Activation Of Ligand-gated Ion Channels Research Articles

Use of Bioluminescence Resonance Energy Transfer (Bret) for Studies of Protein-Protein Interactions Between the GLUD2 Receptor and Intracellular Interaction Partners

The GluD2 receptor is a member of the ionotropic glutamate receptor (iGluR) superfamily and is expressed in cerebellar Purkinje cells (PCs). GluD2 receptors recognize the neurotransmitters D-serine, but lack excitatory ligand-gated ion channel activity. Recently, D-serine activity mediated via GluD2 has been shown to modulate cerebellar long-term-depression (LTD). The intracellular C-terminal domain (CTD) of GluD2 is required for D-serine regulation. A working hypothesis for this regulation is that D-serine binding to the extracellular ligand-binding domain is translated to intracellular rearrangement of the CTD and hereby potentially affects protein-protein interactions (PPIs) with intracellular scaffolding proteins or signaling enzymes involved in expression of cerebellar LTD. Various interacting partners of the GluD2 CTD have been identified; including the PDZ-domain containing scaffolding protein S-SCAM and Delphilin and the tyrosine kinase PTPMEG. Furthermore, the CTD contains multiple phosphorylation sites, which may regulate PPIs.In the present study, our aim is to study the potential regulation of GluD2 PPIs by phosphorylation and D-serine. For this purpose, we have established a bioluminescence resonance energy transfer (BRET) assay that enables measurement of GluD2 PPIs in intact cells by inserting the resonance energy donor Renilla luciferase 8 (Rluc8) at various positions in full-length GluD2 or the isolated CTD and acceptor molecule green fluorescence protein 2 (GFP2) to known GluD2 interaction partners.For PSD95, Delphilin, AP-4 and S-SCAM, it was possible to detect robust BRET between GluD2 and GFP2-tagged full-length interacting partners as well as truncated versions containing the isolated PDZ domain. Hereby, an experimental platform has been created for studying GluD2 PPIs in intact cells. Results from ongoing characterizations of the influence of CTD phosphorylation and D-serine ligand-binding on PPIs will be presented.

Defining the structural relationship between kainate-receptor deactivation and desensitization

Desensitization is an important mechanism that curtails the activity of ligand-gated ion-channels (LGICs). Although the structural basis of desensitization is not fully resolved, it is thought to be governed by the physicochemical properties of the bound ligand. Here, we show the importance of an allosteric cation binding pocket in controlling transitions between activated and desensitized states of rat kainate-type (KAR) ionotropic glutamate receptors (iGluRs). Tethering a positive charge to this pocket sustains KAR activation, preventing desensitization, whereas mutations that disrupt cation binding eliminate channel gating. These different outcomes explain the structural distinction between deactivation and desensitization. Deactivation occurs when the ligand unbinds before the cation, whereas desensitization proceeds if a ligand is bound without cation pocket occupancy. This sequence of events is absent from AMPA-type iGluRs, identifying cations as gatekeepers of KAR gating, a role unique among even closely-related LGICs.

Spontaneous Inhibitory Synaptic Currents Mediated by a G Protein-Coupled Receptor

G protein-coupled receptors (GPCRs) affect many physiological processes by modulating both intrinsic membrane conductances and synaptic transmission. This study describes spontaneous miniature inhibitory postsynaptic currents mediated by vesicular dopamine release acting locally on metabotropic D2 receptors leading to the activation of a G protein-coupled inwardly rectifying potassium conductance. Thus, individual exocytotic events result in spontaneous GPCR-mediated transmission, similar to synaptic activation of classical ligand-gated ion channels.

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes (“catch” and “hold”) that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement (“capping”). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation.

General Anesthetics Predicted to Block the GLIC Pore with Micromolar Affinity

Although general anesthetics are known to modulate the activity of ligand-gated ion channels in the Cys-loop superfamily, there is at present neither consensus on the underlying mechanisms, nor predictive models of this modulation. Viable models need to offer quantitative assessment of the relative importance of several identified anesthetic binding sites. However, to date, precise affinity data for individual sites has been challenging to obtain by biophysical means. Here, the likely role of pore block inhibition by the general anesthetics isoflurane and propofol of the prokaryotic pentameric channel GLIC is investigated by molecular simulations. Microscopic affinities are calculated for both single and double occupancy binding of isoflurane and propofol to the GLIC pore. Computations are carried out for an open-pore conformation in which the pore is restrained to crystallographic radius, and a closed-pore conformation that results from unrestrained molecular dynamics equilibration of the structure. The GLIC pore is predicted to be blocked at the micromolar concentrations for which inhibition by isofluorane and propofol is observed experimentally. Calculated affinities suggest that pore block by propofol occurs at signifcantly lower concentrations than those for which inhibition is observed: we argue that this discrepancy may result from binding of propofol to an allosteric site recently identified by X-ray crystallography, which may cause a competing gain-of-function effect. Affinities of isoflurane and propofol to the allosteric site are also calculated, and shown to be 3 mM for isoflurane and for propofol; both anesthetics have a lower affinity for the allosteric site than for the unoccupied pore.

A rapid agonist application system for fast activation of ligand-gated ion channels

The synaptic delay between neurotransmitter release across the synaptic cleft and activation of neurotransmitter gated ion channels is less than a ms. Nicotinic acetylcholine receptors (nAChRs), like many other classes of ligand-gated ion channels, are comprised of different protein subunits forming a variety of receptors with different activation and desensitization kinetics and pharmacological sensitivities. To measure and fully characterize ligand-gated ion channel currents accurately, one must apply agonists in a fraction of a ms and repeatedly at various concentrations without any prior desensitization of the receptors. In this paper, we describe an economical, easy to assemble and operate rapid drug application system. The drug applicator system consists of a parallel array of three pinch valves, which allow either agonist or wash solution into a theta tube. Solution exchanges of 0.16 ms can be achieved. In transfected cells, ACh elicited α4β2 nicotinic currents with mean rise times of 55 ± 13 ms. We recorded α7 nAChRs, which desensitize very rapidly, and obtained very fast rise times of 19 ± 2 ms. With this novel drug applicator, agonists can be applied repeatedly without any loss of current. Hence, complete dose–response relations can be obtained for even α7 nAChRs, which are very sensitive to desensitization caused by agonist exposure on a ms time scale. The drug application system can also be extended to the study of ligand-gated ion channels in brain slices. The theta tube valve-driven drug applicator system can be applied to study other ligand-gated ion channels including glutamate and GABA receptors.

Effects of protostane-type triterpenoids on the 5-HT 3A receptor-mediated ion current in Xenopus oocytes

Alisol derivatives are unique protostane-type triterpenoid compounds that are isolated from Alismatis rhizoma, which is a well-known traditional medicine in East Asia. In the present study, we investigated the effects of protostane-type triterpenoids (AA, Alisol A; AB, Alisol B; AB-ac, Alisol B 23-acetate; AC-ac, Alisol C 23-aceteate) on 5-HT-induced currents mediated by the human 5-HT 3A receptor expressed in Xenopus laevis oocytes. Co-treatment with triterpenoids regulated the 5-HT-induced inward peak current in a concentration-dependent and reversible manner. In addition, regulation of I 5-HT by triterpenoids occurred in a non-competitive manner. Taken together, these results indicate that triterpenoids may regulate the 5-HT 3A receptors that are expressed in Xenopus oocytes. Furthermore, this regulation of the ligand-gated ion channel activity by triterpenoids may be one of the pharmacological actions of Alismatis rhizoma.

Energetics of glutamate receptor ligand binding domain dimer assembly are modulated by allosteric ions

The activity of many ligand-gated ion channels and cell surface receptors is modulated by small molecules and ions, but an understanding of the underlying molecular mechanisms is scarce. For kainate, but not AMPA subtype glutamate receptors, the binding of Na(+) and Cl(-) ions to discrete, electrostatically coupled sites in the extracellular ligand binding domain (LBD) dimer assembly regulates the rate of entry into the desensitized state, which occurs when the dimer interface ruptures and the channel closes. Studies on glutamate receptors have defined the LBD dimer assembly as a key functional unit that controls activation and desensitization. Here we use analytical ultracentrifugation to probe the energetic effects of allosteric ions on kainate receptor dimer stability in solution, using a GluR6 mutant that desensitizes slowly. Our results show that sodium and chloride ions modulate kainate receptor dimer affinity as much as 50-fold, and that removal of either Cl(-) or Na(+) disrupts the dimer. The applicability of a similar allosteric mechanism for modulation of delta2 glutamate receptors by Ca(2+) was also tested. Our results indicate that ions can contribute substantial free energy to active state stabilization in both these receptors, and provide quantitative measurements of the energetic consequences of allosteric ion binding to a ligand-gated ion channel.

Effects of triterpenoids from Poria cocos Wolf on the serotonin type 3A receptor-mediated ion current in Xenopus oocytes

Poria cocos Wolf ( P. cocos Wolf) is used to treat chronic gastritis, edema, nephrosis, gastric atony, acute gastroenteric catarrh, dizziness, emesis and vomiting. Triterpenoids are a class of natural compounds produced by P. cocos Wolf that contain acyclic 30-carbon precursors. In this study, we investigated the effect of triterpenoids (PA, Pachymic acid; DA, dehydroeburicoic acid; HA, 3β-hydroxylanosta-7,9(11),24-trien-21-oic acid) on human 5-hydroxytryptamine 3A (5-HT 3A) receptor channel activity, which is one of the ligand-gated ion channel families. The two-electrode voltage-clamp technique was used to examine the 5-HT3A mediated current. The inhibitory effect of triterpenoids on 5HT-induced inward current ( I 5-HT) occurred in a concentration dependent and reversible manner. Furthermore, the half-inhibitory concentrations (IC 50) of PA, DA and HA were 3.2 ± 0.2, 5.5 ± 0.6 and 1.4 ± 0.2 µM, respectively. This corresponded to an order of potency for the inhibition of I 5-HT in oocytes expressing human 5-HT 3A receptor of HA > PA > DA. Finally, inhibition of I 5HT by triterpenoids occurred in a non-competitive manner, while inhibition by HA and PA showed more voltage-dependency. Taken together, these results indicate that triterpenoids may regulate the expressed 5-HT 3A receptors in Xenopus oocytes. Furthermore, this regulation of the ligand-gated ion channel activity by triterpenoids may be one of the pharmacological actions of P. cocos Wolf.

A Novel Conotoxin Inhibitor of Kv1.6 Channel and nAChR Subtypes Defines a New Superfamily of Conotoxins,

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated pl14a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. pl14a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an alpha-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of pl14a revealed a novel signal sequence, indicating that pl14a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of pl14a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, pl14a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50 = 1.59 microM) and neuronal (IC50 = 8.7 microM for alpha3beta4) and neuromuscular (IC50 = 0.54 microM for alpha1beta1 epsilondelta) subtypes of the nicotinic acetylcholine receptor (nAChR). Similarities in sequence and structure are apparent between the middle loop of pl14a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.

External Ions Are Coactivators of Kainate Receptors

The activation of ligand-gated ion channels is thought to depend solely on the binding of chemical neurotransmitters. In this study, we demonstrate that kainate (KA) ionotropic glutamate receptors (iGluRs) require not only the neurotransmitter L-glutamate (L-Glu) but also external sodium and chloride ions for activation. Removal of external ions traps KA receptors (KARs) in a novel inactive state that binds L-Glu with picomolar affinity. Moreover, occupancy of KARs by L-Glu precludes external ion binding, demonstrating crosstalk between ligand- and ion-binding sites. AMPA iGluRs function normally in the absence of external ions, revealing that even closely related iGluR subfamilies operate by distinct gating mechanisms. This behavior is interchangeable via a single amino acid residue that operates as a molecular switch to confer AMPA receptor behavior onto KARs. Our findings identify a novel allosteric site that singles out KARs from all other ligand-gated ion channels.

Ubiquilin-1 Regulates Nicotine-induced Up-regulation of Neuronal Nicotinic Acetylcholine Receptors

Chronic exposure to nicotine, as in tobacco smoking, up-regulates nicotinic acetylcholine receptor surface expression in neurons. This up-regulation has been proposed to play a role in nicotine addiction and withdrawal. The regulatory mechanisms behind nicotine-induced up-regulation of surface nicotinic acetylcholine receptors remain to be determined. It has recently been suggested that nicotine stimulation acts through increased assembly and maturation of receptor subunits into functional pentameric receptors. Studies of muscle nicotinic acetylcholine receptors suggest that the availability of unassembled subunits in the endoplasmic reticulum can be regulated by the ubiquitin-proteosome pathway, resulting in altered surface expression. Here, we describe a role for ubiquilin-1, a ubiquitin-like protein with the capacity to interact with both the proteosome and ubiquitin ligases, in regulating nicotine-induced up-regulation of neuronal nicotinic acetylcholine receptors. Ubiquilin-1 interacts with unassembled alpha3 and alpha4 subunits when coexpressed in heterologous cells and interacts with endogenous nicotinic acetylcholine receptors in neurons. Coexpression of ubiquilin-1 and neuronal nicotinic acetylcholine receptors in heterologous cells dramatically reduces the expression of the receptors on the cell surface. In cultured superior cervical ganglion neurons, expression of ubiquilin-1 abolishes nicotine-induced up-regulation of nicotinic acetylcholine receptors but has no effect on the basal level of surface receptors. Coimmunostaining shows that the interaction of ubiquilin-1 with the alpha3 subunit draws the receptor subunit and proteosome into a complex. These data suggest that ubiquilin-1 limits the availability of unassembled nicotinic acetylcholine receptor subunits in neurons by drawing them to the proteosome, thus regulating nicotine-induced up-regulation.

Mechanism of Partial Agonist Action at the NR1 Subunit of NMDA Receptors

Partial agonists produce submaximal activation of ligand-gated ion channels. To address the question of partial agonist action at the NR1 subunit of the NMDA receptor, we performed crystallographic and electrophysiological studies with 1-aminocyclopropane-1-carboxylic acid (ACPC), 1-aminocyclobutane-1-carboxylic acid (ACBC), and 1-aminocyclopentane-1-carboxylic acid (cycloleucine), three compounds with incrementally larger carbocyclic rings. Whereas ACPC and ACBC partially activate the NMDA receptor by 80% and 42%, respectively, their cocrystal structures of the NR1 ligand binding core show the same degree of domain closure as found in the complex with glycine, a full agonist, illustrating that the NR1 subunit provides a new paradigm for partial agonist action that is distinct from that of the evolutionarily related GluR2, AMPA-sensitive receptor. Cycloleucine behaves as an antagonist and stabilizes an open-cleft conformation. The NR1-cycloleucine complex forms a dimer that is similar to the GluR2 dimer, thereby suggesting a conserved mode of subunit-subunit interaction in AMPA and NMDA receptors.

Evidence that the tertiary structure of 20( S)-ginsenoside Rg 3 with tight hydrophobic packing near the chiral center is important for Na + channel regulation

Ginsenosides are the active ingredients of Panax ginseng. Ginsenoside Rg 3 exists as two stereoisomers of carbon-20: 20- S-protopanaxatriol-3-[ O-β- d-glucopyranosyl (1 → 2)-β-glucopyranoside] (20( S)-Rg 3) and 20- R-protopanaxatriol-3-[ O-β- d-glucopyranosyl (1 → 2)-β-glucopyranoside] (20( R)-Rg 3). Recently, we reported that 20( S)-Rg 3 regulates voltage-dependent Ca 2+ channel activity and several types of ligand-gated ion channels, whereas 20( R)-Rg 3 does not have this activity. In this study, we investigated the structure–activity relationship of these two stereoisomers by NMR spectroscopy and by measurement of the current in Xenopus oocytes expressing the mouse cardiac voltage-dependent Na + channel (Na v1.5). We found that 20( S)-Rg 3 but not 20( R)-Rg 3 inhibited Na + channel current in a dose- and voltage-dependent manner. The difference between Rg 3 epimers in voltage-dependent ion channel regulation indicates that the structure of 20( S)-Rg 3 may be geometrically better aligned than that of 20( R)-Rg 3 for interaction with receptor regions in Na + channels. The 1H and 13C NMR chemical shifts, including all hydroxyl protons of 20( S)-Rg 3 and 20( R)-Rg 3, were completely assigned, and their tertiary structures were determined. 20( S)-Rg 3 has more tight hydrophobic packing near the chiral center than 20( R)-Rg 3. Tertiary structures and activities of 20( S)-Rg 3 and 20( R)-Rg 3 indicate that 20( S)-Rg 3 may have stronger interactions with the receptor region in ion channels than 20( R)-Rg 3. This may result in different stereoselectivity of Rg 3 stereoisomers in the regulation of voltage-dependent Na + channel activity. This is the first structural approach to ginsenoside action on ion channel.

In vitro inhibition of recombinant ligand-gated ion channels by high concentrations of milnacipran

Tricyclic antidepressants are known to inhibit various ligand-gated ion-channel (LGIC) receptors, and some of their clinical features may be associated with this activity. The effects of milnacipran, a selective inhibitor of the reuptake of serotonin and noradrenaline, on LGIC receptors have not yet been investigated on such ion-channel receptors. To determine the in vitro effect of milnacipran on four recombinant LGIC receptors, nicotinic acetylcholine, N-methyl-D-aspartate, gamma-amino butyric acid (GABA) and 5-hydroxytryptamine3A receptors. Receptors were expressed in Xenopus oocytes and LGIC activity measured using a two-voltage clamp technique. There was no interaction at the GABA receptor. The results at the other LGIC receptors showed that they could be inhibited by high concentrations of milnacipran. At high concentrations, milnacipran can inhibit certain LGICs. It is, however, unlikely that these interactions have any clinical consequence under normal therapeutic conditions, since the concentrations required are considerably higher than those achieved in plasma of treated patients.

Activation and modulation of ligand-gated ion channels

Ligand-gated ionic channels are integral membrane proteins that enable rapid and selective ion fluxes across biological membranes. In excitable cells, their role is crucial for generation and propagation of electrical signals. This survey describes recent results from studies performed in the Department of Cellular Neurophysiology, Institute of Physiology ASCR, aimed at exploring the conformational dynamics of the acetylcholine, glutamate and vanilloid receptors during their activation, inactivation and desensitization. Distinct families of ion channels were selected to illustrate a rich complexity of the functional states and conformational transitions these proteins undergo. Particular attention is focused on structure-function studies and allosteric modulation of their activity. Comprehension of the fundamental principles of mechanisms involved in the operation of ligand-gated ion channels at the cellular and molecular level is an essential prerequisite for gaining an insight into the pathogenesis of many psychiatric and neurological disorders and for efficient development of novel specifically targeted drugs.

Effects of ginsenosides on GABA(A) receptor channels expressed in Xenopus oocytes.

Ginsenosides, major active ingredients of Panax ginseng, are known to regulate excitatory ligand-gated ion channel activity such as nicotinic acetylcholine and NMDA receptor channel activity. However, it is not known whether ginsenosides affect inhibitory ligand-gated ion channel activity. We investigated the effect of ginsenosides on human recombinant GABA(A) receptor (alpha1beta1gamma2S) channel activity expressed in Xenopus oocytes using a two-electrode voltage-clamp technique. Among the eight individual ginsenosides examined, namely, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2, we found that Rc most potently enhanced the GABA-induced inward peak current (I(GABA)). Ginsenoside Rc alone induced an inward membrane current in certain batches of oocytes expressing the GABA(A) receptor. The effect of ginsenoside Rc on I(GABA) was both dose-dependent and reversible. The half-stimulatory concentration (EC50) of ginsenoside Rc was 53.2 +/- 12.3 microM. Both bicuculline, a GABA(A) receptor antagonist, and picrotoxin, a GABA(A) channel blocker, blocked the stimulatory effect of ginsenoside Rc on I(GABA). Niflumic acid (NFA) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), both Cl- channel blockers, attenuated the effect of ginsenoside Rc on I(GABA). This study suggests that ginsenosides regulated GABA(A) receptor expressed in Xenopus oocytes and implies that this regulation might be one of the pharmacological actions of Panax ginseng.

Molecular determinants for the action of general anesthetics at recombinant alpha(2)beta(3)gamma(2)gamma-aminobutyric acid(A) receptors.

General anesthetics modulate the activity of ligand-gated ion channels including the GABA(A) receptor. Mutational studies mainly on the benzodiazepine-insensitive alpha(2)beta(1(M286W)) and alpha(6)beta(3(N289M))gamma(2) GABA(A) receptors revealed that a serine in transmembrane domain 2 and a methionine in transmembrane domain 3 are essential for the action of most general anesthetics. We investigated whether these residues would similarly be relevant for their action at the benzodiazepine-sensitive GABA receptor subtype, alpha(2)beta(3)gamma(2). We found that not only the N265M but also the M286W mutation nearly abolished the modulatory effect of etomidate. However, the anti-convulsant loreclezole, a structural homologue of etomidate, was inactive on the N265M mutant, but displayed normal modulatory activity on the M286W mutant. Both mutations did not affect the modulatory action of the neurosteroid alphaxalone. The direct action of alphaxalone, however, was dramatically increased in the M286W mutant to about twice the maximal GABA current but not significantly affected in the N265M mutant. These data demonstrate that the structural requirements for modulatory and direct actions of various general anesthetics are distinct. The molecular switches induced by these mutations can be exploited to identify the molecular determinants for the action of general anesthetics.

What the actions of anaesthetics on fast synaptic transmission reveal about the molecular mechanism of anaesthesia

1. Synapses with the brain are important components of the networks responsible for higher nervous function and current evidence suggests that general anaesthetics modulate synaptic transmission in the brain. 2. Analysis of anaesthetic action on these synapses not only defines the cellular mechanisms involved in anaesthesia but also reveals much about the molecular targets of anaesthetic action. 3. It appears that while anaesthetics affect a wide variety of processes, the most sensitive are those which are directly linked to the activity of ligand-gated ion channels. Moreover, both single channel patch clamp studies and the molecular biological investigations of the sub-unit specificity of the sensitivity to anaesthetics indicate that anaesthetics interact directly with these functional proteins.

Neurosteroid modulation of recombinant ionotropic glutamate receptors

Pregnenolone sulfate (PS) is an abundant neurosteroid that can potentiate or inhibit ligand gated ion channel activity and thereby alter neuronal excitability. Whereas PS is known to inhibit kainate and AMPA responses while potentiating NMDA responses, the dependence of modulation on receptor subunit composition remains to be determined. Toward this end, the effect of PS on recombinant kainate (GluR6), AMPA (GluR1 or GluR3), and NMDA (NR1 100+NR2A) receptors was characterized electrophysiologically with respect to efficacy and potency of modulation. With Xenopus oocytes expressing GluR1, GluR3 or GluR6 receptors, PS reduces the efficacy of kainate without affecting its potency, indicative of a noncompetitive mechanism of action. Conversely, with oocytes expressing NR1 100+NR2A subunits, PS enhances the efficacy of NMDA without affecting its potency. Whereas the modulatory efficacy, but not the potency, of PS is increased two-fold by co-injection of NR1 100+NR2A cRNAs as compared with NR1 100 cRNA alone, there is little or no effect of the NR2A subunit on efficacy or potency of pregnanolone (or epipregnanolone) sulfate as an inhibitor of the NMDA response. This suggests that the NR2A subunit controls the efficacy of neurosteroid enhancement, but not inhibition, which is consistent with our previous finding that potentiating and inhibitory steroids act at distinct sites on the NMDA receptor. This represents a first step towards understanding the role of subunit composition in determining neurosteroid modulation of ionotropic glutamate receptor function.