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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2006 Feb 10;103(8):2943–2947. doi: 10.1073/pnas.0511063103

Effects of cyclothiazide on GluR1/AMPA receptors

Sergio Fucile *,†, Ricardo Miledi ‡,†, Fabrizio Eusebi *,§
PMCID: PMC1413821  PMID: 16473938

Abstract

Cyclothiazide (CTZ), a positive allosteric modulator of ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, is used frequently to block the desensitization of both native and heterologously expressed AMPA receptors. Specifically, CTZ is known to produce a fast inhibition of AMPA receptor desensitization and a much slower potentiation of the AMPA current. By using patch-clamp techniques, the effects of CTZ were studied in HEK 293 cells stably transfected with the rat flip GluR1 subunit. Upon CTZ treatment, we found an increased apparent affinity for the agonist, a slow whole-cell current potentiation, a fast inhibition of desensitization, and a lengthening of single-channel openings. Furthermore, we show that CTZ alters the channel gating events modifying the relative contribution of different single-channel classes of conductance (γ), increasing and decreasing, respectively, the contributions of γM (medium) and γL (low) without altering that of the γH (high) conductance channels. We also present a kinetic model that predicts well all of the experimental findings of CTZ action. Finally, we suggest a protocol for standard cell treatment with CTZ to attain maximal efficacy of CTZ on GluR1 receptors.

Keywords: glutamate receptor, Hek 293, receptor potentiation

Cyclothiazide (CTZ) is one of the most potent benzothiadiazides, a class of positive allosteric modulators of non-NMDA-type glutamate receptors (1, 2), that inhibit receptor desensitization with a marked selectivity for the flip splice variants (3) of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (4). For that reason, treating cells with CTZ has become a very useful and widespread tool for investigating the molecular composition of AMPA receptors and the mechanisms of desensitization (for review, see ref. 5).

The AMPA receptors (for review, see ref. 6) are homo- or heterotetrameric assemblies (7) organized in dimer pairs (8, 9), and their rapid desensitization is: (i) controlled by the dimer interface, being reduced by CTZ through dimer interface stabilization (10); (ii) dependent on the receptor subunit composition, including alternative splicing (4, 11, 12); and (iii) characterized by the receptor entry into an inactive state displaying an increased binding affinity (13).

Together with a fast inhibition of desensitization (4, 14–16), CTZ causes many other effects on AMPA-type receptors including: (i) a slow increase in the peak amplitude of whole-cell currents (2, 4, 14); (ii) an enhancement of the current deactivation time (14); (iii) a lengthening of single-channel openings (2); (iv) a decrease in agonist binding (17, 18); and (v) an increase in the apparent affinity for agonists (2, 4, 14).

Several studies have provided important insights into the structural determinants and mechanisms of the interaction of CTZ with AMPA receptors (10, 15, 19–22). Because those studies have been made in different systems, however, a global picture of the various effects of CTZ is still lacking. This study reports some of the effects of CTZ on AMPA receptors, all in the same cell system (HEK 293 cells stably transfected with the rat GluR1 subunit), highlighting the discrepancy between the fast inhibition of current desensitization and the slow current potentiation.

Results and Discussion

Concentration–Response Relationships on CTZ Administration.

HEK 293 cells expressing the rat flip GluR1 subunit (GluR1-HEK cells) responded to AMPA with an inward current that decayed biphasically due to a rapid desensitization leading to a fairly stable phase (Fig. 1A). The same cells treated with CTZ generated AMPA current responses in which the profiles depended on the concentrations of AMPA and CTZ, as well as on the duration of the exposure to CTZ. The AMPA currents were greatly potentiated by CTZ in a dose-dependent manner. For instance, by using 150 μM AMPA, a 4-s pretreatment of the cells with 100 μM CTZ increased the peak AMPA currents by 90-fold (EC50 = 28 μM; nH = 2.8; n = 5), the currents at the end of AMPA application by 636-fold (EC50 = 46 μM; nH = 3.6), and the current integrals by 730-fold (EC50 = 41 μM; nH = 4.5) (Fig. 1 A and B). The potentiation induced by CTZ was accompanied by an increase in the apparent affinity of the receptors for AMPA. For example, in the absence of CTZ, the AMPA dose/current–response curve exhibited an EC50 value of 139 μM (Fig. 2A and C). In contrast, with 50 μM CTZ in the bathing medium (i.e., EC50 value for CTZ with AMPA at 10 μM; data not shown), the AMPA dose/current–response relation shifted to the left with an ≈8-fold decrease in the EC50 value (Fig. 2 B and C). To maximize the effects of CTZ, the subsequent experiments were done with CTZ at 100 μM.

Fig. 1.

Fig. 1.

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Effects of CTZ on whole-cell currents elicited by AMPA. (A) Typical whole-cell currents elicited in a cell by AMPA (solid bars) alone or in the presence of CTZ (hatched bars), applied for 4 s before the coapplication of CTZ plus AMPA. For this and the other figures, the holding potential was −70 mV. (B) The CTZ dose/current–response relationship to AMPA (150 μM). Data (n = 6) represent current amplitudes at peak (•) and at the end of AMPA application (○) and current integrals (▾) normalized to the values obtained without CTZ (mean peak current, 82 ± 20 pA; mean current at the end of AMPA application, 9 ± 3 pA; mean current integral, 40 ± 18 pC) and fitted to Hill equations. The resulting EC50 and nH values were 28, 46, and 48 μM, and 2.8, 3.6, and 4.5, respectively.

Fig. 2.

Fig. 2.

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Effects of CTZ on the AMPA dose/current–response relationship. Typical whole-cell currents induced in two different cells by AMPA alone (A) or by AMPA plus CTZ (different cell from A) (B). (C) AMPA dose/peak current response to AMPA alone (•) or in the presence of 50 μM CTZ (○). Imax = −524 ± 184 pA (control); I = −5.8 ± 1.1 nA with CTZ (n = 5). In the control, EC50 and nH values were 139 μM and 2.65, respectively. With CTZ at the indicated concentration, EC50 and nH values were 18 μM and 1.27, respectively.

AMPA Current Potentiation as a Function of the Duration of CTZ Pretreatment.

CTZ potentiated the AMPA current with increasing efficacy as the duration of CTZ preincubation was increased. Therefore, two different protocols were used to determine the dependence of the AMPA current potentiation on the CTZ pretreatment. One protocol consisted of applying AMPA (150 μM) for 10 s before adding the CTZ (Fig. 3A). Under these conditions, CTZ potentiated the AMPA current, and the corresponding rising phase was best fitted to a single exponential function with a mean time constant of 16 ± 4 s (n = 5), indicating that CTZ needed many seconds to maximally potentiate the AMPA currents. When AMPA and CTZ were applied simultaneously (Fig. 3 A Right), the current rise did not change significantly, indicating that the desensitization induced by AMPA developed faster than the potentiating action of CTZ. Another protocol consisted in monitoring the GluR1 potentiation by making brief applications of AMPA (150 μM) during a continuous application of CTZ (Fig. 3B). Although the current took many seconds to reach the maximal amplitude (4.8 ± 0.6-s time constant; n = 5; Fig. 3C), the inhibition of desensitization was much faster (Fig. 3D). Both, current potentiation and inhibition of desensitization exhibited a slower recovery than their onset (Fig. 3 C and D). Taken together, these findings indicate that the interaction between GluR1 and CTZ develops very quickly, leading to a prompt block of desensitization followed by a delayed current potentiation.

Fig. 3.

Fig. 3.

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Kinetics of CTZ-induced potentiation of AMPA current. (A Left) Typical AMPA induced whole-cell current in the presence of CTZ applied 10 s after AMPA application as indicated. (A Right) Typical whole-cell current induced by simultaneous coapplication of CTZ and AMPA. Both traces recorded were from the same cell. (B) Sample currents elicited by repetitive pulses of AMPA (150 μM for 500 ms; every 5 s) in the continuous presence of CTZ starting 1 s before the first AMPA stimulation. Sample traces are for the first, fifth, and ninth AMPA applications. (C) Time course of current potentiation by CTZ and its recovery by using the same protocol as in B. Each point is the mean ± SEM (n = 5 from five different experiments). Data were normalized to the mean amplitude at the end of CTZ treatment. (D) Time course of desensitization during and after CTZ treatment (as indicated) (n = 5) by using the same protocol as in C. There was a 1-s CTZ pretreatment before the first AMPA pulse. Desensitization are expressed (in percentage) as the ratio between current decay and the current peak, i.e. (Ipeak − Iend)/Ipeak. Note that CTZ inhibited desensitization even at the first AMPA pulse.

CTZ Modulation of GluR1 Single-Channel Activity.

To investigate further the mechanisms underlying the modulation of AMPA currents by CTZ, an analysis was made of the single-channel characteristics of AMPA-activated GluR1 receptors by using the patch-clamp outside-out recording mode. When patch membranes were exposed to AMPA (1 μM), there was an immediate activation of channel openings, with a mean frequency ≈10 Hz (e.g., Fig. 4A; n = 4). Analyses of unitary events disclosed three classes of current levels in each patch, with no obvious transitions between the different conductance levels. The three classes of channel conductance were: a low, γL, at 10.9 ± 0.3 pS; a medium, γM, at 15.2 ± 0.3 pS; and a high conductance, γH, at 21.6 ± 0.6 pS. The AMPA-induced channel openings had a mean open time of 0.63 ± 0.07 ms, whereas the mean burst duration was 1.08 ± 0.09 ms (Fig. 5A and B). When the same excised patch membranes were pretreated for 30 s with 100 μM CTZ and then exposed to AMPA (1 μM) together with CTZ, there was again a rapid activation of channel openings with the same three classes of channel conductance (Fig. 4B); but the mean open time, burst duration, and frequency all increased by ≈ 4–5 fold (Fig. 5 A–C). Moreover, the CTZ modified the relative weight of the conductance classes, increasing γM and decreasing γL, whereas the γH weight was not significantly modified (Fig. 5 D and E). These findings indicate that the potentiation induced by CTZ is mainly due to a combined increase in frequency of activation and burst duration. The shift in the relative weight of conductance classes may be important instead at relatively low agonist concentration because, with high agonist occupancy, AMPA receptors gate at larger conductance levels (7).

Fig. 4.

Fig. 4.

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Effects of CTZ on single-channel currents. (A Upper) Typical single-channel currents elicited by AMPA in the absence of CTZ, representative of four recordings obtained from outside-out patches (holding potential, −70 mV). (A Lower) Time expanded for better temporal resolution. (B) The AMPA sample elicited single-channel currents from the same outside-out patch as in A, starting 30 s after the onset of CTZ treatment.

Fig. 5.

Fig. 5.

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Effects of CTZ on single-channel properties. (A–C) Histograms of mean open time, burst duration, and burst frequency of single-channel currents elicited by AMPA (1 μM) in control (0) and upon CTZ (100) treatment. (D) Histograms of amplitudes of single-channel currents elicited in a typical outside-out patch by AMPA alone (1 μM; holding potential, −70 mV) (Left) or by AMPA in the presence of CTZ (Right). Data were best fitted with the sum (bold line) of three Gaussian functions (thin lines). Note the different amplitude distribution. (E) Relative weight of γL (solid column), γM (open column), and γH (hatched column) in control and upon CTZ treatment (data averaged from four outside-out patches). ∗, statistically significant differences between data sets (P < 0.05).

In summary, we show here, in a single heterologous expression system, multiple effects of CTZ on homomeric GluR1 AMPA receptors. Some of these effects have been described for different AMPA receptor subtypes in native and heterologous systems (2, 4, 14–16; for review, see ref. 5). Thus, CTZ modulates whole-cell AMPA currents, producing: (i) a block of desensitization, (ii) a delayed potentiation of current amplitude, (iii) and an increase of agonist affinity. Furthermore, CTZ induces a marked lengthening of channel openings, and causes a shift in the relative weights of conductance classes, with increased medium and decreased low conductance classes. In view of the known dependence of channel conductance on the number of agonist molecules bound to the receptor (7), the latter observation is more relevant when considering the effects of CTZ on extra-synaptic AMPA receptors in which both agonist concentration and receptor occupancy are very likely low.

To gain some insight on the mechanisms by which CTZ promptly blocks AMPA receptor desensitization and slowly potentiates the AMPA current, we developed a kinetic model in which the effects of CTZ are reproduced by changing the kinetic constants of the transitions between receptor states. This model (see Fig. 6 and Supporting Text, which are published as supporting information on the PNAS web site) accounted well for the following effects of CTZ: (i) a fast block of desensitization, (ii) a slow current potentiation, (iii) a lengthening of the duration of single-channel openings (related to the increase of deactivation time), (iv) a shift in the relative weight of the conductance classes, and (v) an increased apparent affinity for the agonist; giving a clue on the kinetic difference between the block of desensitization and the current potentiation (see Fig. 6). Simulated AMPA currents, closely mimicking the recorded currents, support the hypothesis that CTZ action constrains the receptor, so that if a receptor interacts with CTZ when it resides in a nondesensitized state, it does not desensitize; whereas if a receptor resides in a desensitized state, its transition to a nondesensitized state is very slow. Specifically, we speculate that CTZ binds to AMPA receptors and “freezes” them in their existing state, i.e., nondesensitized or desensitized. Upon agonist application, the nondesensitized receptors shift to their open states, with a very small probability of undergoing desensitization, whereas the desensitized receptors slowly become available for activation, yielding a delayed potentiation. Alternatively, the interaction between CTZ and GluR1 receptors develops slowly over time. Such a hypothesis is unlikely, however, because desensitization would be gradually inhibited; in most whole-cell recordings, as well as in our study, the desensitization of macroscopic AMPA currents is immediately inhibited by CTZ (2, 10, 16). Furthermore, our model is in agreement with the proposed mechanism of CTZ-receptor interaction, locating the CTZ molecule at the subunit dimer interface, where it alters the conformational equilibrium of the channel complex (10, 22).

Although the slow kinetics of the CTZ-induced potentiation of AMPA currents has long been known (2, 4, 14), there is no common protocol for applying CTZ in the many studies in which CTZ is used as a pharmacological tool to potentiate the AMPA current. The results reported here indicate that a preexposure of CTZ for tens of seconds is necessary to reach a maximal AMPA current potentiation, whereas the inhibition of the fast current decay is a rapidly developing process that occurs within 1 s after starting the application of CTZ. Therefore, our data point to a standard protocol to be used, and we suggest the use of a 60-s pretreatment with CTZ to invariably attain a plateau efficacy of CTZ for blocking desensitization and potentiating GluR1 receptor function. Our data may also help to improve the experimental design of studies investigating the therapeutic potential of AMPA receptor potentiators (23).

In conclusion, the results reported here show that the effects exerted by CTZ on GluR1 receptors are not limited to the fast block of desensitization and also involve changes in single-channel kinetics and conductance distribution. These latter actions, in turn, are responsible for the slow-developing current potentiation and probably differ among receptors made up of different subunit combinations. Therefore, further analyses of CTZ actions on AMPA receptors will help to identify receptor subtypes and also help in understanding the structure–function relationship of these receptor channels.

Materials and Methods

Cell Culture.

The human retroviral packaging cell line HEK 293 stably expressing the rat flip variant of wild-type glutamate receptor 1 (GluR1-HEK cells; a gift from P. Bregestovski, The Mediterranean Institute of Neurobiology, Marseille, France) was grown in DMEM with Glutamax-I/10% FBS/1% penicillin/streptomycin/5% CO2 (37°C) supplemented with geneticin (0.5 mg/ml). Cells were plated onto Petri dishes at a density of 104 cells/cm2.

Electrophysiology.

Whole-cell currents were recorded from GluR1-HEK cells 1 to 3 days after plating. Recordings were performed at room temperature (≈25°C) by using borosilicate glass patch pipettes (3 to 6-MΩ tip resistance) connected to an Axopatch 200A amplifier (Axon Instruments, Union City, CA). Data were stored and analyzed on a PC computer by using pclamp8 software (Axon Instruments). Whole-cell capacitance and patch series resistance (5–15 MΩ) were estimated from slow transient compensations, with a series resistance compensation of 70–90%. Cells were voltage-clamped at a holding potential of −70 mV (unless otherwise indicated), continuously superfused by using a gravity-driven system consisting of independent tubes for standard and agonist-containing solutions, positioned 50–100 μm from the patched cell, and connected to a fast exchanger system (RSC-100, Biologique). The solutions were exchanged completely in ≈50 ms, a time adequate for the kinetic analysis of the main events addressed in this study. Data are reported as means ± SEM. Differences between data sets were considered statistically significant when P < 0.05 (ANOVA test). Dose–response curves were constructed by fitting current peak amplitude values obtained at different concentrations, after normalization. The nonlinear fitting routine of sigma plot software (Jandel, San Rafael, CA) was used to fit the data to the Hill equation: I = 1/(1 + (EC50/[A])nH), where I is the normalized current amplitude induced by the agonist at concentration [A], nH is the Hill coefficient, and EC50 is the concentration at which a half-maximum response was induced. Single-channel data were filtered at 2 KHz, sampled at 20 KHz, and analyzed on a PC computer by using pclamp8 software (Axon Instruments). Opening and closing transitions were detected by using a 50% threshold criterion, and the kinetic parameters were apparent because of the filter-cut undetected shuttings and openings. Kinetic analysis was performed on a subset of patches that provided a minimization of noise-induced errors. The critical time (τc), used to identify a burst, was determined as reported in refs. 24 and 25. To analyze the distribution of single-channel conductance levels (Fig. 5 D and E), channel openings shorter than 0.5 ms were not included.

Solutions and Chemicals.

Patch-clamped cells were superfused with an external solution containing 140 mM NaCl/2.8 mM KCl/2 mM CaCl2/2 mM MgCl2/10 mM Hepes-NaOH/10 mM glucose (pH 7.3). The patch pipettes were filled with a solution containing 140 mM CsCl/2 mM MgATP/10 mM Hepes-CsOH/5 mM BAPTA (pH 7.3). Chemicals were purchased from Sigma, except CTZ (Tocris Cookson, Bristol, U.K.). Culture medium and FBS were purchased from GIBCO. l-Glutamine and penicillin/streptomycin were purchased from Euroclone (Wetherby, U.K.). CTZ was stocked at 50 mM in DMSO. It has been shown that concentrations of DMSO higher than those used in this study do not affect the kinetic behavior of GluR1 AMPA receptors expressed in HEK 293 cells (4).

Supplementary Material

Supporting Information
pnas_0511063103_index.html (9.7KB, html)

Acknowledgments

We thank Dr. Quoc Thang Nguyen for the critical reading of the manuscript. This work was supported by Ministero Istruzione Universitá Ricerca–Fondo Investimenti Ricerca di Base.

Abbreviations

CTZ

cyclothiazide

AMPA

α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid.

Footnotes

Conflict of interest statement: No conflicts declared.

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Supplementary Materials

Supporting Information
pnas_0511063103_index.html (9.7KB, html)
pnas_0511063103_1.pdf (95KB, pdf)

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