A charge-inverting mutation in the "linker" region of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors alters agonist binding and gating kinetics independently of allosteric modulators.

AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728-4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.

Modulation of agonist binding to AMPA receptors by 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine (CX546): differential effects across brain regions and GluA1-4/transmembrane AMPA receptor regulatory protein combinations.

Ampakines are cognitive enhancers that potentiate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents and synaptic responses by slowing receptor deactivation. Their efficacy varies greatly between classes of neurons and brain regions, but the factor responsible for this effect remains unclear. Ampakines also increase agonist affinity in binding tests in ways that are related to their physiological action. We therefore examined 1) whether ampakine effects on agonist binding vary across brain regions and 2) whether they differ across receptor subunits expressed alone and together with transmembrane AMPA receptor regulatory proteins (TARPs), which associate with AMPA receptors in the brain. We found that the maximal increase in agonist binding (E(max)) caused by the prototypical ampakine 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine (CX546) differs significantly between brain regions, with effects in hippocampus and cerebellum being nearly three times larger than that in thalamus, brainstem, and striatum, and cortex being intermediate. These differences can be explained at least in part by regional variations in receptor subunit and TARP expression because combinations prevalent in hippocampus (GluA2 with TARPs gamma3 and gamma8) exhibited E(max) values nearly twice those of combinations abundant in thalamus (GluA4 with gamma2 or gamma4). TARPs seem to be critical because GluA2 and GluA4 alone had comparable E(max) and also because hippocampal and thalamic receptors had similar E(max) after solubilization with Triton X-100, which probably removes associated proteins. Taken together, our data suggest that variations in physiological drug efficacy, such as the 3-fold difference previously seen in recordings from hippocampus versus thalamus, may be explained by region-specific expression of GluA1-4 as well as TARPs.

The role of kisspeptin and GPR54 in the hippocampus.

The granule cells of the dentate gyrus form the input stage of the hippocampal trisynaptic circuit and their function is strongly influenced by peptidergic systems. GPR54 is highly and discretely expressed in these cells. We have found that activation of GPR54 with kisspeptin-10 causes a rapid and large increase in the amplitude of excitatory synaptic responses in granule cells, without changing membrane properties. The effect was suppressed by the G-protein inhibitor GDP-beta-S and the calcium chelator BAPTA, and analysis of miniature EPSCs revealed an increase in mean amplitude but not event frequency, indicating that GPR54 and the mechanisms for enhancing EPSCs are postsynaptic, possibly involving changes in AMPA receptor number or conductance. The kisspeptin-induced synaptic potentiation was abolished by inhibitors of ERK1/2, tyrosine kinase, and CaMKII. RT-PCR experiments showed that KiSS-1 is expressed in the dentate gyrus. KiSS-1 mRNA was significantly increased by seizure activity in rats and when neuronal activity in organotypic hippocampal slice cultures was enhanced by kainate or picrotoxin, while mRNA for GPR54 remained essentially unchanged. These results suggest that kisspeptin may be locally synthesized and act as an autocrine factor. In separate experiments, hippocampal KiSS-1 mRNA in male rats was increased after gonadectomy. In summary, kisspeptin is a novel endogenous factor which is dynamically regulated by neuronal activity and which, in marked distinction from other neuropeptides, increases synaptic transmission in dentate granule cells through signaling cascades possibly linked to the MAP kinase system. This novel peptide system may play a role in cognition and in the pathogenesis of epilepsy.

Factors that regulate KiSS1 gene expression in the hippocampus.

Kisspeptin is a C-terminally amidated peptide encoded by the KiSS1 gene. The peptide and its receptor GPR54 are abundant in the hypothalamus and have been implicated as gatekeepers for the onset of puberty and the development of the reproductive system. Interestingly, GPR54 is also highly expressed in granule cells of the hippocampal dentate gyrus, and in a previous study we showed that kisspeptin enhances excitatory synaptic transmission in these cells. The present study examined how expression of KiSS1 and GPR54 is regulated in rat hippocampus, using in vivo and in vitro preparations. In animals, a 3 h period of kainate-induced seizures significantly altered expression of both genes. KiSS1 mRNA showed a 3-4 fold increase which peaked 1-3 days post-seizure and subsided after one week. GPR54 mRNA, on the other hand, was reduced by 20-30% at 6-24 h. In organotypic hippocampal slice cultures, brief exposure to kainate produced a significant increase in KiSS1 mRNA with a time course comparable to that in vivo, and the effect was blocked by tetrodotoxin and CNQX. Chronic (7-day) treatment with picrotoxin, which induced a persistent four-fold increase in spike activity in multi-electrode recordings, caused a similar size but more persistent upregulation in KiSS1 mRNA. As in other studies, kainate and picrotoxin induced an upsurge in BDNF expression, but BDNF mRNA was also significantly increased when slice cultures were treated with kisspeptin. Taken together, KiSS1 expression is upregulated by neuronal activity and activation of GPR54 by kisspeptin may in turn contribute to sustain basal BDNF levels required for hippocampal function. In additional experiments, KiSS1 mRNA was found to be increased after orchidectomy and thus expression may be regulated also by gonadal hormones.

Spontaneous activity in Purkinje cells: multi-electrode recording from organotypic cerebellar slice cultures.

Organotypic cerebellar cultures were maintained on multi-electrode dishes (MED) with an 8x8 array of electrodes and examined for physiological activity. The cultures remained viable for up to seven months and exhibited spontaneous discharges most likely originating from Purkinje cells. Spike frequencies varied but were mostly around 10-30 Hz and were often stable over weeks with average drifts of <20% per week. Spontaneous firing was significantly reduced by blockers of sodium channels (riluzole) and several potassium channels (iberiotoxin, TEA, 4-amino-pyridine), but blockers of calcium channels, GIRK channels, and SK-type potassium channels were ineffective. Inhibitors of excitatory and inhibitory synaptic transmission made spike discharges more regular. Particularly robust changes in spike frequency were produced by agents that increase cGMP. Bromo-cGMP, the NO donor SNAP, the guanylate cyclase activator YC-1, and the phosphodiesterase inhibitor zaprinast greatly reduced spike frequency. Activation of the metabotropic receptor mGluR1 and inhibition of I(h) channels caused a majority of cells to switch from tonic firing to a cyclic activity mode in which intense firing alternated with silence. Agonists for cholinergic, serotonergic, histamine, opiate, and CRF receptors had no effect, but those for adrenergic and adenosine A1 receptors reduced firing. Moreover, brief application of bromocriptine caused a delayed decrease in firing that reached a minimum after 24 to 48 h and recovered after 1-2 weeks. Taken together, our results demonstrate that long-term cultures maintained on multi-electrode arrays retain many essential features of cerebellar physiology and that they provide a test system that is well suited for broad screening of pharmacological agents as well as for studying long-term effects of drugs, tissue factors, and pathogens.

Physiological significance of high- and low-affinity agonist binding to neuronal and recombinant AMPA receptors.

Radioligand binding studies have shown that AMPA receptors exist in two variants that differ about twenty-fold in their binding affinities, with brain receptors being mainly of the low-affinity type and recombinantly expressed receptors having almost exclusively high affinity. However, the physiological correlate of high- and low-affinity binding is not yet known. In this study we examined if physiological experiments similarly reveal evidence for two distinct receptor variants. We therefore measured equilibrium desensitization by glutamate and determined IC(50) values for neuronal receptors and for the homomeric receptors GluR1-4 expressed in HEK293 cells. Contrary to the prediction that these IC(50) values exhibit large differences commensurate with those of high- and low-affinity binding, values for homomeric receptors (1-18 microM) were on an average not different from those of neuronal receptors (3-10 microM). Moreover, simulations with kinetic receptor models suggest that the IC(50) values for neuronal and recombinant receptors correspond to the binding affinity of the low-affinity receptor variant. These findings indicate that the high-affinity binding measured in heterologous expression systems represents an immature receptor variant that does not contribute to the currents recorded from these cells, and that the functional low-affinity receptors are present in such small number that they are effectively masked in binding assays by the high-affinity receptors. Thus, in order to compare experimentally determined saturation binding profiles with those predicted by kinetic receptor models and with dose-response curves from physiological studies, it will be imperative to develop methods for isolating first the low-affinity receptors.

The fast kinetics of AMPA GluR3 receptors is selectively modulated by the TARPs gamma 4 and gamma 8.

Transmembrane AMPA receptor-associated regulatory proteins (TARPs) modulate the kinetics of AMPA receptors and increase their surface expression. This study compared the effects of the TARPs gamma2, gamma3, gamma4 and gamma8 on the AMPA receptor subunits GluR1-3. We found that the deactivation and desensitization time constants of GluR3 receptors were greatly increased by gamma4 and gamma8 (approximately 3 fold) whereas gamma2 and gamma3 caused minimal changes. This stands in contrast to the effects on GluR1 and GluR2 receptors which were similar across all four TARPs. Other response parameters like the current density, the EC(50) for glutamate-induced peak currents, and kainate-induced currents were in general more effectively modulated by gamma2 and gamma3 in all subunits, including GluR3. All TARPs were effective in increasing the unitary conductance. The differential TARP effects on deactivation time constants of the GluR3 subunit are likely to have a significant impact on synaptic responses across different neurons in the brain.

Mutations in ionotropic AMPA receptor 3 alter channel properties and are associated with moderate cognitive impairment in humans.

Ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (iGluRs) mediate the majority of excitatory synaptic transmission in the CNS and are essential for the induction and maintenance of long-term potentiation and long-term depression, two cellular models of learning and memory. We identified a genomic deletion (0.4 Mb) involving the entire GRIA3 (encoding iGluR3) by using an X-array comparative genomic hybridization (CGH) and four missense variants (G833R, M706T, R631S, and R450Q) in functional domains of iGluR3 by sequencing 400 males with X-linked mental retardation (XLMR). Three variants were found in males with moderate MR and were absent in 500 control males. Expression studies in HEK293 cells showed that G833R resulted in a 78% reduction of iGluR3 due to protein misfolding. Whole-cell recording studies of iGluR3 homomers in HEK293 cells revealed that neither iGluR3-M706T (S2 domain) nor iGluR3-R631S (near channel core) had substantial channel function, whereas R450Q (S1 domain) was associated with accelerated receptor desensitization. When forming heteromeric receptors with iGluR2 in HEK293 cells, all four iGluR3 variants had altered desensitization kinetics. Our study provides the genetic and functional evidence that mutant iGluR3 with altered kinetic properties is associated with moderate cognitive impairment in humans.

Factors affecting guanine nucleotide binding to rat AMPA receptors.

Glutamate receptors are competitively inhibited by guanine nucleotides. Insight into the physiological function of this inhibition would be greatly advanced if nucleotide binding could be eliminated through mutations without altering other aspects of receptor function, or if compounds were discovered that selectively prevent nucleotide binding. It was previously reported that a lysine in the chick kainate binding protein (cKBP) is specifically involved in guanine nucleotide binding. In the present study we mutated the equivalent lysine in the rat AMPA receptor subunit GluR1 flip to alanine (K445A) and assessed changes in nucleotide affinity from the displacement of [(3)H]fluorowillardiine. As in the cKBP, the affinity for nucleotides was greatly reduced while the binding affinity for agonists remained unchanged. The reduction in affinity was largest for GTP (factor of 5.8) and GDP (4.4) and minor for GMP and guanosine. This suggests that K445 is involved in stabilizing the second phosphate of the nucleotide. Given that bulkier analogs like GDP-fucose are also accommodated at this site, it seems likely that nucleotides bind in such a way that their phosphates project out of the cleft. In excised-patch recordings using short pulses of glutamate, the K445A mutation increased the EC(50) for the peak response 1.8-fold and accelerated desensitization and deactivation. This indicates that the effects of this mutation are not as specific as previously suggested. Efforts to selectively eliminate inhibition by nucleotides may therefore depend on mapping out further the docking site. In a first attempt using point mutations we ruled out several amino acids around the cleft as being involved in nucleotide binding. Also, the AMPA receptor modulator PPNDS which competitively inhibits nucleotide binding to purinergic receptors did not affect nucleotide inhibition, suggesting that there are major differences in the topography between purinergic and glutamate receptors. Thus new approaches, including crystallography, may be called for to identify residues uniquely involved in nucleotide binding.

Use of [3H]fluorowillardiine to study properties of AMPA receptor allosteric modulators.

Compounds which modulate AMPA receptor function through allosteric mechanisms were examined for their effect on the binding of the agonist [3H]fluorowillardiine (FW). Benzamide-type positive modulators (ampakinestrade mark) under all experimental circumstances increased [3H]FW binding to native receptors in rat brain membranes. Benzothiadiazide drugs had more variable effects ranging from large reductions with cyclothiazide and JM-13 to increases produced by more recent compounds like PEPA, D1 and LY392098. These effects on binding were moderately influenced by the assay conditions, including temperature and the presence or absence of thiocyanate. Significant changes in agonist binding were also produced by other modulatory agents such as noncompetitive blockers (GYKI 53655, SYM 2206), polycationic compounds (spermine, Naspm, philanthotoxin) and polyanionic compounds (Evans Blue, suramin, PPNDS). EC50 values usually were similar to those from physiological studies, which validates using binding tests to assess drug potencies. Moreover, direction and magnitude of the binding change (Emax) provide information about which kinetic aspects are affected by a drug. For example, the magnitude of the binding increase produced by positive modulators was strongly correlated with their ability to slow response deactivation in excised patch recordings. Binding also provides a reliable method to examine whether interactions between agents are competitive. Thus, thiocyanate did not significantly influence the EC50 of cyclothiazide, suggesting distinct sites of action. Taken together, [3H]FW binding can yield important information about drug-receptor and drug-drug interactions for a wide range of modulatory agents. One potential limitation of [3H]FW is a large preference for subunits GluR1 and GluR2 (KD 4-10 nM) over GluR3 and GluR4 (160-600 nM) which implies that tests with brain membranes preferentially reveal drug effects produced at the former two subunits. Lastly, data are shown which highlight the importance of optimizing experimental conditions in filtration assays, for instance by always including thiocyanate in wash buffers.

Cancer metastasis-suppressing peptide metastin upregulates excitatory synaptic transmission in hippocampal dentate granule cells.

Metastin is an antimetastatic peptide encoded by the KiSS-1 gene in cancer cells. Recent studies found that metastin is a ligand for the orphan G-protein-coupled receptor GPR54, which is highly expressed in specific brain regions such as the hypothalamus and parts of the hippocampus. This study shows that activation of GPR54 by submicromolar concentrations of metastin reversibly enhances excitatory synaptic transmission in hippocampal dentate granule cells in a mitogen-activated protein (MAP) kinase-dependent manner. Synaptic enhancement by metastin was suppressed by intracellular application of the G-protein inhibitor GDP-beta-S and the calcium chelator BAPTA. Analysis of miniature excitatory postsynaptic currents (mEPSCs) revealed an increase in the mean amplitude but no change in event frequency. This indicates that GPR54 and the mechanism responsible for the increase in EPSCs are postsynaptic. Metastin-induced synaptic potentiation was abolished by 50 microM PD98059 and 20 microM U0126, two inhibitors of the MAP kinases ERK1 and ERK2. The effect was also blocked by inhibitors of calcium/calmodulin-dependent kinases and tyrosine kinases. RT-PCR experiments showed that both KiSS-1 and GPR54 are expressed in the hippocampal dentate gyrus. Metastin is thus a novel endogenous factor that modulates synaptic excitability in the dentate gyrus through mechanisms involving MAP kinases, which in turn may be controlled upstream by calcium-activated kinases and tyrosine kinases.

C-terminal truncation affects kinetic properties of GluR1 receptors.

GluR1flop receptors in which the C-terminal 52 amino acids had been recombinantly removed were characterized with whole-cell recording and binding assays. Compared to wildtype GluR1, truncated receptors showed faster desensitization and deactivation and they recovered more slowly from desensitization. The EC50 for glutamate was increased 2-fold. In binding tests, K(D)s for [3H]fluorowillardiine were 1.5 times larger for truncated receptors. According to receptor simulations, most differences can be explained if the C-terminal domain is assumed to stabilize the ligand-bound closed and open states. The effects on response waveforms are different from those caused by phosphorylation, suggesting that the C-terminus influences receptor function in multiple ways. Truncated forms of GluR1 identical or similar to the one examined here may also be generated by calcium-activated proteases during intense synaptic activity. The lowered affinity and faster inactivation of these receptors suggests that their presence does not represent a risk for neuronal viability.

Positive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators have different impact on synaptic transmission in the thalamus and hippocampus.

Earlier studies showed that positive modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors enhance synaptic responses and facilitate synaptic plasticity. Those studies focused mainly on hippocampal functions. However, AMPA receptors have regionally distinct subunit compositions and thus potencies and efficacies of modulators may vary across the brain. The present study compared the effects of CX546 [1-(1,4-benzodioxan-6-ylcarbonyl) piperidine], a benzamide-type modulator, on synaptic transmission in neurons of the reticular thalamic nucleus (RTN), which regulates the firing mode of relay cells in other thalamic nuclei, and on hippocampal CA1 pyramidal cells. CX546 greatly prolonged synaptic responses in CA1 pyramidal cells, but at the same concentration it had only weak modulatory effects in RTN neurons. Effects on miniature excitatory postsynaptic currents (EPSCs) were similar to those on EPSCs in both regions, suggesting that variations in neuronal morphology and transmitter release kinetics do not account for the differences. Relay cells in the ventrobasal thalamus also exhibited weak modulatory effects that were comparable with those in RTN neurons. Regionally different effects on response duration were also observed with CX516 [BDP-12, 1-(quinoxalin-6-ylcarbonyl)piperidine], a second benzamide drug. In contrast, 100 microM cyclothiazide produced comparable synaptic enhancements in hippocampus and RTN. The regional selectivity of benzamide drugs (ampakines) may be explained, at least in part, by a lower potency at thalamic AMPA receptors, perhaps due to the prevalence of the subunits GluR3 and 4. Although regional preferences of the ampakines were modest in their extent, they may be sufficient to be of relevance when considering future therapeutic applications of such compounds.

Divergent effects of the purinoceptor antagonists suramin and pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.

Suramin is a large naphthyl-polysulfonate compound that inhibits an array of receptors and enzymes, and it has also been reported to block currents mediated by glutamate receptors. This study shows that suramin and several structurally related compounds [8,8'-[carbonylbis(imino-3,1-phenylenecarbonylamino)]bis-(1,3,5-naphthalenetrisulfonic acid), 6Na (NF023), 8,8'-(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis-1,3,5-naphthalenetrisulfonic acid, Na (NF279), and 4,4',4'',4'''-[carbonyl-bis[imino-5,1,3-benzenetriyl-bis-(carbonylimino)]]tetrakis-benzene-1,3-disulfonic acid, 8Na (NF449)] reduce binding of [3H]alpha-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 microM. 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 microM) 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 microM 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.

A novel mechanism for the facilitation of theta-induced long-term potentiation by brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) contributes to the induction of long-term potentiation (LTP) by theta-pattern stimulation, but the specific processes underlying this effect are not known. Experiments described here, using BDNF concentrations that have minor effects on baseline responses, show that the neurotrophin both reduces the threshold for LTP induction and elevates the ceiling on maximal potentiation. The enhanced LTP proved to be as stable and resistant to reversal as that recorded under control conditions. BDNF markedly increased the facilitation of burst responses that occurs within a theta train. This suggests that the neurotrophin acts on long-lasting events that (1) are set in motion by the first burst in a train and (2) regulate the amplitude of subsequent bursts. Whole-cell recordings established that BDNF causes a rapid reduction in the size of the long-lasting afterhyperpolarization (AHP) that follows individual theta bursts. Apamin, an antagonist of type 2 small-conductance Ca2+-activated potassium (SK2) channels, also reduced hippocampal AHPs and closely reproduced the effects of BDNF on theta-burst responses and LTP. The latter results were replicated with a newly introduced, highly selective inhibitor of SK2 channels. Immunoblot analyses indicated that BDNF increases SK2 serine phosphorylation in hippocampal slices. These findings point to the conclusion that BDNF-driven protein kinase cascades serve to depress the SK2 component, and possibly other constituents, of the AHP. It is likely that this mechanism, acting with other factors, promotes the formation and increases the magnitude of LTP.

Integrins regulate NMDA receptor-mediated synaptic currents.

Synapses contain high concentrations of integrins, adhesion receptors known to influence the operation of neighboring transmembrane proteins. Evidence that integrins are important for consolidation of long-term potentiation suggests that these adhesion proteins may modulate activities of synaptic glutamate receptors. The present study provides a first test of the possibility that integrins modulate synaptic N-methyl-d-aspartate (NMDA)-type glutamate receptor activities. Excitatory postsynaptic currents (EPSCs) were recorded with whole cell clamp from hippocampal slices in which AMPA-type glutamate receptors and GABA(A) receptors were pharmacologically blocked. Microperfusion of the peptide integrin ligand gly-arg-gly-asp-ser-pro (GRGDSP) caused an approximately twofold increase in the amplitude and duration of NMDA receptor-gated synaptic currents. Control peptides had no effect. Paired-pulse facilitation was unchanged, indicating that the ligand did not modify neurotransmitter release probabilities. Infusion of the Src kinase antagonist PP2 but not the control drug 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine eliminated the enhancing effect of GRGDSP. Integrins regulate Src kinases that are known to phosphorylate NMDA receptors. It is concluded that integrins act through this route to exert potent modulatory effects on the operation of NMDA receptors.

Benzamide-type AMPA receptor modulators form two subfamilies with distinct modes of action.

CX516 (BDP-12) and CX546, two first-generation benzamide-type AMPA receptor modulators, were compared with regard to their influence on AMPA receptor-mediated currents, autaptic responses in cultured hippocampal neurons, hippocampal excitatory postsynaptic currents, synaptic field potentials, and agonist binding. The two drugs exhibited comparable potencies in most tests but differed in their efficacy and in their relative impact on various response parameters. CX546 greatly prolonged the duration of synaptic responses, and it slowed 10-fold the deactivation of excised-patch currents following 1-ms pulses of glutamate. The effects of CX516 on those measures were, by comparison, small; however, the drug was equally or more efficacious than CX546 in increasing the amplitude of synaptic responses. This double dissociation suggests that amplitude and duration of synaptic responses are governed by different aspects of receptor kinetics, which are differentially modified by the two drugs. These effects can be reproduced in receptor simulations if one assumes that CX516 preferentially accelerates channel opening while CX546 slows channel closing. In binding tests, CX546 caused an approximately 2-fold increase in the affinity for radiolabeled agonists, whereas CX516 was ineffective. More importantly, even millimolar concentrations of CX516 did not influence the dose-response relation for CX546, suggesting the possibility that they bind to different sites. Taken together, the evidence suggests that benzamide modulators from the Ampakine family form two subgroups with different modes and sites of action. Of these, CX516-type drugs may have the greater therapeutic utility because of their limited efficacy in prolonging synaptic responses and in attenuating receptor desensitization.

Effects of 5'-alkyl-benzothiadiazides on (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor biophysics and synaptic responses.

Alkyl-substituted benzothiadiazides (BTDs) were tested for their effects on (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. In excised patches, the 5'-ethyl derivative "D1" blocked the desensitization of AMPA receptor currents during prolonged application of glutamate (EC(50), 36 microM), and it slowed deactivation of responses elicited by 1-ms glutamate pulses greater than 10-fold. [(3)H]Fluorowillardiine binding to rat synaptic membranes was increased by D1 by a factor of 3.6 (EC(50), 17 microM) with a Hill coefficient near 2. In hippocampal slices, the compound reversibly increased excitatory postsynaptic currents and field excitatory postsynaptic potentials (EPSPs) with thresholds around 10 microM. The size of the alkyl substituent influenced both the potency and nature of the drug effect on synaptic currents: 5'-methyl compounds had a 2-fold greater effect on response amplitude than on response duration, whereas 5'-ethyl compounds like D1 caused greater increases in duration than amplitude. In tests with recombinantly expressed AMPA receptor subunits, D1 preferred the glutamate receptor (GluR) subunit GluR4 flip (0.64 microM) over GluR4 flop (5.3 microM); similar affinities but with smaller flip-flop differences were obtained for GluR1 through 3. These results show that D1 and congeners are significantly more potent than the parent compound IDRA-21 and that they differ in two fundamental aspects from cyclothiazide, the most widely studied BTD: 1) D1 markedly increases the agonist affinity of AMPA receptors and 2) it has immediate and large effects on field EPSPs. The large gain in potency conferred by alkyl substitution suggests that the 5' substituent is in intimate contact with the receptor, with the size of the substituent determining the way in which receptor kinetics is changed.

5'-alkyl-benzothiadiazides: a new subgroup of AMPA receptor modulators with improved affinity.

AMPA receptors form a major subdivision of the glutamate receptor family that mediates excitatory synaptic transmission in the brain. Currents through AMPA receptors can be up- or down-regulated by compounds that allosterically modulate receptor kinetics through binding sites distinct from that for glutamate. One of those modulators is the benzothiadiazide IDRA-21 which has been reported to enhance synaptic transmission and be effective in behavioral tests, but typically requires threshold concentrations of at least 100 microM to be active in vitro. In this study, new benzothiadiazides were developed with IDRA-21 as lead compound and examined for their potency in modulating AMPA receptor kinetics. A significant increase in drug affinity was obtained by alkyl substitution at the 5'-position of IDRA-21; substitutions at other positions of the benzothiadiazide core generally did not yield a further gain in affinity and in some cases abolished drug binding. The 5'-ethyl derivative exhibited an EC(50) value in the order of 22 microM which represents about a 30-fold gain in affinity over that of IDRA-21. The EC(50) value is comparable to that of cyclothiazide, the most potent benzothiadiazide drug, but the effects on AMPA receptors differed substantially between these two compounds in that the 5'-ethyl derivative of IDRA-21 greatly increased the binding affinity for receptor agonists whereas cyclothiazide is known to reduce agonist binding. The structure--activity relationships reported here thus offer to provide new insights how receptor kinetics is linked to particular aspects of receptor--drug interactions.