Agonist and blocking actions of choline and tetramethylammonium on human muscle acetylcholine receptors

Choline has been used widely as an agonist for the investigation of gain-of-function mutants of the nicotinic acetylcholine receptor. It is useful because it behaves like a partial agonist. The efficacy of choline is difficult to measure because choline blocks the channel at concentrations about four times lower than those that activate it. We have fitted activation mechanisms to single-channel activity elicited from HEK-expressed human recombinant muscle nicotinic receptors by choline and by tetramethylammonium (TMA). Channel block by the agonist was incorporated into the mechanisms that were fitted, and block was found not to be selective for the open state. The results also suggest that channel block is very fast and that the channel can shut almost as fast as normal when the blocker was bound. Single-channel data are compatible with a mechanism in which choline is actually a full agonist, its maximum response being limited only by channel block. However, they are also compatible with a mechanism incorporating a pre-opening conformation change ('flip') in which choline is a genuine partial agonist. The latter explanation is favoured by concentration jump experiments, and by the fact that only this mechanism fits the TMA data. We propose that choline, like TMA, is a partial agonist because it is very ineffective (approximately 600-fold less than acetylcholine) at eliciting the initial, pre-opening conformation change. Once flipping has occurred, all agonists, even choline, open the channel with similar efficiency.     

Activation of NR1/NR2B NMDA receptors

N-methyl-D-aspartate (NMDA) receptors are highly expressed in the central nervous system and are involved in excitatory synaptic transmission as well as synaptic plasticity. Despite considerable structural and biophysical research, the mechanism behind activation of the NMDA receptor is still poorly understood. By analyzing patch clamp recordings of one channel activated by glutamate, we determined the burst structure and open probability for recombinant rat NR1/NR2B receptors. We used partial agonists at the glutamate and glycine binding sites to show that at least two kinetically distinct subunit-associated conformational changes link co-agonist binding to the opening of the NMDA receptor pore. These data suggest that NR1 and NR2B subunits, respectively, undergo a fast and slow agonist-dependent conformational change that precedes opening of the pore. We propose a new working model of receptor activation that can account for macroscopic as well as microscopic NMDA receptor properties.     

Activation and block of mouse muscle-type nicotinic receptors by tetraethylammonium

We have studied the activation and inhibition of the mouse muscle adult-type nicotinic acetylcholine receptor by tetraethylammonium (TEA) and related quaternary ammonium derivatives. The data show that TEA is a weak agonist of the nicotinic receptor. No single-channel clusters were observed at concentrations as high as 5 mM TEA or in the presence of a mutation which selectively increases the efficacy of the receptor. When coapplied with 1 mM carbamylcholine (CCh), TEA decreased the effective opening rate demonstrating that it acts as a competitive antagonist of CCh-mediated activation. Kinetic analysis of currents elicited by CCh and TEA allowed an estimate of receptor affinity for TEA of about 1 mM, while an upper limit of 10 s⁻¹ could be set for the wild-type channel-opening rate constant for receptors activated by TEA alone. At millimolar concentrations, TEA inhibited nicotinic receptor currents by depressing the single-channel amplitude. The effect had an IC₅₀ of 2–3 mM, depending on the conditions of the experiment, and resembled a standard open-channel block. However, the decrease in channel amplitudes was not accompanied by an increase in the mean burst duration, indicating that a linear open-channel blocking mechanism is not applicable. Upon studying block by other nicotinic receptor ligands it was found that block by CCh, tetramethylammonium and phenyltrimethylammonium can be accounted for by the sequential blocking mechanism while block in the presence of methyltriethylammonium, ethyltrimethylammonium or choline was inconsistent with such a mechanism. A mechanism in which receptors blocked by TEA can close would account for the experimental findings.     

Evaluation of a proposed mechanism of ligand-gated ion channel activation in the GABAA and glycine receptors

Ligand-gated ion channels (LGICs) mediate rapid chemical neurotransmission in the mammalian brain. This gene superfamily includes the nicotinic acetylcholine (nAChR), GABA(A), 5-hydroxytryptamine type 3, and glycine receptors. Upon agonist binding these receptors undergo a rapid allosteric transition from the closed to open state. The molecular mechanism of coupling between agonist binding and channel gating remains poorly understood, in part due to the lack of a high-resolution structure of the entire receptor. Miyazawa, Fujiyoshi, and Unwin published a 4A resolution structure of the nAChR, and proposed that a single residue--valine 44 in Loop 2 of the extracellular domain--functions as a critical determinant of a "pin-into-socket" mechanism for receptor activation in nAChR. Here we examined whether this proposed "pin-into-socket" mechanism also contributes to channel activation in the GABA(A) and glycine receptors. We mutated residues corresponding to nAChR valine 44 in the GABA(A) (alpha(1) histidine 56 and beta(2) valine 53) and glycine (alpha(1) threonine 54) receptors. The results obtained in this study do not support a simple "pin-into-socket" mechanism of activation for the activation of GABA(A) and glycine receptors. This conclusion is consistent with other recent reports in which mutations of residues distributed throughout several loops of nAChR, GABA(A) and glycine receptors had large effects on gating behavior.     

Contributions of the non-α subunit residues (loop D) to agonist binding and channel gating in the muscle nicotinic acetylcholine receptor

The agonist binding site of the nicotinic acetylcholine receptor has a loop-based structure, and is formed by residues located remotely to each other in terms of primary structure. Amino acid residues in sites δ57 and δ59, and the equivalent residues in the ε subunit, have been identified as part of the agonist binding site and designated as loop D. The effects of point mutations in sites δ57, δ59, ε55 and ε57 on agonist binding and channel gating were studied. The mutated receptors were expressed transiently in HEK 293 cells and the currents were recorded using the cell-attached single-channel patch clamp technique. The results demonstrate that the mutations mainly affect channel gating with the major portion of the effect due to a reduction in the channel opening rate constant. For both the δ57/ε55 and the δ59/ε57 site, a mutation in the ε subunit had a greater effect on channel gating than a mutation in the δ subunit. In all instances, agonist binding was affected to a lesser degree than channel gating. Previous data have placed the δ57 and δ59 residues in or near the agonist binding pocket. The data presented here suggest that these two residues (and the homologous sites in the ε subunit) are not involved in specific interactions with the nicotinic agonist and that they affect the activation of the nicotinic receptor by shaping the overall structure of the agonist binding site.     

Nicotinic receptor: an allosteric protein specialized for intercellular communication

The nicotinic acetylcholine receptor is the prototype of a superfamily of ligand-gated channels and shares with the other members of the family, such as glycine, GABA and the 5HT₃receptor, many structural features including its transmembrane organization. They are integral membrane proteins made up of the assembly of five subunits each of them spanning four times the membrane. Up to now, 9 α, 3 β, 1 γ, 1 δ and 1 ϵ subunits have been identified and cloned. Sequence homologies suggest that they derive from a common ancestor. Strong evidence, however, supports the view that other subunits whose function is unknown are still missing. Affinity labeling and site-directed mutagenesis of the agonist binding site reveal that the agonist binds at the interface between two adjacent α-and non-α subunits. Structural and functional studies point to the major contribution of the transmembrane segment M2 to the formation of the ionic pore, the properties of which depend critically on amino acid residues which face the channel lumen and are aligned along the meridian of an α-helix. Assembly of differents subunits into a pseudo-symmetrical heteropentamer results in a large number of combinations of receptors which may differ by their physiological and/or pharmacological properties. Several regulatory properties of the nicotinic acetylcholine receptors (as well as those of other ligand-gated channels) can be accounted for by an adapted version of the allosteric model. According to this model, binding of the agonist stabilizes one (or more) conformational state(s), and thusindirectlycauses channel opening and/or desensitization. Short-term modulation of the efficacy of these receptors can also be explained on the basis of this model assuming that modulatory ligands act as allosteric effectors which preferentially stabilize defined conformational states when they bind at allosteric sites topologically distinct from the agonist binding site.     

Activation of heteroliganded mouse muscle nicotinic receptors

The activation of the mouse muscle-type nicotinic acetylcholine receptor was studied in the presence of carbachol, and in the simultaneous presence of carbachol and choline. The channel currents were recorded under steady-state conditions using cell-attached single-channel patch clamp, and during transient exposures to the agonists using a piezo-driven fast application system. The presence of choline resulted in inhibition of currents elicited by carbachol. The inhibitory effect of choline manifested as a reduction in the effective opening rate (increase in the mean intracluster closed time duration) in single-channel recordings. In the fast application experiments, the peak current amplitude was reduced and the current rise time increased when choline was co-applied with carbachol. The data were analysed according to a model in which receptor interactions with carbachol and choline resulted in three types of ligation: receptors occupied by two carbachol molecules, receptors occupied by two choline molecules, and receptors in which one agonist binding site was occupied by carbachol and the other by choline, i.e. heteroliganded receptors. All three agonist-bound receptor populations could open albeit with different efficacies. The affinity of the resting receptor to choline was estimated to be 1–2 m m , and heteroliganded receptors opened with an opening rate constant of ∼3000 s⁻¹. The results of the analysis suggest that the presence of choline in the neuromuscular junction in vivo has little effect on the time course of synaptic currents. Nevertheless, the contribution of heteroliganded receptors should be taken into consideration when the receptor is exposed simultaneously to two or more agonists.     

Single-channel study of the spasmodic mutation α1A52S in recombinant rat glycine receptors

Inherited defects in glycine receptors lead to hyperekplexia, or startle disease. A mutant mouse, spasmodic , that has a startle phenotype, has a point mutation (A52S) in the glycine receptor α1 subunit. This mutation reduces the sensitivity of the receptor to glycine, but the mechanism by which this occurs is not known. We investigated the properties of A52S recombinant receptors by cell-attached patch-clamp recording of single-channel currents elicited by 30–10000 μ m glycine. We used heteromeric receptors, which resemble those found at adult inhibitory synapses. Activation mechanisms were fitted directly to single channel data using the HJCFIT method, which includes an exact correction for missed events. In common with wild-type receptors, only mechanisms with three binding sites and extra shut states could describe the observations. The most physically plausible of these, the 'flip' mechanism, suggests that preopening isomerization to the flipped conformation that follows binding is less favoured in mutant than in wild-type receptors, and, especially, that the flipped conformation has a 100-fold lower affinity for glycine than in wild-type receptors. In contrast, the efficacy of the gating reaction was similar to that of wild-type heteromeric receptors. The reduction in affinity for the flipped conformation accounts for the reduction in apparent cooperativity seen in the mutant receptor (without having to postulate interaction between the binding sites) and it accounts for the increased EC ₅₀ for responses to glycine that is seen in mutant receptors. This mechanism also predicts accurately the faster decay of synaptic currents that is observed in spasmodic mice.     

Kinetic Determinants of Agonist Action at the Recombinant Human Glycine Receptor

The amino acids glycine, β-alanine and taurine are all endogenous agonists of the glycine receptor. In this study, a combination of rapid agonist application onto macropatches and steady-state single-channel recordings was used to compare the actions of glycine, β-alanine and taurine upon homomeric α₁ human glycine receptors transiently expressed in human embryonic kidney (HEK 293) cells. The 10–90 % rise times determined from rapid application of 100 μ m of each agonist were indistinguishable, indicating each agonist has a similar association rate. At saturating concentrations (30 m m ) the rise time for glycine (0.26 ms) was 1.8-fold faster than that for β-alanine (0.47 ms) and 3.9-fold faster than that for taurine (1.01 ms), indicating clear differences in the maximum opening rate between agonists. The relaxation following rapid removal of agonist was fitted with a single exponential for β-alanine (3.0 ms) and taurine (2.2 ms), and two exponential components for glycine with a weighted mean time constant of 27.1 ms. This was consistent with differences in dissociation rates estimated from analysis of bursts, with taurine > β-alanine > glycine. Exponential fits to the open period distributions gave time constants that did not differ between agonists and the geometric distribution for the number of openings per burst indicated that all three agonists had a significant component of single-opening bursts. Based upon these data, we propose a kinetic scheme with three independent open states, where the opening rates are dependent upon the activating agonist, while the closing rates are an intrinsic characteristic of the receptor.     

Activation of α6-containing GABAA receptors by pentobarbital occurs through a different mechanism than activation by GABA

The GABA_A receptors are ligand-gated chloride channels which are the targets for many clinically used sedatives, including the barbiturates. The barbiturate pentobarbital acts through multiple sites on the GABA_A receptor. At low concentrations (μM), it acts as a positive allosteric modulator while at higher concentrations it can directly activate the receptor. This agonist action is influenced by the subunit composition of the receptor, and pentobarbital is a more effective agonist than GABA only at receptors containing an α6 subunit. The conformational change that translates GABA binding into channel opening is known to involve a lysine residue located in an extracellular domain between the 2nd and 3rd transmembrane domains. Mutations of this residue disrupt activation of the channel by GABA and have been linked to inherited epilepsy. Pentobarbital binds to the receptor at a different agonist site than GABA, but could use a common signal transduction mechanism to gate the channel. To address this question, we compared the effect of a mutating the homologous lysine residue in the α1 or α6 subunits (K278 or K277, respectively) to methionine on direct activation of recombinant GABA_A receptors by GABA or pentobarbital. We found that this mutation reduced GABA sensitivity for both α1 and α6 subunits, but affected pentobarbital sensitivity only for the α1 subunit. This suggests that pentobarbital acts through a distinct signal transduction pathway at the α6 subunit, which may account for its greater efficacy compared to GABA at receptors containing this subunit.     

Receptor-ion channel interactions in Torpedo electric organ: Influence of thiol group modification

The influence of sulfhydryl and disulfide reagents on nicotinic cholinergic receptors and ion channels and their interactions was investigated using specific probes for receptor and channel binding sites in electric organs from Torpedo californica. N-ethylmaleimide, a sulfhydryl alkylating agent, did not alter receptor or ion channel binding, or receptor-mediated ion channel binding activation or desensitization. Alteration of receptor-ion channel coupling produced by treatment with an organic sulfhydryl, dithiothreitol, could be accounted for on the basis of decreases in receptor affinity for agonists. These changes were reversed by exposure to an oxidizing agent. Following reduction by dithiothreitol, N-ethylmaleimide treatment produced large decreases in the extent and affinity of both receptor and channel binding. No evidence for a direct role of thiol groups in ion channel function was obtained.     

Alterations in leukocyte beta-receptor affinity with aging. A potential explanation for altered beta-adrenergic sensitivity in the elderly

The elderly have reduced beta-adrenergic sensitivity to agonists, but no change in receptor density. We investigated the relation between beta-receptor affinity for agonists and age in beta-receptors on lymphocytes from 20 healthy men 21 to 74 years old. As an index of beta-receptor affinity for agonists, we determined the IC50 of isoproterenol--the concentration of isoproterenol required to inhibit 50 per cent of iodohydroxybenzylpindolol binding in vitro. We found that receptor affinity for agonists was correlated with age and plasma norepinephrine concentration. Twelve subjects (six 21 to 29 years old and six 55 to 74 years old) were also studied in both the supine and upright positions. In samples obtained in the supine position, the proportion of receptors binding agonist with a high affinity was decreased in the older subjects as compared with the young subjects (22 +/- 1 per cent vs. 38 +/- 3 per cent; P less than 0.05). With upright posture and the associated acute elevation of endogenous plasma catecholamines, the proportion of receptors binding agonist with a high affinity was reduced in the young; no such changes were seen in the older subjects. The data suggest that reduced beta-receptor affinity for agonists may be an explanation for altered beta-adrenergic sensitivity in the elderly.     

Calcium channel effectors are potent non-competitive blockers of acetylcholine receptors

Nicardipine and other calcium channel effectors (CCEs) were studied for their effects on nicotinic acetylcholine receptor (nAChR) activity. While CCEs had no effect on frog (Rana pipiens) skeletal muscle contractions resulting from nerve stimulation or direct stimulation of the muscle, nicotinic agonist-induced contractures were blocked. Nicardipine did not affect nAChR single-channel open time or amplitude, corroborating data from endplate currents (EPCs); EPC amplitudes and decays were unaffected. All the CCEs tested, however, non-competitively blocked nAChRs. The block of nAChRs resulted in a shortened agonist-induced depolarization and thus a diminished contracture response. An increase in cultured mouse skeletal muscle (C-2) cell single-channel closed times was observed with the intracellular addition of nicardipine, verifying a direct block of nAChRs. Using single-channel analysis, nicardipine's site of action, or at least access to its site of action, was determined to be at the intracellular side of the receptor. A direct action of the CCEs on the nAChR was also shown by their ability to block phencyclidine (PCP) binding to Torpedo nobiliana membranes. All the CCEs blocked specific binding of [³H]-PCP to its binding site on the nAChR-channel complex, with bepridil and nicardipine being the most potent. These data are compatible with a model in which nicardipine and other CCEs, at concentrations which do not alter nAChR channel open time or conductance, block the effects of superfused nicotinic agonist on nAChRs either by stabilizing the formation of the nAChR desensitized state or by effecting a slow channel block.     

Channel gating of NMDA receptors

Glutamate receptors specifically activated by N-methyl-D-aspartate (NMDA receptors) are ion channels that play multiple fundamental roles in the physiology of vertebrate nervous systems. The mechanisms that control the opening and closing, or gating, of the channel of NMDA receptors are among the most basic determinants of receptor function, and yet are not well understood. Here we consider current understanding of the link between agonist binding and NMDA receptor channel gating, of the conformational changes that occur during gating, and of the location of the channel gate. Information is drawn from studies of NMDA receptors themselves, of other types of glutamate receptors, and of more distantly related potassium channels.     

Cholinergic actions of false neurotransmitters: acetyldiethylcholine

Acetyldiethylcholine (AcDECh), a false transmitter at cholinergic synapses, binds to central muscarinic receptors with 14-fold lower affinity than acetylcholine (AcCh). The properties of this binding, including the limited extent of regional specificity and binding activation by N-ethylmaleimide, are those associated with weak agonists and antagonists. Intracerebroventricular injection of AcDECh or AcCh produces an increase in arterial blood pressure which is blocked by prior administration of atropine (10 nmol). While AcDECh is 17-fold less potent tha AcCh in producing the pressor response, the maximum pressure changes and time courses are comparable. AcDECh also binds to nicotinic receptors of Torpedo electric organs thereby inducing conformational changes in the receptor-ion channel complex that are associated with postsynaptic activation. In these nicotinic actions, however, AcDECh is 320-fold less active than AcCh.     

Reduced nicotinic receptor function in sympathetic ganglia is responsible for the hypothermia in the acetylcholinesterase knockout mouse

Cholinergic signalling in the sympathetic ganglia (SG) contributes to non-shivering thermogenesis by relaying the activation signal from the brain to SG neurons which activate many peripheral tissues to produce heat. Paradoxically, acetylcholinesterase (AChE) inhibitors, which should enhance cholinergic signalling, induce hypothermia. To understand the mechanism of how cholinergic signalling in the SG controls thermoregulation, we analysed infant AChE knockout mice, which are known to show hypothermia by postnatal day 15. Nicotinic receptor currents were reduced in acutely dissociated SG neurons of the AChE-deficient mice by over 40% compared with wild-type mice. When wild-type neurons were treated for 1 h with either oxotremorine-M, a muscarinic agonist, or nicotine, the amplitude of nicotinic receptor currents was also decreased by 40%. The hypothermia in AChE mutant mice was fully rescued by a peripheral injection of both ivermectin, which increases nicotinic receptor currents, and methyl-atropine, a muscarinic antagonist. Our results demonstrate that the hypothermia induced by the lack of AChE activity is primarily caused by a downregulation of nicotinic receptors via prolonged stimulation of muscarinic and nicotinic receptors in SG neurons. The stationary noise analysis of the nicotinic receptor current traces showed that the properties of single-channel activities were not different between the two genotypes, suggesting that the primary reason for downregulation of nicotinic receptors is due to a reduction of the receptors on the surface.     

Properties of the human muscle nicotinic receptor, and of the slow-channel myasthenic syndrome mutant εL221F, inferred from maximum likelihood fits

The mechanisms that underlie activation of nicotinic receptors are investigated using human recombinant receptors, both wild type and receptors that contain the slow channel myasthenic syndrome mutation, εL221F. The method uses the program HJCFIT, which fits the rate constants in a specified mechanism directly to a sequence of observed open and shut times by maximising the likelihood of the sequence with exact correction for missed events. A mechanism with two different binding sites was used. The rate constants that apply to the diliganded receptor (opening, shutting and total dissociation rates) were estimated robustly, being insensitive to the exact assumptions made during fitting, as expected from simulation studies. They are sufficient to predict the main physiological properties of the receptors. The εL221F mutation causes an approximately 4-fold reduction in dissociation rate from diliganded receptors, and a smaller increase in opening rate and mean open time. These are sufficient to explain the approximately 6-fold slowing of decay of miniature synaptic currents seen in patients. The distinction between the two binding sites was less robust, the estimates of rate constants being dependent to some extent on assumptions, e.g. whether an extra short-lived shut state was included or whether the EC₅₀ was constrained. The results suggest that the two binding sites differ by roughly 10-fold in the affinity of the shut receptor for ACh in the wild type, and that in the εL221F mutation the lower affinity is increased so the sites become more similar.     

Maximum likelihood fitting of single channel NMDA activity with a mechanism composed of independent dimers of subunits

Steady-state single channel activity from NMDA receptors was recorded at a range of concentrations of both glutamate and glycine. The results were fitted with several plausible mechanisms that describe both binding and gating. The mechanisms we have tested were based on our present understanding of receptor structure, or based on previously proposed mechanisms for these receptors. The steady-state channel properties appear to have virtually no dependence on the concentration of either ligand, other than the frequency of channel activations. This limited the ability to discriminate detail in the mechanism, and, along with the persistence of open–shut correlations in high agonist concentrations, suggests that NMDA channels, unlike other neurotransmitter receptors, cannot open unless all binding sites are occupied. As usual for analyses of NMDA channels, the applicability of our results to physiological observations is limited by uncertainties in synaptic zinc and hydrogen ion concentrations, both of these being known to affect the receptor. The mechanism that we propose, on the basis of steady-state single channel recordings, predicts with fair accuracy the apparent open and shut-time distributions in different concentrations of agonists, correlations between open and shut times, and both the rising and falling phases of the macroscopic response to concentration jumps, and can therefore account for the main features of synaptic currents.     

Horizontal cells isolated from catfish retina contain two types of excitatory amino acid receptors

1. Inward going membrane currents elicited by N-methyl-D-aspartate (NMDA), glutamate (GLU), and glutamate analogues were recorded from isolated catfish (Ictalurus punctatus) cone horizontal cells using the patch-clamp technique in the whole-cell mode. 2. Currents elicited by the N-methyl-D-aspartate receptor agonists NMDA, L-aspartate (ASP) or L-homocysteate (L-HCA) in nominally Mg-free saline were completely blocked by 100 microM 2-amino-5-phosphonovalerate (AP-5) but responses to non-NMDA receptor agonists kainate (KA), quisqualate (QA), or alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) in normal Mg2+ saline were unaffected. Responses to GLU were partially blocked. Kynurenic acid (1 mM) effectively blocked responses to all agonists. 3. Concentration-response curves obtained from measured responses in the presence of different NMDA receptor agonists gave Hill coefficients of near 1 indicating a single binding site for channel activation. The rank order of agonist affinity at the NMDA receptor is L-HCA greater than NMDA greater than ASP. Glycine potentiates responses to NMDA in horizontal cells. 4. The NMDA-activated channel is blocked in a voltage-dependent manner by Mg2+, Ni2+, and Co2+ and in a voltage-independent manner by Zn2+. Both the NMDA- and KA-activated channel were permeable to monovalent cations but the NMDA-activated channel appeared to have a greater permeability to Ca2+ than the KA-activated channel. 5. Concentration-response curves measured from responses to the non-NMDA receptor agonists QA, KA, and AMPA gave Hill coefficients of approximately 1.5 suggesting multiple binding sites for channel activation and cooperativity. The rank-order affinity was QA greater than AMPA greater than GLU greater than KA. KA was the most efficacious of the agonists resulting in the largest Imax. Rapid desensitization was observed only in the presence of QA, AMPA, or GLU and this desensitization could be removed by pretreatment with conconavalin A (Con A). 6. Single-channel conductance and mean open time were measured from the fluctuations in current noise in the presence of the agonists. The single-channel conductance estimated from the slope of a linear regression obtained from a plot of the variance of the conductance versus the whole-cell conductance measured in NMDA and ASP was 4.7 pS. The mean channel open time was 1.3 ms. These same parameters measured for KA and QA were 5.7 and 5.9 pS and 1.1 to 1.3 ms, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential effect of troponin T mutations on the inotropic responsiveness of mouse hearts – role of myofilament Ca2+ sensitivity increase

The agonist-binding domain of ionotropic glutamate receptors (iGluRs) has recently been crystallized as two polypeptide chains with a linker region. Although work on the structure of this isolated ligand-binding core has been invaluable, there is debate over how it relates to conformations adopted by intact receptors. iGluR crystals are proposed to represent the activated state as their degree of domain closure correlates well with agonist efficacy. However, iGluR crystals exhibit high agonist affinity that more closely matches that of desensitized receptors. Consequently, conformations adopted by iGluR crystals may represent this state. To test this, we have employed the plant lectin, concanavalin-A (Con-A) to report conformational changes elicited by kainate (KA) iGluR agonists during desensitization. When GluR6 KA receptors (KARs) were pre-incubated with Con-A, equilibrium responses evoked by the full agonist, l -glutamate ( l -Glu), increased almost 30-fold. However, in the continued presence of l -Glu, Con-A exerted no effect suggesting that it has restricted access to its binding sites when the agonist is bound. However, Con-A does not discriminate well between agonist-bound or -unbound states with the weak partial agonist, domoate. Accessibility experiments with KA were intermediate in nature consistent with its equilibrium efficacy at GluR6 KARs. Our results suggest that full and partial agonists elicit distinct conformational changes in KARs during desensitization. This finding can be reconciled with crystallographic data if the agonist-binding domain adopts the same conformation in the activated and desensitized states. However, other interpretations are possible suggesting future work is required if this issue is to be resolved.