Cyclothiazide decreases [3HAMPA binding to rat brain membranes: evidence that AMPA receptor desensitization increases agonist affinity

doi:10.1016/0006-8993(93)90978-V

Brain Research

Volume 628, Issues 1–2, 19 November 1993, Pages 345-348

https://doi.org/10.1016/0006-8993(93)90978-V Get rights and content

Abstract

The effects of cyclothiazide, a drug which blocks AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor desensitization, were tested on binding of [³HAMPA to rat brain membranes. Cyclothiazide reduced [³HAMPA binding by lowering the apparent affinity of the AMPA receptor. The magnitude of the decrease was temperature dependent and greater for membrane-bound than for solubilized receptors. These data provide evidence that desensitization increases the affinity of the AMPA receptor for agonists and indicate that a significant percentage of AMPA receptors in conventional equilibrium binding assays are in a desensitized state.

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Cited by (48)

Distinct Structural Features of Cyclothiazide Are Responsible for Effects on Peak Current Amplitude and Desensitization Kinetics at iGluR2
2009, Journal of Molecular Biology
Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission. Upon glutamate application, 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptors undergo rapid and almost complete desensitization that can be attenuated by positive allosteric modulators. The molecular mechanism of positive allosteric modulation has been elucidated previously by crystal structures of the ligand-binding core of iGluR2 in complex with, for example, cyclothiazide (CTZ). Here, we investigate the structure and function of CTZ and three closely related analogues NS1493, NS5206, and NS5217 at iGluR2, by X-ray crystallography and fast application patch-clamp electrophysiology. CTZ was the most efficacious and potent modulator of the four compounds on iGluR2(Q)_i [E_max normalized to response of glutamate: 754% (CTZ), 490% (NS1493), 399% (NS5206), and 476% (NS5217) and EC₅₀ in micromolar: 10 (CTZ), 26 (NS1493), 43 (NS5206), and 48 (NS5217)]. The four modulators divide into three groups according to efficacy and desensitization kinetics: (1) CTZ increases the peak current efficacy twice as much as the three analogues and nearly completely blocks receptor desensitization; (2) NS5206 and NS5217 have low efficacy and only attenuate desensitization partially; (3) NS1493 has low efficacy but nearly completely blocks receptor desensitization. A hydrophobic substituent at the 3-position of the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system is important for compound efficacy, with the following ranking: norbornenyl (bicyclo[2.2.1]hept-2-ene) > cyclopentyl > methyl. The replacement of the norbornenyl moiety with a significantly less hydrophobic cyclopentane ring increases the flexibility of the modulator as the cyclopentane ring adopts various conformations at the iGluR2 allosteric binding site. The main structural feature responsible for a nearly complete block of desensitization is the presence of an NH hydrogen bond donor in the 4-position of the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system, forming an anchoring hydrogen bond to Ser754. Therefore, the atom at the 4-position of CTZ seems to be a major determinant of receptor desensitization kinetics.
Measurement of Conformational Changes accompanying Desensitization in an Ionotropic Glutamate Receptor
2006, Cell
The canonical conformational states occupied by most ligand-gated ion channels, and many cell-surface receptors, are the resting, activated, and desensitized states. While the resting and activated states of multiple receptors are well characterized, elaboration of the structural properties of the desensitized state, a state that is by definition inactive, has proven difficult. Here we use electrical, chemical, and crystallographic experiments on the AMPA-sensitive GluR2 receptor, defining the conformational rearrangements of the agonist binding cores that occur upon desensitization of this ligand-gated ion channel. These studies demonstrate that desensitization involves the rupture of an extensive interface between domain 1 of 2-fold related glutamate-binding core subunits, compensating for the ca. 21° of domain closure induced by glutamate binding. The rupture of the domain 1 interface allows the ion channel to close and thereby provides a simple explanation to the long-standing question of how agonist binding is decoupled from ion channel gating upon receptor desensitization.
Use of [<sup>3</sup>H]fluorowillardiine to study properties of AMPA receptor allosteric modulators
2006, Brain Research
Citation Excerpt :
As expected, the drug increased the apparent affinities for the radioligands about three-fold but did not significantly alter Bmax values (receptor densities). Similar tests with other drugs, including CTZ, found that the drug-induced change in binding in each instance reflects a change in agonist affinity and not in Bmax (Hall et al., 1993a; Suzuki et al., 2004). 2,3-Benzodiazepines and related compounds, which include GYKI 52466, GYKI 53655 and SYM2206 (Fig. 1), effectively inhibit AMPA receptor currents, but their site of action and the nature of their impact on receptor kinetics are not yet clear.
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 (ampakines™) 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 (E_max) 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 (K_D 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.
Cyclothiazide binding to functionally active AMPA receptor reveals genuine allosteric interaction with agonist binding sites
2004, Neurochemistry International
The agonist, [ $^{3} H$ ](−)[S]-1-(2-amino-2-carboxyethyl)-5-fluoro-pyrimidine-2,4-dione ([ $^{3} H$ ](S)F-Willardiine) binding to functional alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors of resealed plasma membrane vesicles and nerve endings freshly isolated from the rat cerebral cortex displayed two binding sites (K_D1=33±7 nM, B_MAX1=1.6±0.3 pmol/mg protein, K_D2=720±250 nM and B_MAX2=7.8±4.0 pmol/mg protein). The drug which impairs AMPA receptor desensitisation, 6-chloro-3,4-dihydro-3-(2-norbornene-5-yl)-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide, CTZ) fully displaced the [ $^{3} H$ ](S)F-Willardiine binding at a concentration of 500 μM. In the presence of 100 μM CTZ (K_I(CTZ)=60±6 μM), both the antagonist [ $^{3} H$ ]-1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(F)quinoxaline-7-sulfonamide ([ $^{3} H$ ]NBQX: K_D=24±4 nM, B_MAX=12.0±0.1 pmol/mg protein) and the high-affinity agonist binding showed similar affinity reduction ([ $^{3} H$ ](S)F-Willardiine: K_D=140±19 nM, B_MAX=2.9±0.5 pmol/mg protein; [ $^{3} H$ ]NBQX: K_D=111±34 nM, B_MAX=12±3 pmol/mg protein). To disclose structural correlates underlying genuine allosteric binding interactions, molecular mechanics calculations of CTZ-induced structural changes were performed with the use of PDB data on extracellular GluR2 binding domain dimeric crystals available by now. Hydrogen-bonding and root mean square (rms) values of amino acid residues recognising receptor agonists showed minor alterations in the agonist binding sites itself. Moreover, CTZ binding did not affect dimeric subunit structures significantly. These findings indicated that the structural changes featuring the non-desensitised state could possibly occur to a further site of the extracellular GluR2 binding domain. The increase of agonist efficacy on allosteric CTZ binding may be interpreted in terms of a mechanism involving AMPA receptor desensitisation sequential to activation.
Interactions between recording technique and AMPA receptor modulators
2002, Brain Research
Citation Excerpt :
This is not entirely unexpected. Binding studies have shown that AMPA receptor kinetics, and the degree to which they are affected by cyclothiazide [16], are uncommonly sensitive to membrane variables (e.g. Ref. [25]). As hypothesized above, changes in the activity of protein kinases and/or phosphatases brought about by whole cell clamp recording could affect AMPA receptors.
Whole cell recording (EPSCs) and extracellular recording (field EPSPs) were compared in hippocampal field CA1 with regard to the effects of experimental treatments that increase AMPA receptor gated currents. Cyclothiazide, which maintains AMPA receptors in the sensitized state, caused a rapid and pronounced increase in EPSCs but only minor changes in field EPSPs. This difference was evident in recordings carried out at 22 and 32 °C and with different solutions in the clamp pipette. The larger effect of cyclothiazide on EPSCs was unaffected by blockade of GABA and NMDA receptors. Two-dimensional current source density analyses derived from 64 recording sites were used to provide extracellular estimates of AMPA receptor mediated synaptic currents. With this method, cyclothiazide again had much smaller effects than were obtained with whole cell clamp. Differences between whole cell and extracellular recordings were present, although not as pronounced, for the ampakines, a class of drugs that slow both deactivation and desensitization of AMPA receptors. Additionally, increases in synaptic responses produced by frequency facilitation, a manipulation that enhances the number of bound receptors, were not qualitatively different between recording techniques. These results support the conclusion that the whole cell clamp technique may alter AMPA receptors in such a way as to increase the relative importance of desensitization.
5′-Alkyl-benzothiadiazides: A new subgroup of AMPA receptor modulators with improved affinity
2002, Bioorganic and Medicinal Chemistry
Citation Excerpt :
This may also account for the observations that cyclothiazide and D1 produced very different changes in many receptor properties. For one, D1 increased binding of [3H]AMPA nearly 3-fold whereas cyclothiazide reduces binding under the same assay conditions by up to 90%.34 D1 also prolonged the rate at which responses to 1-ms glutamate pulses deactivate receptors by more than 4-fold, whereas cyclothiazide had relatively small effects.24
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 μM 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 EC₅₀ value in the order of 22 μM which represents about a 30-fold gain in affinity over that of IDRA-21. The EC₅₀ 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.

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: This work was supported by a Grant from the Air Force Office of Scientific Research (AFOSR 92-J-0307) to G.L.

^**: The authors wish to thank Eli Lilly Corp. for a generous donation of cyclothiazide, Dr. Gary Rogers for graciously providing the initial cyclothiazide samples, and Jackie Porter and Marla Lay for secretarial assistance.

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Cyclothiazide decreases [3HAMPA binding to rat brain membranes: evidence that AMPA receptor desensitization increases agonist affinity

Abstract

Trends Neurosci.

Neuron

Neuron

Neuron

Evidence that high- and low-affinity AMPA binding sites reflect membrane-dependent states of a single receptor

J. Neurochem.

Thiocyanate equally increases affinity for two AMPA receptor states

Mol. Pharmacol.

Cyclothiazide decreases [³HAMPA binding to rat brain membranes: evidence that AMPA receptor desensitization increases agonist affinity