Effects of Cyclothiazide on Glur1 AMPA Receptors

Effects of cyclothiazide on GluR1͞AMPA receptors

Sergio Fucile*†, Ricardo Miledi†‡, and Fabrizio Eusebi*§

*Istituto Pasteur–Fondazione Cenci Bolognetti, Dipartimento di Fisiologia Umana e Farmacologia, Centro di Eccellenza BEMM, Universita’ di Roma ‘‘La Sapienza,’’ Piazzale Aldo Moro 5, I00185 Rome, Italy; ‡Laboratory of Cellular and Molecular Neurobiology, Department of Neurobiology and Behavior, University of California, Irvine, CA 92697-4550; and §Neuromed Instituto di Ricovero e Cura a Carattere Scientiﬁco, Via Atinese 18, I86077 Isernia, Italy

Contributed by Ricardo Miledi, December 22, 2005 Cyclothiazide (CTZ), a positive allosteric modulator of ionotropic Results and Discussion ␣ -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)- Concentration–Response Relationships on CTZ Administration. HEK type glutamate receptors, is used frequently to block the desen- 293 cells expressing the rat flip GluR1 subunit (GluR1-HEK sitization of both native and heterologously expressed AMPA cells) responded to AMPA with an inward current that decayed receptors. Specifically, CTZ is known to produce a fast inhibition of biphasically due to a rapid desensitization leading to a fairly AMPA receptor desensitization and a much slower potentiation of stable phase (Fig. 1A). The same cells treated with CTZ gen- the AMPA current. By using patch-clamp techniques, the effects erated AMPA current responses in which the profiles depended of CTZ were studied in HEK 293 cells stably transfected with the rat on the concentrations of AMPA and CTZ, as well as on the flip GluR1 subunit. Upon CTZ treatment, we found an increased duration of the exposure to CTZ. The AMPA currents were apparent affinity for the agonist, a slow whole-cell current poten- greatly potentiated by CTZ in a dose-dependent manner. For tiation, a fast inhibition of desensitization, and a lengthening of instance, by using 150 ␮M AMPA, a 4-s pretreatment of the cells single-channel openings. Furthermore, we show that CTZ alters the with 100 ␮M CTZ increased the peak AMPA currents by 90-fold channel gating events modifying the relative contribution of (EC50 ϭ 28 ␮M; nH ϭ 2.8; n ϭ 5), the currents at the end of ␥ different single-channel classes of conductance ( ), increasing and AMPA application by 636-fold (EC50 ϭ 46 ␮M; nH ϭ 3.6), and ␥ ␥ decreasing, respectively, the contributions of M (medium) and L the current integrals by 730-fold (EC50 ϭ 41 ␮M; nH ϭ 4.5) (Fig. (low) without altering that of the ␥H (high) conductance channels. 1 A and B). The potentiation induced by CTZ was accompanied We also present a kinetic model that predicts well all of the by an increase in the apparent affinity of the receptors for

experimental findings of CTZ action. Finally, we suggest a protocol AMPA. For example, in the absence of CTZ, the AMPA NEUROSCIENCE for standard cell treatment with CTZ to attain maximal efficacy of dose͞current–response curve exhibited an EC50 value of 139 ␮M CTZ on GluR1 receptors. (Fig. 2 A and C). In contrast, with 50 ␮M CTZ in the bathing medium (i.e., EC50 value for CTZ with AMPA at 10 ␮M; data glutamate receptor ͉ Hek 293 ͉ receptor potentiation not shown), the AMPA dose͞current–response relation shifted to the left with an Ϸ8-fold decrease in the EC50 value (Fig. 2 B yclothiazide (CTZ) is one of the most potent benzothiadia- and C). To maximize the effects of CTZ, the subsequent ␮ Czides, a class of positive allosteric modulators of non- experiments were done with CTZ at 100 M. NMDA-type glutamate receptors (1, 2), that inhibit receptor desensitization with a marked selectivity for the flip splice AMPA Current Potentiation as a Function of the Duration of CTZ variants (3) of ␣-amino-3-hydroxy-5-methylisoxazole-4- Pretreatment. CTZ potentiated the AMPA current with increas- propionic acid (AMPA) receptors (4). For that reason, treating ing efficacy as the duration of CTZ preincubation was increased. cells with CTZ has become a very useful and widespread tool for Therefore, two different protocols were used to determine the investigating the molecular composition of AMPA receptors and dependence of the AMPA current potentiation on the CTZ pretreatment. One protocol consisted of applying AMPA (150 the mechanisms of desensitization (for review, see ref. 5). ␮ The AMPA receptors (for review, see ref. 6) are homo- or M) for 10 s before adding the CTZ (Fig. 3A). Under these heterotetrameric assemblies (7) organized in dimer pairs (8, 9), conditions, CTZ potentiated the AMPA current, and the cor- responding rising phase was best fitted to a single exponential and their rapid desensitization is: (i) controlled by the dimer Ϯ ϭ interface, being reduced by CTZ through dimer interface sta- function with a mean time constant of 16 4s(n 5), indicating bilization (10); (ii) dependent on the receptor subunit compo- that CTZ needed many seconds to maximally potentiate the sition, including alternative splicing (4, 11, 12); and (iii) char- AMPA currents. When AMPA and CTZ were applied simulta- acterized by the receptor entry into an inactive state displaying neously (Fig. 3 A Right), the current rise did not change an increased binding affinity (13). significantly, indicating that the desensitization induced by Together with a fast inhibition of desensitization (4, 14–16), AMPA developed faster than the potentiating action of CTZ. Another protocol consisted in monitoring the GluR1 potentia- CTZ causes many other effects on AMPA-type receptors in- tion by making brief applications of AMPA (150 ␮M) during a cluding: (i) a slow increase in the peak amplitude of whole-cell continuous application of CTZ (Fig. 3B). Although the current currents (2, 4, 14); (ii) an enhancement of the current deacti- took many seconds to reach the maximal amplitude (4.8 Ϯ 0.6-s vation time (14); (iii) a lengthening of single-channel openings time constant; n ϭ 5; Fig. 3C), the inhibition of desensitization (2); (iv) a decrease in agonist binding (17, 18); and (v) an increase was much faster (Fig. 3D). Both, current potentiation and in the apparent affinity for agonists (2, 4, 14). inhibition of desensitization exhibited a slower recovery than Several studies have provided important insights into the their onset (Fig. 3 C and D). Taken together, these findings structural determinants and mechanisms of the interaction of indicate that the interaction between GluR1 and CTZ develops 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 Conflict of interest statement: No conflicts declared. reports some of the effects of CTZ on AMPA receptors, all in Abbreviations: CTZ, cyclothiazide; AMPA, ␣-amino-3-hydroxy-5-methylisoxazole-4-propi- the same cell system (HEK 293 cells stably transfected with the onic acid. rat GluR1 subunit), highlighting the discrepancy between the †To whom correspondence may be addressed. E-mail: [email protected] or fast inhibition of current desensitization and the slow current [email protected]. potentiation. © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0511063103 PNAS ͉ February 21, 2006 ͉ vol. 103 ͉ no. 8 ͉ 2943–2947 Downloaded by guest on September 27, 2021 Fig. 1. 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 Ϫ70 mV. (B) The CTZ dose͞current–response relationship to AMPA (150 ␮M). Data (n ϭ 6) represent current amplitudes at peak (F) and at the end of AMPA application (E) and current integrals () normalized to the values obtained without CTZ (mean peak current, 82 Ϯ 20 pA; mean current at the end of AMPA application, 9 Ϯ 3 pA; mean current integral, 40 Ϯ 18 pC) and ﬁtted to Hill equations. The resulting EC50 and nH values were 28, 46, and 48 ␮M, and 2.8, 3.6, and 4.5, respectively.

Fig. 2. Effects of CTZ on the AMPA dose͞current–response relationship. very quickly, leading to a prompt block of desensitization Typical whole-cell currents induced in two different cells by AMPA alone (A) followed by a delayed current potentiation. or by AMPA plus CTZ (different cell from A)(B). (C) AMPA dose͞peak current response to AMPA alone (F) or in the presence of 50 ␮M CTZ (E). Imax ϭϪ524 Ϯ ϭϪ Ϯ ϭ CTZ Modulation of GluR1 Single-Channel Activity. To investigate 184 pA (control); I 5.8 1.1 nA with CTZ (n 5). In the control, EC50 and ␮ further the mechanisms underlying the modulation of AMPA nH values were 139 M and 2.65, respectively. With CTZ at the indicated concentration, EC and n values were 18 ␮M and 1.27, respectively. currents by CTZ, an analysis was made of the single-channel 50 H characteristics of AMPA-activated GluR1 receptors by using the patch-clamp outside-out recording mode. When patch mem- system, multiple effects of CTZ on homomeric GluR1 AMPA branes were exposed to AMPA (1 ␮M), there was an immediate receptors. Some of these effects have been described for differ- activation of channel openings, with a mean frequency Ϸ10 Hz ent AMPA receptor subtypes in native and heterologous systems (e.g., Fig. 4A; n ϭ 4). Analyses of unitary events disclosed three (2, 4, 14–16; for review, see ref. 5). Thus, CTZ modulates classes of current levels in each patch, with no obvious transitions whole-cell AMPA currents, producing: (i) a block of desensiti- between the different conductance levels. The three classes of zation, (ii) a delayed potentiation of current amplitude, (iii) and ␥ Ϯ channel conductance were: a low, L, at 10.9 0.3 pS; a medium, an increase of agonist affinity. Furthermore, CTZ induces a ␥ Ϯ ␥ Ϯ M, at 15.2 0.3 pS; and a high conductance, H, at 21.6 0.6 marked lengthening of channel openings, and causes a shift in pS. The AMPA-induced channel openings had a mean open time the relative weights of conductance classes, with increased of 0.63 Ϯ 0.07 ms, whereas the mean burst duration was 1.08 Ϯ medium and decreased low conductance classes. In view of the 0.09 ms (Fig. 5 A and B). When the same excised patch known dependence of channel conductance on the number of membranes were pretreated for 30 s with 100 ␮M CTZ and then agonist molecules bound to the receptor (7), the latter obser- exposed to AMPA (1 ␮M) together with CTZ, there was again vation is more relevant when considering the effects of CTZ on a rapid activation of channel openings with the same three extra-synaptic AMPA receptors in which both agonist concen- classes of channel conductance (Fig. 4B); but the mean open tration and receptor occupancy are very likely low. time, burst duration, and frequency all increased by Ϸ 4–5 fold To gain some insight on the mechanisms by which CTZ (Fig. 5 A–C). Moreover, the CTZ modified the relative weight of promptly blocks AMPA receptor desensitization and slowly the conductance classes, increasing ␥M and decreasing ␥L, potentiates the AMPA current, we developed a kinetic model in whereas the ␥H weight was not significantly modified (Fig. 5 D which the effects of CTZ are reproduced by changing the kinetic and E). These findings indicate that the potentiation induced by constants of the transitions between receptor states. This model CTZ is mainly due to a combined increase in frequency of (see Fig. 6 and Supporting Text, which are published as support- activation and burst duration. The shift in the relative weight of ing information on the PNAS web site) accounted well for the conductance classes may be important instead at relatively low following effects of CTZ: (i) a fast block of desensitization, (ii) agonist concentration because, with high agonist occupancy, a slow current potentiation, (iii) a lengthening of the duration of AMPA receptors gate at larger conductance levels (7). single-channel openings (related to the increase of deactivation In summary, we show here, in a single heterologous expression time), (iv) a shift in the relative weight of the conductance

2944 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0511063103 Fucile et al. Downloaded by guest on September 27, 2021 Fig. 3. 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 NEUROSCIENCE 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 ␮M 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 Ϯ SEM (n ϭ 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 ϭ 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 Ϫ Iend)͞Ipeak. Note that CTZ inhibited desensitization even at the first AMPA pulse.

classes, and (v) an increased apparent affinity for the agonist; support the hypothesis that CTZ action constrains the receptor, giving a clue on the kinetic difference between the block of so that if a receptor interacts with CTZ when it resides in a desensitization and the current potentiation (see Fig. 6). Simu- nondesensitized state, it does not desensitize; whereas if a lated AMPA currents, closely mimicking the recorded currents, receptor resides in a desensitized state, its transition to a

Fig. 4. 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, Ϫ70 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.

Fucile et al. PNAS ͉ February 21, 2006 ͉ vol. 103 ͉ no. 8 ͉ 2945 Downloaded by guest on September 27, 2021 Fig. 5. 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 ␮M) 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 ␮M; holding potential, Ϫ70 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 ␥L (solid column), ␥M (open column), and ␥H (hatched column) in control and upon CTZ treatment (data averaged from four outside-out patches). *, statistically signiﬁcant differences between data sets (P Ͻ 0.05).

nondesensitized state is very slow. Specifically, we speculate that subtypes and also help in understanding the structure–function CTZ binds to AMPA receptors and ‘‘freezes’’ them in their relationship of these receptor channels. existing state, i.e., nondesensitized or desensitized. Upon agonist application, the nondesensitized receptors shift to their open Materials and Methods states, with a very small probability of undergoing desensitiza- Cell Culture. The human retroviral packaging cell line HEK 293 tion, whereas the desensitized receptors slowly become available stably expressing the rat flip variant of wild-type glutamate for activation, yielding a delayed potentiation. Alternatively, the receptor 1 (GluR1-HEK cells; a gift from P. Bregestovski, The interaction between CTZ and GluR1 receptors develops slowly Mediterranean Institute of Neurobiology, Marseille, France) over time. Such a hypothesis is unlikely, however, because was grown in DMEM with Glutamax-I͞10% FBS͞1% penicillin/ desensitization would be gradually inhibited; in most whole-cell streptomycin͞5% CO2 (37°C) supplemented with geneticin (0.5 recordings, as well as in our study, the desensitization of mac- mg͞ml). Cells were plated onto Petri dishes at a density of 104 roscopic AMPA currents is immediately inhibited by CTZ (2, 10, cells͞cm2. 16). Furthermore, our model is in agreement with the proposed mechanism of CTZ-receptor interaction, locating the CTZ mol- Electrophysiology. Whole-cell currents were recorded from ecule at the subunit dimer interface, where it alters the confor- GluR1-HEK cells 1 to 3 days after plating. Recordings were mational equilibrium of the channel complex (10, 22). performed at room temperature (Ϸ25°C) by using borosilicate Although the slow kinetics of the CTZ-induced potentiation glass patch pipettes (3 to 6-M⍀ tip resistance) connected to an of AMPA currents has long been known (2, 4, 14), there is no Axopatch 200A amplifier (Axon Instruments, Union City, CA). common protocol for applying CTZ in the many studies in which Data were stored and analyzed on a PC computer by using CTZ is used as a pharmacological tool to potentiate the AMPA PCLAMP8 software (Axon Instruments). Whole-cell capacitance current. The results reported here indicate that a preexposure of and patch series resistance (5–15 M⍀) were estimated from slow CTZ for tens of seconds is necessary to reach a maximal AMPA transient compensations, with a series resistance compensation current potentiation, whereas the inhibition of the fast current of 70–90%. Cells were voltage-clamped at a holding potential of decay is a rapidly developing process that occurs within1safter Ϫ70 mV (unless otherwise indicated), continuously superfused starting the application of CTZ. Therefore, our data point to a by using a gravity-driven system consisting of independent tubes standard protocol to be used, and we suggest the use of a 60-s for standard and agonist-containing solutions, positioned pretreatment with CTZ to invariably attain a plateau efficacy of 50–100 ␮m from the patched cell, and connected to a fast CTZ for blocking desensitization and potentiating GluR1 re- exchanger system (RSC-100, Biologique). The solutions were ceptor function. Our data may also help to improve the exper- exchanged completely in Ϸ50 ms, a time adequate for the kinetic imental design of studies investigating the therapeutic potential analysis of the main events addressed in this study. Data are of AMPA receptor potentiators (23). reported as means Ϯ SEM. Differences between data sets were In conclusion, the results reported here show that the effects considered statistically significant when P Ͻ 0.05 (ANOVA test). exerted by CTZ on GluR1 receptors are not limited to the fast Dose–response curves were constructed by fitting current peak block of desensitization and also involve changes in single- amplitude values obtained at different concentrations, after channel kinetics and conductance distribution. These latter normalization. The nonlinear fitting routine of SIGMA PLOT actions, in turn, are responsible for the slow-developing current software (Jandel, San Rafael, CA) was used to fit the data to the n potentiation and probably differ among receptors made up of Hill equation: I ϭ 1͞(1 ϩ (EC50͞[A]) H), where I is the normal- different subunit combinations. Therefore, further analyses of ized current amplitude induced by the agonist at concentration CTZ actions on AMPA receptors will help to identify receptor [A], nH is the Hill coefficient, and EC50 is the concentration at

2946 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0511063103 Fucile et al. Downloaded by guest on September 27, 2021 which a half-maximum response was induced. Single-channel KCl͞2 mM CaCl2͞2 mM MgCl2͞10 mM Hepes-NaOH͞10 mM data were filtered at 2 KHz, sampled at 20 KHz, and analyzed glucose (pH 7.3). The patch pipettes were filled with a solution on a PC computer by using PCLAMP8 software (Axon Instru- containing 140 mM CsCl͞2mMMgATP͞10 mM Hepes- ments). Opening and closing transitions were detected by using CsOH͞5 mM BAPTA (pH 7.3). Chemicals were purchased from a 50% threshold criterion, and the kinetic parameters were Sigma, except CTZ (Tocris Cookson, Bristol, U.K.). Culture apparent because of the filter-cut undetected shuttings and medium and FBS were purchased from GIBCO. L-Glutamine openings. Kinetic analysis was performed on a subset of patches and penicillin͞streptomycin were purchased from Euroclone that provided a minimization of noise-induced errors. The (Wetherby, U.K.). CTZ was stocked at 50 mM in DMSO. It has ␶ critical time ( c), used to identify a burst, was determined as been shown that concentrations of DMSO higher than those reported in refs. 24 and 25. To analyze the distribution of used in this study do not affect the kinetic behavior of GluR1 single-channel conductance levels (Fig. 5 D and E), channel AMPA receptors expressed in HEK 293 cells (4). openings shorter than 0.5 ms were not included. We thank Dr. Quoc Thang Nguyen for the critical reading of the Solutions and Chemicals. Patch-clamped cells were superfused manuscript. This work was supported by Ministero Istruzione Universita´ with an external solution containing 140 mM NaCl͞2.8 mM Ricerca–Fondo Investimenti Ricerca di Base.

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