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2 Subunit Specificity of Cyclothiazide Inhibition on Glycine Receptors

Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China (X.-B.Z., G.-C.S., L.-Y.L., F.Y., T.-L.X.); and School of Life Sciences, University of Science and Technology of China, Hefei, China (X.-B.Z., T.-L.X.)

Received October 12, 2007; accepted December 27, 2007

	Abstract

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In the mammalian cortex, 2 subunit-containing glycine receptors (GlyRs) mediate tonic inhibition, but the precise functional role of this type of GlyRs is difficult to establish because of the lack of subtype-selective antagonist. In this study, we found that cyclothiazide (CTZ), an epileptogenic agent, potently inhibited GlyR-mediated current (I_Gly) in cultured rat hippocampal neurons. The inhibition was glycine concentration-dependent, suggesting a competitive mechanism. Note that GlyRs containing the 2 but not 1 or 3 subunits, when being heterologously expressed in human embryonic kidney 293T cells, were inhibited by CTZ, indicating subunit specificity of CTZ action. In addition, the degree of CTZ inhibition on I_Gly in rat spinal neurons declined with time in culture, in parallel with a decline of 2 subunit expression, which is known to occur during spinal cord development. Furthermore, site-directed mutagenesis indicates that a single-amino acid threonine at position 59 near the N terminus of the 2 subunit confers the specificity of CTZ action. Thus, CTZ is a potent and selective inhibitor of 2-GlyRs, and threonine at position 59 plays a critical role in the susceptibility of GlyR to CTZ inhibition.

Cyclothiazide (CTZ) was originally developed as a diuretic for the treatment of hypertension (Antlitz and Valle, 1967). In the early 1990s, CTZ was reported to enhance the activity of the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptors by suppressing their desensitization (Patneau et al., 1993). Recent evidence indicates that CTZ also exerts an inhibitory effect on GABA_A receptors (Deng and Chen, 2003). Therefore, CTZ interacts oppositely with both AMPA and GABA_A receptors and dramatically elevates the overall neuronal activity in the brain by these two independent mechanisms. More recently, it was found that CTZ is a novel epileptogenic agent that induces robust epileptiform activity in the hippocampus (Qi et al., 2006). In this study, we demonstrated that in addition to AMPA and GABA_A receptors, the inhibitory GlyR is also a target of CTZ. We found that CTZ efficiently reduced I_Gly in cultured neurons from embryonic rat hippocampus and spinal cord. Most notably, CTZ exerted an 2 subunit-specific inhibition on GlyRs. These results add a new dimension to the mechanism underlying CTZ-induced epileptogenesis (Qi et al., 2006) and highlight a role of CTZ in the investigation of 2-GlyR function in the CNS.

	Materials and Methods

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Primary Neuronal Cultures. The care and use of animals in these experiments followed the guidelines and protocols approved by the Care and Use of Animals Committee of the Institute of Neuroscience. Hippocampal and spinal dorsal horn (SDH) neurons from 15- to 18-day-old embryonic Sprague-Dawley rats were isolated by a standard enzyme treatment protocol (Gao et al., 2005). In brief, rat hippocampi and SDH were dissociated in calcium-free saline with sucrose (20 mM) and plated (1-5 x 10⁵ cell/ml) on poly-D-lysine (Sigma-Aldrich, St. Louis, MO)-coated cover glasses. The neurons were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) with L-glutamine plus 10% fetal bovine serum (Invitrogen) and 10% F12 Nutrient mixtures (Invitrogen). Neurobasal medium (1.5 ml; Invitrogen) with 2% B27 serum-free supplements (Invitrogen) was replaced every 3 to 4 days. Treatment with 5-fluoro-5'-deoxyuridine (20 µg/ml; Sigma-Aldrich) on the 3rd day after plating was used to block cell division of non-neuronal cells, which helped to stabilize the cell population. The cultures were maintained at 37°C in a 5% CO₂ humidified atmosphere. Cells were used for electrophysiological recording 5 to 21 days after plating.

Mutagenesis. Mutations of receptor cDNA were performed using a commercially available QuikChange site-directed mutagenesis kit (Stratagene, Shanghai, China) with commercially produced mutagenic primers (Invitrogen). All mutants were verified by DNA sequencing (Invitrogen).

Expression of Recombinant GlyRs. The human 1 and β subunit cDNA were obtained from Dr. Yu-tian Wang (University of British Columbia, Vancouver, BC, Canada). The human 2 subunit cDNA was kindly provided by Dr. Heinrich Betz (Department of Neurochemistry, Max Planck Institute for Brain Research, Frankfurt, Germany). The rat 3 subunit cDNA was donated by Dr. Jochen Meier (Department of Developmental Physiology, Johannes-Mueller Center of Physiology, Charite University Medicine, Berlin, Germany). In brief, HEK293T cells were cultured at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 2 mM L-glutamine, 10% fetal bovine serum, and 100 units/ml penicillin/streptomycin (all from Invitrogen). Transient transfection of HEK293T cells was carried out using calcium phosphate precipitation protocol. Cotransfection with a green fluorescent protein expression vector, pEGFP-N1, was used to enable identification of transfected cells for patch clamping by monitoring green fluorescent protein fluorescence in some experiments. When cotransfecting the GlyR and β subunits, their respective cDNAs were combined in a ratio of 1:3 to ensure the formation of functional heteroligomers. After exposure to transfection solution for 8 h, cells were washed twice with the culture medium. Electrophysiological recording was performed 24 to 48 h after calcium phosphate treatment.

Electrophysiological Recordings. The cells were observed using a fluorescent microscope, and currents were measured by the conventional whole-cell patch-recording configuration under voltage-clamp conditions. The cells were perfused by the standard external solution contained: 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose, 10 mM HEPES, with the pH adjusted to 7.3 with Tris base. The osmolarity of all bath solutions was adjusted to 325 to 330 mOsM with sucrose (3300; Advanced Instruments, Norwood, MA). Patch pipettes were pulled from glass capillaries with an outer diameter of 1.5 mm on a two-stage puller (PP-830; Narishige, Tokyo, Japan). The resistance of pipettes was 3 to 5 M when filled with the patch pipette solution, which contained: 120 mM KCl, 30 mM NaCl, 1 mM MgCl₂, 0.5 mM CaCl₂, 5 mM EGTA, 2 mM Mg-ATP, and 10 mM HEPES, with the pH adjusted to 7.2 with Tris base. When the I-V relationships for I_Gly were examined, 300 nM tetrodotoxin and 100 µM CdCl₂ were added to the standard external solution, and K⁺ was replaced with Cs⁺ in the pipette solution. Membrane currents were measured using a patch-clamp amplifier (Axon 200B; Molecular Devices, Sunnyvale, CA), filtered with a cut-off frequency of 2 kHz, sampled at a rate of 100 kHz, and analyzed using a Digidata 1320A interface and a personal computer with Clampex and Clampfit software (version 9.0.1; Molecular Devices). Unless otherwise noted, the membrane potential was held at -60 mV throughout the experiment. All the experiments were carried out at room temperature (22-25°C). The average access resistances for neurons and HEK293T cells used in the experiments are all approximately 10 M. The average whole-cell capacitance for the hippocampal neurons, the spinal cord neurons, and HEK293T cells is approximately 25, 20, and 30 pF, respectively.

Drugs. All drugs were purchased from Sigma-Aldrich. Picrotoxin (PTX) and CTZ were initially dissolved as concentrated stocks solutions in dimethyl sulfoxide and subsequently diluted to the desired concentration in standard external solution. The final dimethyl sulfoxide concentration was lower than 0.1%. Other drugs were first dissolved in ion-free water and then diluted to the final concentrations in the standard external solution just before use or dissolved directly in the standard external solution. Drugs were applied using a rapid application technique termed the "Y-tube" method throughout the experiments. This system allows a complete exchange of external solution surrounding a neuron within 20 ms (Murase et al., 1990). Throughout the experiment, the bath was superfused continuously with the standard external solution.

Data Analysis. The software Clampfit 9.0.1 (Molecular Devices) was used for data analysis. The continuous theoretical curves for concentration-response relationships of glycine in the presence or absence of CTZ were drawn according to a modified Michaelis-Menten equation by the method of least squares (the Newton-Raphson method) after normalizing the amplitude of the response: I = I_max C^nH/(C^nH + EC₅₀^nH), where I is the normalized value of the current, I_max is the maximal response, C is the drug concentration, EC₅₀ is the agonist concentration producing a half-maximal response, and n_H is the apparent Hill coefficient. The curve for the effect of CTZ on I_Gly was fitted by the following equation: I = I_max (IC₅₀)^nH / (C^nH + IC₅₀^nH), where IC₅₀ represents the antagonist concentration producing a half-maximal inhibitory effect, and the others are the same as described above. All data were calculated as the mean ± S.E.M. When quantification analysis was made, statistical comparison was carried out using Student's t test for the comparison of two groups. Statistically significant differences were assumed as P < 0.05 for all data. P and n represent the value of significance and the number of neurons, respectively.

	Results

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CTZ Effects on Cultured Rat Hippocampal Neurons. Glycine and taurine are the major endogenous ligands of GlyRs in mammalian CNS (Mori et al., 2002); therefore, we first examined the effects of CTZ on both glycine- and taurine-evoked currents (I_Gly and I_Tau, respectively) in cultured rat hippocampal neurons. At a holding potential of -60 mV under the whole-cell voltage clamp mode, bath application of glycine (100 µM) and taurine (500 µM in the presence of 10 µM bicuculline for blocking GABA_A receptor) evoked inward currents in all tested neurons under our experimental conditions (161 and 153 mM Cl^- in the external and internal solutions, respectively) (Fig. 1A). These currents were strychnine-sensitive (data not shown). Addition of CTZ (100 µM) resulted in a rapid reduction of the current amplitude (Fig. 1, A and B), although CTZ itself produced no detectable responses. The effect was reversible because both I_Gly and I_Tau were recovered after 2 min of wash-off. The concentration dependence of CTZ inhibition was investigated with 30 and 100 µM glycine. Figure 1C shows that CTZ caused a concentration-dependent reduction of I_Gly, and the IC₅₀ value of CTZ for the current induced by 30 µM glycine is 22.2 ± 4.2 µM and significantly increased to 95.7 ± 2.0 µM for that induced by 100 µM glycine. This significant alteration of the CTZ concentration-response relationship by glycine concentration suggests the competitive interaction between glycine and CTZ.

View larger version (24K): [in this window] [in a new window]   Fig. 1. CTZ reduces both glycine- and taurine-evoked currents in cultured rat hippocampal neurons. A, representative recordings showing 100 µM Gly-evoked current (IGly) and 500 µM Tau-evoked current (ITau) in the absence or presence of 100 µM CTZ. Note the rapid recovery of responses after washing out of CTZ. B, histograms show the percentage inhibition of IGly and ITau by 100 µM CTZ (n = 8-13). C, concentration-response relationship for CTZ-induced reduction of current activated by 30 () or 100 () µM glycine. All responses were normalized to the peak IGly induced by 100 µM glycine alone (n = 8-14). Continuous line was the result of fitting to the Hill equation (see Materials and Methods). D, typical recordings obtained from a single neuron illustrating the effect of 100 µM CTZ on IGly activated by various concentrations of glycine. E, concentration-response curves for Gly alone () or glycine plus 100 µM CTZ () (n = 6-8). All responses were normalized to the peak current evoked by 100 µM glycine alone (*). Note the rightward shift of the curve in the presence of CTZ.

To elucidate whether CTZ interferes with glycine binding to the GlyR, we recorded I_Gly in the absence or presence of CTZ. We found that the inhibition of CTZ depended on glycine concentrations (Fig. 1D). As shown in Fig. 1E, the EC₅₀ of I_Gly without CTZ was 34.1 ± 6.5 µM, and in the presence of 100 µM CTZ, this was increased to 105.4 ± 19.9 µM. In addition, CTZ did not significantly alter the Hill coefficient of I_Gly (without CTZ, 1.1 ± 0.2; with CTZ, 1.2 ± 0.3). The apparent increase of the EC₅₀ by CTZ further supports a competitive nature of its inhibition.

To investigate the voltage sensitivity of CTZ inhibition, we measured the I-V relationships of I_Gly. CTZ equally inhibited I_Gly at either positive or negative voltages, as revealed by the similar reduction of I_Gly at -30 mV (47.9 ± 5.0%) and +30 mV (45.2 ± 4.0%), respectively. In addition, CTZ did not significantly alter the reversal potential of I_Gly. The potentials (without CTZ, 1.8 ± 1.4; with CTZ, 1.5 ± 1.5) were all close to the theoretical Cl^- equilibrium potential (E_Cl) of 1.3 mV calculated from the given extra- and intracellular Cl^- concentrations with the Nernst equation. Therefore, these results indicate that CTZ inhibition of I_Gly is voltage independence.

Use Independence of CTZ Inhibition. The concentration dependence of CTZ effect suggests a competitive manner of CTZ inhibition. We tested this hypothesis further by comparing the inhibition of I_Gly with four different CTZ application protocols. As shown in Fig. 2, A and B, pretreatment of CTZ followed by coapplication of CTZ and glycine produced the most marked inhibition, although prolonged pretreatment of CTZ did not further the effect. However, the inhibition caused by the sequential application of CTZ and glycine (Fig. 2, Ab and B) was indistinguishable from that obtained with coapplication (Fig. 2, Ac and B). That CTZ interacts with GlyR before glycine application suggests that CTZ inhibition of I_Gly is independent of channel opening, favoring the idea that CTZ competes with the agonist at the GlyR.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Effects of CTZ on IGly with different drug application protocols. A, sample recordings illustrating CTZ inhibitory effect on IGly with four different CTZ application protocols. A, a, current evoked by glycine alone; A, b, sequential application of CTZ and glycine. CTZ was preapplied for 10 s. A, c, coapplication of CTZ and glycine. A, d, 10-s pretreatment of CTZ followed by coapplication of CTZ and glycine. A, e, 60-s pretreatment of CTZ followed by coapplication of CTZ and glycine. B, histograms showing relative IGly with four different CTZ application as indicated in A. ***, P < 0.001, compared with the currents induced by 100 µM glycine alone (dashed line). NS, no significant difference. n = 6. C, three repeated CTZ pulses during continuous application of glycine reduced IGly to a similar extent, suggesting a use-independent manner of inhibition. D, summary data showing normalized inhibition of IGly by three separate CTZ pulses. NS, no significant difference, compared with the inhibition induced by the first pulse of CTZ. n = 5.

Additive Effect of Picrotoxin and CTZ. To make sure that CTZ really inhibits GlyRs independent of channel opening, we further examined the mutual effect of CTZ and PTX, a representative open-channel blocker of GlyR and GABA_A receptor chloride channels (Yoon et al., 1993; Wang et al., 2006), on I_Gly. As shown in Fig. 3, the current induced by 100 µM glycine was inhibited to 79 ± 3 and 50 ± 3% of control by PTX (100 µM) and CTZ (300 µM, the saturating concentration), respectively. When PTX (100 µM) and CTZ (300 µM) were applied together, the I_Gly was further depressed to 29 ± 4% of control. In addition, the I_Gly was inhibited to 22 ± 2% of control by the maximally efficacious concentration of PTX (1 mM) and was further inhibited to 9 ± 2% of control by adding 100 µM CTZ. If PTX and CTZ share a common mechanism of inhibition, the maximal inhibition induced by the maximally efficacious concentration of PTX should occlude further inhibition of the glycine response by CTZ. Thus, the additive effect of CTZ and PTX inhibition suggests that the two compounds may block different populations of GlyRs or exert their effect through distinct binding sites on the GlyR.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Additive inhibition of CTZ and PTX on IGly. A, representative traces showing IGly in the absence or presence of various concentrations of CTZ and PTX. The maximally efficacious concentration for PTX used in the experiments is 1 mM. B, relative IGly in the presence of various concentrations of PTX, CTZ, or PTX plus CTZ. ***, P < 0.001, compared with the currents induced by 100 µM glycine alone (dashed line). ##, P < 0.01, compared with the currents induced by PTX plus CTZ. n = 7.

CTZ Inhibition Was 2 Subunit-Specific. Previous studies have shown that hippocampal neurons express multiple GlyR isoforms (Malosio et al., 1991). To determine the subunit specificity of CTZ inhibition, we examined the CTZ effect in HEK293T cells expressing the 1, 2, and 3 subunits of the GlyR, either alone or together with the β subunit. The EC₅₀ and n_H values for each of the six receptor subtypes are summarized in Table 1. As shown in Fig. 4A, the currents activated by 100 µM glycine were recorded in HEK293T cells expressing different homomeric (1, 2, or 3) and heteromeric (1β, 2β, or 3β) GlyRs. Furthermore, CTZ inhibited I_Gly mediated by homomeric 2- and heteromeric 2β-GlyRs in a concentration-dependent manner (Fig. 4B). To further compare the difference of CTZ modulation on 1, 2, and 3 GlyR, we test the effects of CTZ on I_Gly induced by an equally efficacious concentration of glycine (EC₇₀, 50 µM for 1-GlyR, 100 µM for 2-GlyR, and 150 µM for 1-GlyR) in these homomeric GlyR. In contrast to 2-containing GlyRs, I_Gly induced by EC₇₀ glycine concentration in 1- and 3-GlyRs were essentially unaffected by CTZ (Fig. 4C). Coexpressing and β subunits in HEK293T cells with an excess of β subunit leads to the near exclusive formation of functional β heteromers (Pribilla et al., 1992; Bormann et al., 1993). The characteristic reduction in PTX sensitivity was used to test the successful incorporation of β subunit to functional receptors. The IC₅₀ value of PTX for homomeric 1, 2, and 3 GlyR was 8.2 ± 1.4, 3.6 ± 0.9, and 10.6 ± 1.9 µM, respectively. After incorporation of the β subunit, the IC₅₀ value of PTX was increased to 232 ± 25, 38.3 ± 3.8, and 218 ± 32 µM, respectively. Thus, the significant reduction of PTX sensitivity confirmed that heteromeric GlyRs were functionally expressed in HEK293T cells. We found that the CTZ sensitivity of 2β heteromers (IC₅₀ = 76 ± 37 µM, n = 5-7) was indistinguishable from that of 2 homomers (IC₅₀ = 51 ± 21 µM, n = 5-10).

View larger version (33K): [in this window] [in a new window]   Fig. 4. Subunit specificity of CTZ inhibition of GlyRs. A, representative traces showing the effect of CTZ (100 µM) on IGly mediated by homomeric 1-, 2-, 3-, and heteromeric 1β-, 2β-, and 3β-GlyRs expressed in HEK293T cells. B, concentration-response relationship of CTZ inhibition on various GlyR subtypes. Each point represents mean ± S.E.M. normalized to the corresponding peak IGly (dashed line) before CTZ application (n = 5-10). C, histograms showing the relative IGly activated by EC70 glycine concentration (50 µM for 1-GlyR, 100 µM for 2-GlyR, and 150 µM for 3-GlyR) in the presence of CTZ in HEK293T cells expressed with homomeric 1-, 2-, or 3-GlyRs. ***, P < 0.001, compared with IGly before CTZ application (dashed line). n = 6.

View larger version (29K): [in this window] [in a new window]   Fig. 5. The effects of CTZ on IGly mediated by mutant homomeric 1 (A52T), 2 (T59A), and 3 (A52T) GlyRs. A, part of amino acid sequence of 1, 2, and 3 subunits in the extracellular N terminus shows the difference of Thr59 in the 2 subunit with A52 in the 1 and 3 subunits. B, representative traces showing the effect of CTZ (100 µM) on IGly mediated by mutant homomeric 1 (A52T), 2 (T59A), and 3 (A52T) GlyRs expressed in HEK293T cells. C, concentration-response curves for IGly in HEK293T cells expressing WT 2 and mutant 2 (T59A) GlyR subunits (n = 6-12). D, percent inhibition of CTZ on IGly induced by EC70 glycine concentration [100 µM for WT 2-GlyR and 80 µM for mutant 2 (T59A)-GlyR] in HEK293T cells expressing WT 2 and mutant 2 (T59A) GlyR. (n = 6-12). E, histograms showing the relative IGly in the presence of CTZ (100 µM) in HEK293T cells transfected with 1 (A52T), 2 (T59A), or 3 (A52T) GlyR subunits (n = 6-12). ***, P < 0.001, compared with IGly before CTZ application (dashed line).

Threonine at Position 59 Conferred the Specificity of CTZ Inhibition on 2-GlyR.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Effects of CTZ on IGly in spinal cord and hippocampal neurons of various times in culture. A, typical traces showing IGly in the absence or presence of CTZ in DIV 5 to 7, 12 to 14, and 19 to 21 neurons from SDH and hippocampus (HIP), respectively. B, summary data showing the degree of CTZ inhibition of IGly with time in culture. *, P < 0.05; ***, P < 0.001 compared with IGly before CTZ application (dashed line). ###, P < 0.001; NS, no significant difference. n = 7-10.

CTZ Inhibition Was Developmentally Regulated in Spinal Cord. A developmental switch from 2 homomers to 1β heteromers has been reported for spinal GlyRs (Becker et al., 1988). This differential expression pattern promotes us to examine whether CTZ inhibition of I_Gly declined with time in cultured spinal neurons. As shown in Fig. 6, CTZ markedly inhibited I_Gly in neurons 5 to 7 days in vitro (DIV), whereas in neurons of DIV 12 to 14 and 19 to 21, the inhibition became largely attenuated. Consistent with the predominant expression of the 2 subunit in the hippocampus throughout the developmental stage, no significant difference about CTZ inhibition was observed in cultured hippocampal neurons during development. This specific decline of CTZ inhibition on I_Gly in cultured spinal neurons further supports the notion that the CTZ action is 2 subunit-specific.

	Discussion

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The main finding of this study was that CTZ, an epilepigenic agent that interacts with both AMPA (Patneau et al., 1993) and GABA_A receptors (Deng and Chen, 2003), specifically inhibited 2-GlyRs in both CNS neurons and HEK293T cells. The inhibition was glycine concentration-dependent, suggesting a competitive mechanism. Furthermore, a single mutation of T59A in the 2 subunit markedly reduced the inhibitory effect of CTZ. Because the 2-GlyR represents the major component of GlyRs in adult cortical neurons (Malosio et al., 1991), CTZ is useful to explore the role of GlyRs in the brain.

Several lines of evidence suggest it is very unlikely that CTZ acts as an open channel blocker. Unlike open channel blockers, brief exposure to CTZ before glycine application can result in inhibition of I_Gly. Furthermore, the CTZ effect on GlyR was independent of membrane voltage and was use-independent. In addition, the action of CTZ differs from that of the known open channel blocker PTX in that CTZ blocks homomeric and heteromeric 2-containing receptors, whereas PTX only blocks homomeric GlyRs (Yoon et al., 1993; Wang et al., 2006).

The mutation of A52S in 1 subunit was shown to decrease glycine sensitivity on GlyRs (Saul et al., 1994). In contrast, our results indicate the mutation of T59A, the corresponding site in the 2 subunit, only slightly altered the glycine sensitivity. However, T59A mutation largely eliminated the inhibitory effect of CTZ on 2-GlyR. Therefore, threonine in position 59 of the 2 subunit is critical for CTZ inhibition. Further support for Thr59 as the key residue conferring CTZ specificity comes from the results that CTZ became effective in inhibiting GlyRs containing 1 or 3 subunits when the site corresponding to Thr59 of the 2 subunit was changed to threonine (A52T). These data support the idea that a single amino acid at the extracelluar N terminus can dramatically affect the regulatory properties of GlyRs (Mascia et al., 1996).

Through site-directed mutagenesis combined with homology modeling based on the crystal structure of the acetylcholine binding protein, the ligand binding residues of 1 GlyR were investigated by previous studies (Lynch, 2004; Grudzinska et al., 2005). Ala52 of the 1 subunit is not one of the binding sites that were identified through homology modeling. Thus, Thr59 in the 2 subunit, the corresponding residue of Ala52 in the 1 subunit, may also not be the binding sites for glycine binding sites. However, Ala52 is one of the closest residues to the putative glycine binding sites as revealed by homology modeling (Speranskiy et al., 2007). In addition, the mutation of A52S in 1 subunit was shown to decrease glycine sensitivity of GlyRs (Saul et al., 1994). These studies suggest that Ala52 in 1 GlyR is relevant to the glycine binding site. Accordingly, Thr59 in the 2 subunit, the corresponding residue of Ala52 in the 1 subunit, may play a similar role in glycine binding through which CTZ exerts the competitive inhibition of GlyRs.

Previous studies have indicated that the expression of GlyR subunits in rat spinal cord neurons in vivo was developmentally regulated (Malosio et al., 1991; Watanabe and Akagi, 1995). The 1 and β subunits are expressed at very low levels in embryonic rat spinal cord, with increasing expression during the first 2 postnatal weeks and the sustained high level thereafter. The 3 subunit is expressed after the 3rd postnatal week and only at a low level. In contrast, 2 expression is high and widespread in the embryonic nervous system and decreases gradually after birth. Similar developmental regulation of GlyR subunits expression has also been observed in primary cultures of spinal cord neurons; mRNA for 1 increased by 1.5- to 2-fold, whereas that for 2 decreased substantially over the first 10 days in culture (Bechade et al., 1996). Consistent with the selective inhibition of CTZ on 2-GlyRs expressed in HEK293T cells, we found that CTZ significantly inhibited I_Gly in DIV 5 to 7 spinal dorsal horn neurons, but the inhibition was greatly reduced after DIV 12. These results show that CTZ inhibition of GlyRs in spinal cord is developmentally regulated, reflecting the 2-to-1 switch of GlyR subunit (Bechade et al., 1996; Aguayo et al., 2004) and support the notion of 2 subunit specificity of the CTZ action.

Unlike the high level of GlyR 1 subunit expression in the mature spinal cord, the 2- rather than 1-containing GlyR is the predominant form in the hippocampus (Malosio et al., 1991; Thio et al., 2003). The physiological consequences of this distinct distribution pattern are difficult to establish because of the lack of specific antagonists. Thus, CTZ may be a useful tool to study the molecular organization and function of GlyRs in the hippocampus. Furthermore, recent studies have shown that the activation of 2-GlyRs is excitatory in developing cortex (Sturman, 1993; Flint et al., 1998) and plays an important role in rod photoreceptor development (Young and Cepko, 2004) and interneuron differentiation (McDearmid et al., 2006). However, genetic deletion of the 2 subunit in mice has not yielded clear developmental deficiency (Young-Pearse et al., 2006). This may be accounted for the compensatory expression of other subunits in these 2 knockout mice (Kling et al., 1997). Therefore, the acute pharmacological inhibition of 2-GlyRs by CTZ may be helpful for studying the role of 2-GlyRs in neuronal development.

The endogenous amino acids, glycine, taurine and β-alanine, mediate tonic activation of 2-GlyRs, which maintains inhibitory tone in the hippocampus (Mori et al., 2002). Moreover, the synaptic transmission in the rat hippocampus was also shown to be depressed by activation of GlyRs (Chattipakorn and McMahon, 2003; Song et al., 2006). These findings indicate that GlyRs may play an important role in mediating inhibitory neurotransmission in the hippocampus. Recent studies have provided evidence that, in addition to enhancing the function of glutamate receptors, CTZ affects the output of neural networks by reducing GABAergic inhibition (Deng and Chen, 2003; Qi et al., 2006). Our present study showed that CTZ inhibited both glycine- and taurine-evoked currents in cultured rat hippocampal neurons. These results thus suggest that CTZ may inhibit the tonic activation of 2-GlyRs by taurine and glycine and further accelerate the overall neuronal activity within the hippocampus, an aspect not considered in previous studies on the CTZ action in enhancing hippocampal excitability. Given its enhancing effect on AMPA receptors and inhibitory effect on glycine or GABA_A receptors, CTZ might be a superior epileptogenic agent. Note that CTZ-induced epileptiform activity in cultured hippocampal neurons is not a transient change but rather a permanent alteration of neural networks (Qi et al., 2006). Because 2-GlyRs may promote interneuron development (McDearmid et al., 2006), it is possible that the long-lasting effect of CTZ on overall network output in developing neural network may result from the impairment of interneuron differentiation as a result of its inhibition of 2-GlyRs. In addition, previous studies also indicated that the GlyR 2 subunit mRNA as well as functional GlyRs are expressed in other forebrain areas, such as hypothalamus, dorsal striatum, and amygdala (McCool and Farroni, 2001; Sergeeva and Haas, 2001). Therefore, CTZ may also modulate neuronal excitability through inhibiting GlyR in these forebrain regions.

Both inhibitory glycine and GABA_A receptors belong to the cysteine loop superfamily of ligand-gated ion channels. The homology between these two receptors may account for the similar inhibitory effect of CTZ. Unraveling the molecular mechanisms underlying the CTZ action on both glycine and GABA_A receptors may yield critical insight into the specificity and mechanism of drug-receptor interactions.

	Acknowledgements

 
We thank Peng Jiang for assistance in the electrophysiological recording and Yu Ding and Bo Duan for primary neuron preparations. We also thank James Celentano and Muming Poo for helpful discussion.

	Footnotes

 
This study was supported by the National Natural Science Foundation of China (grant 30621062), by the National Basic Research Program of China (grant 2006CB500803), and by the Knowledge Innovation Project from the Chinese Academy of Sciences (grant KSCX2-YW-R-35).

ABBREVIATIONS: GlyR, glycine receptor; CNS, central nervous system; CTZ, cyclothiazide; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; SDH, spinal dorsal horn; HEK, human embryonic kidney; PTX, picrotoxin; WT, wild type; DIV, day(s) in vitro.

Address correspondence to: Tian-Le Xu, Institute of Neuroscience, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China. E-mail: tlxu{at}ion.ac.cn

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