PPNDS, another member of this drug family, has recently been identified as having even greater affinity for P2X receptors and a marked selectivity toward the P2X1 receptor subtype with a nanomolar inhibition constant (Lambrecht et al., 2000). Yet the two sulfonate groups and the nitro substituent in this compound occupy the same positions as the sulfonate groups in suramin (Fig. 1) and thus the possibility of an interaction with glutamate receptors cannot be dismissed.
The present study was intended to examine whether suramin inhibits binding to AMPA receptors, to clarify the nature of such inhibition, and to test whether the structurally related compounds have similar actions. Binding tests with radiolabeled agonists were used initially because they can readily distinguish between different subclasses of antagonists and their mode of inhibition. For instance, if suramin were to act as a competitive antagonist then it should completely displace radiolabeled agonists such as [3H]AMPA from their binding site, as it is the case for quinoxaline compounds such as CNQX (Honore and Drejer, 1988). In contrast, 2,3-benzodiazepines such as GYKI52466 block AMPA receptor currents through a distinct site because they do not inhibit the binding of agonists (Kessler et al., 1996), except under specific circumstances (Arai et al., 2002b). Guanine nucleotides constitute a third group of AMPA receptor blockers that also reduce agonist binding but through an as yet unidentified mechanism (Monahan et al., 1988; Baron et al., 1989; Dev et al., 1996). Last, if suramin were to act as a channel blocker then binding of agonists would presumably remain unaffected. During examination of the structural analogs described above, it was also found that PPNDS affected AMPA receptors in ways that differed substantially from those of suramin, both in physiological and binding experiments. Thus, the second goal of this project was to identify which aspects of receptor kinetics are most likely influenced by PPNDS. The findings with both suramin and PPNDS have been incorporated into a working model that suggests that both drugs bind to a domain near the agonist-binding site yet with opposite consequences for the stability of the agonist-bound state.
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Materials and Methods |
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) involving homogenization in isotonic sucrose and differential centrifugation to obtain a P2 pellet fraction, followed by an osmotic lysis and repeated washing by centrifugation. For binding tests, membranes were suspended in HEPES-buffered saline (HBS; 150 mM NaCl, 20 mM HEPES, and 0.1 mM EGTA, pH 7.4). Binding studies with recombinant receptors used a preparation of permeabilized and washed cells. Most receptor subunits examined here are stably expressed in HEK293 cells (Hennegriff et al., 1997; Arai et al., 2000) and periodically confirmed by Western blots. HEK293 cells were collected into HBS containing in addition 2 mM EGTA and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The cells were centrifuged at least four times (2000g; 10 min) and resuspended in HBS without additions; after the first centrifugation, 0.1% saponin was included in the HBS to permeabilize the membranes, and the cell suspension was left at 25°C for 3 min. All other steps were carried out at 0 to 4°C. The cells were stored on ice for up to 1 month without evident change in receptor properties and washed on each test day before use. For some tests, the permeabilized cells were extensively sonicated with an ice-cooled tip, and the resulting membrane fragments were collected by high-speed centrifugation (15 min; 60,000g).
Unless mentioned otherwise, binding tests were conducted using the following protocols. Aliquots of rat brain membranes or permeabilized cells (10-20 µg of protein) were incubated at 0°C with radioligand and appropriate additions in 50-µl volume. Where indicated (+SCN), incubation media contained 50 mM KSCN (potassium thiocyanate), which increases affinity for [3H]AMPA about 10-fold. Incubations were terminated after 30 to 60 min by filtration through GF/A or GF/C filters after diluting the sample in 5 ml of ice-cold buffered saline containing 50 mM KSCN (wash buffer); the filters were rapidly rinsed with 15 ml of additional wash buffer. In some assays with brain membranes, incubations were terminated by 15-min centrifugation in a Beckman JA 18.1 rotor with rotor temperature calibrated to match incubation temperature. The pellet was quickly rinsed with ice-cold wash buffer. Stock solutions of test drugs were prepared in HBS. Nonspecific binding was determined with 5 mM glutamate and subtracted from total binding. The drugs in general had no significant effect on nonspecific binding and did not cause quenching of scintillation counting. Protein content was determined according to Bradford (1976) with serum albumin as standard. Binding data were usually normalized and averaged across experiments and plotted as mean and S.E.M. Binding curves were fitted through nonlinear regression using the Prism program from GraphPad Software Inc. (San Diego, CA).
Excised Patch Currents Mediated by GluR2 Receptors. Currents mediated by homomeric GluR2(R607Q) flop or flip receptors were measured in HEK293 cells transiently transfected with the respective cDNAs in a pRK5 vector, using LipofectAMINE (Invitrogen, Carlsbad, CA). Cells were submitted to experimentation 72 to 96 h after transfection. Patch pipettes had a resistance of 2 to 5 M
and were filled with a solution of 130 mM CsF, 10 mM EGTA, 2 mM ATP Mg2+ salt, and 10 mM HEPES, pH 7.3. The extracellular solution contained 140 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 10 mM HEPES, pH 7.3. Patches were excised from the cells and positioned in front of a double pipet delivering a constant flow of background medium (without or with test drug) in one flow line and medium containing 1 mM L-glutamate in the second flow line. The patch was initially placed in the background flow line. A piezo device then moved the double pipet in a fraction of a millisecond into the glutamate flow line (Arai et al., 1996). In general, 10 responses were collected at 5-s intervals and averaged to give one trial. When testing PPNDS, several control trials were collected and then the background flow line was changed to medium containing 50 µM PPNDS. After three to four drug trials, the background medium was switched back to control medium. In tests with suramin, trials were repeatedly alternated between control condition and the various drug concentrations. The holding potential was -70 mV. Data were acquired with a patch amplifier (AxoPatch-1D) at a filter frequency of 5 kHz and digitized at 10 kHz with PClamp/Digidata 1322A (Axon Instruments Inc., Union City, CA).
Materials. S-[3H]AMPA (40.8 Ci/mmol) was obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). [3H]Fluorowillardiine (36 Ci/mmol), PPNDS, PPADS, NF023, NF279, and NF449 were obtained from Tocris Cookson Inc. (Ballwin MO). Suramin and common laboratory reagents were from Sigma-Aldrich.
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The less than complete inhibition seen in some assays suggested that the interaction between suramin and agonists is not competitive. In the remainder of Fig. 2 (D-F), we examined the nature of these interactions in more detail for [3H]AMPA binding. These tests were carried out in the presence of KSCN so that near saturating concentrations of [3H]AMPA could be reached. Figure 2D shows that suramin progressively shifted saturation curves for [3H]AMPA to the right with only insignificant changes (<12%) in the Bmax value. A similar right-shift was seen in inverse concentration-effect experiments in which suramin inhibition curves were constructed for different [3H]AMPA concentrations (IC50 values of 44, 109, and 531 µM at 2, 30, and 180 nM [3H]AMPA, respectively; Fig. 2E). Both right-shifts are basically compatible with a competitive interaction. However, a second notable change in Fig. 2E was that the lower asymptote progressively increased with the result that less than 50% of binding was inhibited at 180 nM [3H]AMPA. The obvious similarity with [3H]FW inhibition curves thus makes it seem likely that the nature of the inhibition is the same for both radioagonists, i.e., that suramin reduces affinity for both agonists through an allosteric mechanism, the main difference being that the reduction in affinity is larger for AMPA than for FW. The last panel of Fig. 2 shows dissociation of bound [3H]AMPA upon adding high concentrations of glutamate, suramin, or a combination of both. If suramin reduces agonist affinity through an independent site, then dissociation would be expected to be accelerated by the drug. This was not the case, however, because dissociation time courses were rather similar for all three conditions, which in essence suggests that suramin can only occupy its site after glutamate has dissociated from its site. Although this may seem to be typical for competitive interactions, other interpretations are possible, as discussed later.
Polysulfonate analogs of suramin have recently been developed that possess orders of magnitude higher affinity for specific subtypes of purinergic receptors. The experiments of Fig. 3 examined the effects of three of these analogs, NF023, NF279, and NF449 on AMPA receptor binding. All compounds inhibited agonist binding to GluR2o and the IC50 values were similar to that of suramin, with NF023 being the most potent (4.5 µM). The most unusual finding was that inhibition by NF279 was governed by a Hill coefficient of a magnitude not previously seen in binding tests with any other AMPA receptor ligands (Fig. 3A). The coefficient ranged between 2.5 and 5.3 with an average of 4.1 and was similar across subunits (not shown). Tests with native AMPA receptors in rat brain membranes (Fig. 3B) similarly revealed a larger nH value for NF279 (1.8) than for NF023 (0.9). In these experiments, maximal inhibition was again far from complete with lower asymptote values near 50%.
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PPADS has been widely used as a P2X receptor antagonist but is now increasingly supplanted by more potent and selective inhibitors, one of them being PPNDS. Both of these compounds contain a polysulfonated ring element, are much smaller than suramin, and lack mirror-symmetry (Fig. 1). PPNDS, however, possesses a naphthyl-disulfonate component that is almost identical to the terminal ring elements in suramin. As shown in Fig. 4A, PPADS was a weak inhibitor of [3H]FW binding, which accords with the reported lack on AMPA receptor currents. In contrast, PPNDS produced a severalfold increase in [3H]FW binding to rat brain membranes (Fig. 4A) and thus had effects that are fundamentally different from those of the other compounds. The drug was potent with an EC50 of 4.1 µM at 0 and 25°C, and the effect was highly reproducible over a wide range of incubation times (not shown). PPNDS also enhanced [3H]fluorowillardiine binding if thiocyanate was included in the assay (2.3-fold; Fig. 4A); thiocyanate itself had only modest effects on [3H]FW binding (60% increase at the near saturating concentration of 50 mM), in accordance with Hawkins et al. (1995). These observations allow us to dismiss the possibility that PPNDS mimicked the action of thiocyanate. Effects of PPNDS on recombinant GluR2 flop receptors (Fig. 4B) were similar to those on brain receptors with EC50 values of 3 to 5 µM. Scatchard tests showed that binding affinity for [3H]FW was increased 8-fold after addition of 40 µM PPNDS (Fig. 4B, inset). An increase in agonist affinity was also observed in equivalent Scatchard tests with [3H]AMPA (without SCN), but the magnitude of the increase was smaller (2.9-fold; not shown; n = 2). Binding to other AMPA receptor subunits (GluR1 flip, GluR1 flop, and GluR4 flip) was increased in similar manners by PPNDS (not shown).
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Unusual results were also obtained when measuring association and dissociation of [3H]FW. In the absence of drug, binding to rat brain membranes at 25°C reached a level close to equilibrium within seconds (rate constant estimated at >15 min-1; Fig. 4C). When 100 µM PPNDS was added together with the radioagonist, binding again reached 85% of its plateau value within the first minute (rate constant 6 min-1). However, when membranes were first preincubated with PPNDS, the subsequent association of [3H]FW was drastically slowed and equilibrium was not reached for almost 1 h, the rate constant for the dominant component (66%) being 0.07 min-1. Dissociation of [3H]FW was similarly slowed more than 10 times if membranes had been equilibrated with the radioagonist in the presence of 100 µM PPNDS (Fig. 4D). These observations suggest that PPNDS can greatly slow association and dissociation of agonists and also that some aspects of drug action may change with longer exposure to PPNDS.
Figures 5 and 6 show the effects of PPNDS and suramin on AMPA receptor-mediated responses to glutamate pulses. These tests were carried out with outside-out patches excised from HEK293 cells transiently transfected with GluR2(R607Q) flip or flop receptors and used a piezo-driven application system to allow submillisecond resolution of the receptor current. The effects of 50 µM PPNDS on these responses were remarkably small and consisted mainly in a reduction in the amplitude (Fig. 5). This reduction was most prominent in the first trial after infusion of the drug and amounted to about 20 to 40%. Continued infusion of the drug reduced the peak amplitude further but at a lower rate. Other response parameters such as the rise time, the decay time constant and the steady-state current usually showed an increase during drug infusion but typically by a factor of less than 2. Steady-state currents in some experiments increased by a larger factor, but they always remained small compared with the respective peak currents (left) and hence responses with both receptor subtypes continued to exhibit strong desensitization even after infusion of the drug, in clear distinction from the effects of typical AMPA receptor up-modulators such as CX546, which essentially abolished desensitization (Arai et al., 2002b). PPNDS effects were not readily reversible and most response parameters did not return to baseline after drug offset, even after washout times of several minutes. The effects of suramin in the same recording system differed substantially from those of PPNDS in that currents were completely blocked (Fig. 6) and readily reversed after drug washout. However, like PPNDS, suramin did not have a significant effect on the waveform of the response (Fig. 6, inset). The IC50 value was on the order of 20 to 60 µM and thus agreed with those obtained from binding tests.
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Figure 7 illustrates one possible interpretation for the effects of PPNDS in the framework of a previously used elementary receptor model (Patneau and Mayer, 1991; Ambros-Ingerson and Lynch, 1993; Arai et al., 2002b). The equation given in the figure caption shows how binding affinity is related to rate constants in this particular model. A plausible first scenario would be that PPNDS slows both association and dissociation of the agonist without affecting channel gating and desensitization. As shown by the simulations of receptor currents (Fig. 7, center), a 20-fold slowing of these rate constants (condition 2) leads to a reduction in the peak current similar to that observed with modest or no changes in steady-state currents, rise times, and decay time constants. With these modifications, however, binding affinity is not altered and hence there must be additional changes in receptor kinetics. One such possibility is that PPNDS causes a stabilization of the bound-closed state with the consequence that dissociation from this state is slowed more than the association (condition 3 in Fig. 7). This would lead to a commensurate increase in binding affinity because the KD value is in first approximation given by the ratio k-1/k1 x k-2/k2. If we assume that the rate of dissociation is slowed 200 times, then the response wave form remains similar to that of condition 2, but the binding KD is greatly reduced and hence binding measured at any fixed [3H]agonist concentration would be increased; small changes in other rate constants may account for the observed minor changes in the steady-state current. These properties are reminiscent of those observed with PPNDS and thus we tentatively propose that the primary effect of this drug is to slow agonist association and dissociation and that it either stabilizes the bound-closed receptor state or destabilized the unbound state.
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The effects of PPNDS were unexpected yet highly consistent across preparations and assay conditions. Binding affinity for FW was increased 8-fold and accompanied by a slowing of both association and dissociation. In physiological tests, PPNDS reduced the peak amplitude of responses to glutamate but had conspicuously weak impact on other response parameters. This pattern of effects is unique and clearly different from that previously reported for other modulatory factors. Thus, thiocyanate, which also increases binding affinity for [3H]AMPA, accelerated desensitization in patch experiments (Arai et al., 1995) and lowered the steady-state current in oocyte recordings (Bowie and Smart, 1993), both of which pointed to the conclusion that it facilitates transition into the desensitized state. PPNDS did not promote desensitization and its effects on binding were not occluded by thiocyanate. Agonist binding is also increased in the presence of many drugs referred to as up-modulators. Although structurally and functionally diverse, these compounds augment the current passed through AMPA receptors by increasing the peak current and/or slowing the decay of the current after glutamate pulses (Arai and Lynch, 1998; Arai et al., 2002a,b). Increases in agonist binding produced by these drugs were usually less than 100%, with only a few exceptions (Arai et al., 2002a,b). The latter compounds, however, also caused a dramatic slowing of response deactivation and desensitization, and it has been argued that binding increase and response prolongation are related (Arai et al., 2002b). These effects are again clearly distinct from those of PPNDS. Based on such considerations, it seems unlikely that the primary effect of PPNDS is to influence receptor functions involving desensitization (k2/k-2 in Fig. 7) or channel gating (k5/k-5). Simulations with the five-state kinetic receptor model indicated instead that most observed effects can be reproduced if association and dissociation rates for agonists are reduced with the latter being more extensive. Similar conclusions were obtained when using the two-ligand glutamate receptor model of Jonas et al. (1993) (not shown). The marked slowing of association shown in Fig. 2 has indeed been a distinctive feature of the PPNDS effect. It should also be noted, however, that the associations and dissociations measured in those binding tests involve multiple kinetic transitions, including those related to desensitization, and thus the magnitude of changes in k1 and k-1 can not be directly inferred from those measurements.
A further question is whether the actions of PPNDS and suramin involve separate sites and mechanisms. One plausible way to explain the slowing of association and dissociation caused by PPNDS would be to assume that the drug restricts passage to the agonist site. The latter is indeed buried deep within a cleft between the S1 and S2 domains that form the two lobes of the glutamate receptor "flytrap" mechanism (Stern-Bach et al., 1994; Mano et al., 1996). By binding to the outer part of the cleft, PPNDS could thus potentially reduce the rate with which agonists move into and out of the cleft, without occupying the agonist-binding site itself. It is of interest in this regard that guanine nucleotides also seem to bind in this region. In the chicken kainate binding protein, mutation of a lysine residue at the outer edge of the upper lip of the flytrap reduced the potency of GDP to inhibit kainate binding (Paas et al., 1996). Given that guanine nucleotides inhibit [3H]AMPA binding (Dev et al., 1996) and that suramin and PPNDS inhibit proteins with nucleotide-binding sites (see Introduction), we conjecture that the compounds examined in this study mimic guanine nucleotides and that suramin and PPNDS bind to the same receptor domain yet differ in the specifics of their docking mode and the impact on receptor kinetics. The proposed modes of action are sketched in Fig. 8. PPNDS, by binding to the outer lips of the S1-S2 domain, stabilizes the receptor in a "closed-cleft" conformation that is characteristic for the "agonist-bound" state. If agonists and PPNDS are applied together, the agonist presumably binds first and then becomes stabilized when PPNDS associates with the receptor. As a consequence, equilibrium binding affinity is increased and dissociation of the agonist is slowed. If PPNDS is applied before the agonist, as in some association experiments, the receptor is shifted into the closed-cleft conformation even if the agonist site is not occupied. This greatly slows subsequent penetration of the agonist into the cleft and hence association with the receptor. In physiological tests, responses to glutamate pulses are accordingly reduced in amplitude because glutamate is not able to effectively reach its site in short time. According to this scenario the downstream conformational changes associated with channel opening and desensitization kinetics would not be influenced in a major way by PPNDS.
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Binding of suramin would have opposite consequences. Perhaps because of its larger molecular dimensions and the presence of two polyanionic domains, it may act like a wedge that prevents the S1 and S2 domains to close around the bound glutamate (Fig. 8, right). It would thus hold the receptor in a conformation nearer that of the unbound state and thereby lower the affinity for agonist in binding tests. In studies using an S1-S2 model system, cleft-closure upon agonist binding was shown to involve a rotation of the two lobes relative to each other. The degree of this rotation was smaller for FW than for AMPA because of the larger molecular size of FW (Jin et al., 2003). It is thus conceivable that the "open-cleft" state stabilized by suramin can still bind FW with only modest loss of affinity, whereas the smaller AMPA molecule may not be able to make sufficient contact with the two lobes. This could explain the much larger reduction in affinity seen with AMPA as radioligand (a similar, although inverse, rationale may explain why PPNDS produced a larger gain in affinity for [3H]FW than for [3H]AMPA). In addition, if suramin binds to the open-cleft state, it may not be able to bind as long as glutamate is holding the receptor in a closed-cleft conformation. This could explain the apparent competitive behavior observed in the dissociation experiments, which suggested that glutamate must leave before suramin can bind.
An assumption underlying many of the above-mentioned deliberations has been that the polysulfonated ring elements represent the critical pharmacophore. However, several polysulfonated azo-dyes also inhibit AMPA receptor currents, and it has been argued that their action mainly involves hydrophobic interactions mediated by the aromatic backbone. This was based on observations such as that currents were almost completely blocked by 1 µM Evans blue (Fig. 1), whereas Chicago sky blue, which differs only in a methoxy substituent on the central benzidine moiety, was ineffective at this concentration (Keller et al., 1993; Price and Raymond, 1996). In our binding tests, however, Evans blue and Chicago sky blue exhibited comparable potencies for inhibiting [3H]FW binding, and the IC50 values were in the 100 µM range (not shown). Thus, the purported "nanomolar" physiological actions of Evans blue, which are of a kind that is evidently not detectable in our binding tests, perhaps involve different pharmacophores than our "micromolar" actions of suramin, PPNDS, and Evans blue, which we believe to depend rather on the polysulfonated naphthyl structures.
In conclusion, the drugs examined here produced stimulatory and inhibitory effects on agonist binding that are unique and differ in major ways from those produced by other groups of AMPA receptor modulators. The drug effects exhibit mixed competitive and noncompetitive traits that can be adequately understood if one assumes that they do not physically occupy the binding domain of the agonists yet control access for agonists and compete for, or stabilize, certain receptor conformations. This would lead to an effective functional antagonism in the case of suramin yet perhaps to a more controlled and partial response attenuation in the presence of PPNDS. Although PPNDS is unlikely to cross the blood-brain barrier, compounds acting like it might offer new avenues to treat conditions such as epilepsy in which a limited reduction of phasic AMPA receptor activation is desired. This highlights further the need to determine the pharmacophores responsible for its action.
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Footnotes |
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ABBREVIATIONS: AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-D-aspartate; PPADS, pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; CNQX, 6-cyano-2,3-dihydroxy-7-nitroquinoxaline; PPNDS, pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate); HEK, human embryonic kidney; HBS, HEPES-buffered saline; SCN, thiocyanate anion; GluR, glutamate receptor; FW, fluorowillardiine; NF023, 8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis-(1,3,5-naphthalenetrisulfonic acid), 6Na; NF279, 8,8'-(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis-1,3,5-naphthalenetrisulfonic acid, Na; NF449, 4,4',4'',4'''-[carbonyl-bis[imino-5,1,3-benzenetriyl-bis-(carbonylimino)]]tetrakis-benzene-1,3-disulfonic acid, 8Na; GYKI52466, 4-(8-methyl-9H-1,3-dioxolo[4,5-h][2,3]benzodiazepin-5-yl)-benzeneamine dihydrochloride.
Address correspondence to: Dr. M. Kessler, Department of Pharmacology, Southern Illinois University School of Medicine, MC 9629, 801 N. Rutledge St., Springfield, IL 62702. E-mail: mkessler{at}siumed.edu
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References |
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