Introduction

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Fluoxetine (Prozac) is widely used in the treatment of anxiety-related symptoms through its ability to inhibit the transporter for the neurotransmitter serotonin (5-hydroxytryptamine). Whereas many antidepressants are considered proconvulsant, and therefore are not recommended for use by patients with epilepsy (Rosenstein et al., 1993), fluoxetine has commonly been reported to exhibit anticonvulsant activity. In human studies, adjunctive treatment of epileptic patients with fluoxetine was found to reduce or eliminate the occurrence of seizures (Favale et al., 1995). In animal studies, fluoxetine increased the effectiveness of several antiepileptic drugs (Leander, 1992) and was shown to suppress seizure activity and reduce convulsion intensity in both normal and epilepsy-prone rats (Dailey et al., 1992; Pasini et al., 1992; Prendiville and Gale, 1993). The mechanism underlying this anticonvulsant activity is not known, but it has been suggested that it may occur through modulation of neurotransmitter systems beyond direct effects on serotonin signaling. One possibility is that it reduces neuronal activity by enhancing GABAergic transmission.

Most fast inhibitory neurotransmission in the mammalian central nervous system is mediated through the GABA_A receptors (GABARs), which contain an intrinsic, chloride-permeable ion channel. Many drugs used clinically as anticonvulsants act by increasing GABAR activity (Korpi et al., 2002). The GABARs have a very complex structure, with 7 different subunit families and 16 subunit subtypes [(1-6), (1-3), (1-3), , , , and ]. The pharmacological properties of GABARs are determined in large part by their subunit composition (Korpi et al., 2002). Previous studies have suggested both positive and negative regulation of GABAR activity by fluoxetine (Tunnicliff et al., 1999; Matsubara et al., 2000). However, there is little current evidence of a direct effect on the GABAR by fluoxetine or of the possible role of GABAR subunit composition in this modulation.

We examined the effect of fluoxetine and its metabolite norfluoxetine on the activity of recombinant GABARs in a mammalian expression system and found that they are positive modulators at most GABARs. We also examined the subunit subtype dependence and the effect of GABA concentration and membrane voltage on the interaction of fluoxetine with the GABAR.

	Materials and Methods

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Transfection of Mammalian Cells. Full-length wild-type cDNAs in the pCMVNeo (Dr. Robert Macdonald, Vanderbilt University) or pcDNA1.1/Amp (Invitrogen, Carlsbad CA) expression vectors were transfected into the mouse fibroblast cell line L929 (American Type Culture Collection, Manassas, VA) or the human embryonic kidney cell line HEK-293T (GenHunter, Nashville, TN). The results shown were combined from experiments using each of these cell types. Both these lines are widely used to study recombinant GABARs. Studies of other pharmacological and functional properties in our laboratory have shown no differences among GABARs expressed in these two lines. For selection of transfected cells, the plasmid pHook-1 (Invitrogen) containing cDNA encoding the surface antibody sFv was also transfected into the cells. Cells were maintained in Dulbecco's modified Eagle's medium plus 10% heat-inactivated horse serum (L929) or fetal bovine serum (HEK-293T), 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were passaged by a 5-min. incubation with 0.05% trypsin/0.02% EDTA solution in phosphate-buffered saline (10 mM Na₂HPO₄, 150 mM NaCl, pH 7.3).

Electrophysiological Recording Solutions and Techniques. For whole-cell recordings the external solution consisted of 142 mM NaCl, 8.1 mM KCl, 6 mM MgCl₂, 1 mM CaCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4, and osmolarity adjusted to 295 to 305 mOsm. Recording electrodes were filled with an internal solution of 153 mM KCl, 1 mM MgCl₂, 5 mM K-EGTA, 2 mM MgATP, and 10 mM HEPES, pH 7.4, and osmolarity adjusted to 295 to 305 mOsm. These solutions provided a chloride equilibrium potential near 0 mV. GABA and low concentrations of fluoxetine and norfluoxetine (100 µM) were diluted into external solution from stocks frozen in water. High concentrations of fluoxetine (>100 µM) were diluted from freshly made stocks in dimethyl sulfoxide. Patch pipettes were pulled from borosilicate glass with an internal filament (World Precision Instruments, Sarasota FL) on a two-stage puller (Narishige, Tokyo, Japan) to a resistance of 5 to 10 M. Drugs were applied to cells using a stepper solution exchanger with a complete exchange time of <50 ms (open tip, SF-77B; Warner Instruments, Hamden, CT). There was a continuous flow of external solution through the chamber. Currents were recorded with an Axon 200B (Axon Instruments, Inc., Union City, CA) patch-clamp amplifier and stored on a hard drive for off-line analysis. All experiments were performed at room temperature (near 25°C).

Construction of Mutated Subunit cDNAs. Point mutations were generated using the QuikChange mutagenesis procedure and products (Stratagene, La Jolla, CA). Oligonucleotide primers were synthesized by the University of South Carolina DNA core facility (Columbia, SC). Single amino acid changes were created using two nucleotide primers complementary to one another and encoding the desired amino acid mutation. Incorporation of the mutation was verified with DNA sequencing (University of South Carolina DNA core).

Analysis of Whole-Cell Currents. Whole-cell currents were analyzed using the programs Clampfit (pClamp8 suite; Axon Instruments) and Prism (GraphPad Software Inc., San Diego, CA). Concentration-response data were fit with a four-parameter logistic equation (current = [minimum current + (minimum current  maximum current)]/1+(10^(log EC₅₀  log [GABA]) · n), where n represents the Hill number. All fits were made to normalized data with current expressed as a percentage of the response to GABA alone. Paired t tests and Tukey-Kramer multiple comparisons statistical tests were performed using the Instat program (GraphPad) with a significance level of p < 0.05.

	Results

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Fluoxetine Potentiates GABAR Activity. To determine whether fluoxetine altered the activity of recombinant GABARs, 1 µM to 1 mM fluoxetine was coapplied for 5 s with 10 µM GABA to cells expressing the 132L isoform (Fig. 1). Fluoxetine increased the response to GABA in a concentration-dependent manner, with an average EC₅₀ of 134.3 ± 35.1 µM (n = 4). At lower fluoxetine concentrations, the current often continued to increase throughout the 5-s application. Because the peak current was likely not reached in these cases, the measured EC₅₀ for fluoxetine represents an estimation of the actual EC₅₀. The potentiation by 1 mM fluoxetine was not readily reversible. Fluoxetine did not act as an agonist at the GABAR, as application of 100 µM fluoxetine in the absence of GABA produced an average peak current of 4.0 ± 0.3 pA at 50 mV (n = 3), comparable with the variation produced by baseline noise. The average response to 10 µM GABA in these cells was 867.0 ± 363 pA.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Fluoxetine enhances the response to GABA. A, 10 µM GABA or 10 µM GABA + fluoxetine was applied for 5 s (bar) to cells expressing the 132L receptor isoform. Cells were voltage-clamped at 50 mV in the whole-cell configuration. All traces shown are from the same cell. B, the concentration-response relationship was constructed by measuring the peak current with coapplication of fluoxetine as a percentage of the response to 10 µM GABA alone. Symbols indicate mean ± S.E.M. from four cells and were fit with a four-parameter logistic equation (solid line). The EC50 from the fit of the averaged data is 128.1 µM with a Hill number of 1.4.

Fluoxetine Potentiation Depends upon the  Subunit Subtype. The activity of many GABAR modulators depends upon the subunit composition of the receptor (for review, see Korpi et al., 2002). The  subunit family is the most diverse of the GABAR families, with six different subtypes (₁-₆). To determine whether fluoxetine sensitivity is affected by the  subtype, we examined receptors containing each of the  subtypes coexpressed with the same  (₃) and  (_2L) subunits (Fig. 2). The GABA concentration was submaximal (EC_20-30) for each isoform. With the exception of the ₅-containing receptors, enhancement of the GABA-activated current by 100 µM fluoxetine was similar for all these combinations (p > 0.05 compared among isoforms). Only the 532L receptor isoform was not significantly potentiated by 100 µM fluoxetine (p < 0.001 compared with the 132L receptor).

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Only the 5 subunit confers a reduced sensitivity to fluoxetine. A, representative whole-cell current traces from cells transfected with different combinations of GABAR subunits in response to GABA and GABA plus 100 µM fluoxetine. Drugs were applied for 5 s (solid line). The current in response to GABA alone is the smaller in all cases. Cells were voltage-clamped at 50mV. B, average potentiation of the response to GABA by 100 µM fluoxetine.  subunits were coexpressed with 3 and 2L. The current in response to GABA + fluoxetine was normalized to the response to GABA alone. Bars show the mean ± S.E.M., and n is given by the number in parentheses. The dotted line represents the response to GABA alone. (p < 0.01) or  (p < 0.05) indicates a significant difference from the response to GABA alone for each isoform (paired t test).

Fluoxetine Potentiation Does Not Depend upon the  or  Subunit Subtype. Three different GABAR  (₁-₃) and  (₁-₃) subtypes have been described in mammalian species. We examined the sensitivity to fluoxetine of receptors containing each of the  subtypes in combination with ₁ and _2L (Fig. 2). The responses of the 112L, 122L, and 132L isoforms to 100 µM fluoxetine were not significantly different from one another (p > 0.6).

Fluoxetine Potentiation Is GABA Concentration-Dependent. To determine whether fluoxetine could increase the maximal response to GABA concentration, we coapplied 100 µM fluoxetine with 100 µM or 1 mM GABA (Fig. 3). The amount of potentiation decreased with increasing GABA concentrations and fluoxetine did not significantly enhance the response to a maximal concentration (1 mM GABA). This suggests that, like the benzodiazepines, fluoxetine acts by increasing the sensitivity of the receptor for GABA without affecting the peak current (see Möhler et al., 2002).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of GABA concentration on potentiation by fluoxetine. A, representative traces from cells expressing the 132L isoform in response to varying GABA concentrations alone or with 100 µM fluoxetine. Drugs were applied for 5 s as indicated by the solid line. Cells were voltage-clamped at 50mV. B, effect of 100 µM fluoxetine on the peak current response to different GABA concentrations. Bars indicate mean ± S.E.M., and n is given by the number in parentheses. The dotted line represents the response to GABA alone. (p < 0.01) indicates a significant difference from the response to GABA alone for each GABA concentration (paired t test).

Effect of Voltage on Potentiation by Fluoxetine. The lipid solubility of fluoxetine and the gradual increase in potentiation seen throughout the 5-s application period suggest that it may act at a site within the membrane. Therefore, we examined the voltage sensitivity of its action by comparing the response at holding potentials of +50 and 50 mV. The 132L receptor exhibits outward rectification in its response to GABA, due to a voltage-dependent shift in sensitivity to GABA (Fisher, 2002a). Whereas significant potentiation by 100 µM fluoxetine was still observed at +50 mV, the amount of potentiation was decreased compared with 50 mV (Fig. 4). This is consistent with the finding that the amount of potentiation depends upon the GABA concentration. Since GABA sensitivity is increased at positive potentials, 10 µM GABA represents a higher effective concentration (~EC₅₀) at +50 mV than at 50 mV (~EC_20-30). Therefore, the potentiation by fluoxetine does not appear to be altered by membrane voltage.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effect of membrane voltage on potentiation by fluoxetine. A, representative whole-cell traces from cells expressing the 132L isoform at membrane potentials of 50 mV or +50 mV. GABA (10 µM) or GABA + 100 µM fluoxetine was applied for 5 s as indicated by the solid line. All traces are from the same cell. B, effect of 100 µM fluoxetine on the peak current response at different holding potentials. Bars indicate mean ± S.E.M., and n is given by the number in parentheses. The dotted line represents the response to GABA alone. (p < 0.01) or  (p < 0.05) indicates a significant difference from the response to GABA alone at each potential (paired t test).

The Active Metabolite Norfluoxetine Is a More Potent Modulator Than Fluoxetine. Fluoxetine is metabolized to produce norfluoxetine, which is an even more potent inhibitor of the serotonin transporter (Sanchez and Hyttel, 1999) and voltage-gated K⁺ channels (Choi et al., 2001) than fluoxetine. Plasma levels of norfluoxetine and fluoxetine are similar to those in patients treated with fluoxetine (Orsulak et al., 1988; Pato et al., 1991). Therefore, we determined whether this active metabolite also affected the activity of GABARs.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Norfluoxetine is a more potent modulator of GABAR activity. A, 10 µM GABA or 10 µM GABA + norfluoxetine was applied for 5 s (bar) to cells expressing the 132L receptor isoform. Cells were voltage-clamped at 50 mV in the whole-cell configuration. All traces shown are from the same cell. B, the concentration-response relationship was constructed by measuring the peak current with coapplication of norfluoxetine as a percentage of the response to 10 µM GABA alone. Symbols indicate mean ± S.E.M. from three cells and were fit with a four-parameter logistic equation (solid line). The EC50 from the fit of the averaged data is 0.7 µM with a Hill number of 0.5.

Structural Determinants of Fluoxetine Sensitivity. The ₅ subunit appears to confer a unique response in its lower sensitivity to fluoxetine. To begin to elucidate the structures within the ₅ subunit responsible for this property, we examined amino acid residues previously shown to be important in determining its pharmacological properties. The ₅ subunit is also distinctive in its higher sensitivity to positive modulation by several benzodiazepine derivatives (Liu et al., 1996; Quirk et al., 1996; Strakhova et al., 2000), and an isoleucine residue (I215) in the extracellular domain was identified as important for this characteristic (Strakhova et al., 2000; Casula et al., 2001). This subunit also has a high sensitivity to inhibition by zinc, a property conferred by a unique histidine residue (H195) found only in the ₅ subunit (Fisher, 2002b). Since these residues have been shown to contribute to properties associated with the ₅ subunit, we examined whether they are also important in fluoxetine sensitivity.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Histidine195 and isoleucine215 of the 5 subunit do not influence fluoxetine sensitivity. Cells were transfected with mutated 5(H195D) or 5(I215V) subunits along with wild-type 3 and 2L, and the peak current response to GABA and GABA + 100 µM fluoxetine was measured. Bars indicate mean ± S.E.M., and n is given by the number in parentheses. The dotted line represents the response to GABA alone. (p < 0.01) indicates a significant difference from the response to GABA alone for each receptor isoform (paired t test).

	Discussion

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We have demonstrated that the antidepressant fluoxetine directly potentiates the activity of recombinant GABARs. The response to submaximal GABA concentrations was increased, but the maximal current response was unaffected. Fluoxetine showed an interesting and unique dependence on the GABAR subunit composition. Only receptors containing an ₅ subtype were insensitive to potentiation. Receptors containing any of the other six  subtypes were equally enhanced. The nature of the  or  subtype had no effect on sensitivity, and a  subunit was unnecessary for responsiveness, because and receptors were also potentiated. Fluoxetine did not directly activate the GABAR, and its effect was not altered by membrane voltage. Norfluoxetine, a metabolic product of fluoxetine, was even more potent in its enhancement of GABAR activity. Mutations of amino acid residues in the ₅ subunit that regulate its sensitivity to modulation by zinc and benzodiazepines did not alter its insensitivity to fluoxetine.

Many other allosteric sites have been described for the GABAR, and the affinity or efficacy of most of these depends upon the subunit composition of the receptor (for review, see Korpi et al., 2002). Although several benzodiazepine derivatives selectively enhance ₅-containing receptors, fluoxetine is the first positive modulator reported for which the ₅ subunit is selectively insensitive. In addition, the activity of the benzodiazepines also depends upon the nature of the  subunit, whereas the activity of fluoxetine does not. Other modulators that do not require a  subunit for activity, such as the barbiturates or loreclezole, do not share this -subtype dependence. This suggests that fluoxetine acts through a unique site on the GABAR.

The ₅ subunit apparently confers a unique lack of sensitivity to fluoxetine modulation. Therefore, this modulatory site may be exploited for the development of drugs designed to spare this population of receptors. The ₅ subunit is expressed primarily in the CA1 and CA3 regions of the hippocampus, and its production decreases with development (Laurie et al., 1992; Wisden et al., 1992). The physiological importance of these subunits in hippocampal function is unknown. However, changes in expression of the ₅ subunit have been suggested to play a role in learning and memory (Collinson et al., 2002), seizure development (Houser and Esclapez, 1996; Rice et al., 1996; Fritschy et al., 1999), benzodiazepine tolerance (Li et al., 2000), and reward conditioning (June et al., 2001).

In addition to the direct effect shown here, fluoxetine may also increase GABAR activity indirectly, by enhancing the synthesis of neuroactive steroids that positively modulate these receptors. Fluoxetine treatment increases the concentration of allopregnanalone (Uzunova et al., 1998), likely through a direct effect on the activity of the synthetic enzymes responsible for its production (Griffin and Mellon, 1999).

The modulation of the GABAR we observed may not be particularly surprising because fluoxetine and its metabolites have been found to have direct effects on a wide variety of channels. These include the structurally related nicotinic acetylcholine (García-Colunga et al., 1997) and 5-hydroxytryptamine₃ serotonin receptors (Fan, 1994; Breitinger et al., 2001) as well as several Cl channels (Maertens et al., 1999) and voltage-gated Ca²⁺ and K⁺ channels (Tytgat et al., 1997; Deák et al., 2000; Choi et al., 2001; Thomas et al., 2002). These effects generally occur within a concentration range of 1 to 10 µM. In contrast to the GABAR, however, fluoxetine was found to inhibit channel activity in all these cases. At both the nAChR and 5-hydroxytryptamine₃ receptors fluoxetine reduced the response in a noncompetitive manner. For the nAChR, the inhibition was voltage-dependent and consistent with an open channel block mechanism.

Plasma fluoxetine and norfluoxetine concentrations in patients are typically reported near 1 µM (Orsulak et al., 1988; Pato et al., 1991). Although this level of fluoxetine would have little effect on the GABAR isoforms studied here, accumulation of fluoxetine in the brains of chronically treated patients has been reported to increase the concentration nearly 20-fold compared with plasma levels (Karson et al., 1993). Therefore, in the central nervous system, levels can be reached that might enhance the activity of most GABARs. In addition, the more potent metabolite norfluoxetine would effectively modulate GABARs at these concentrations. It may be that much of the anti-convulsant effect of fluoxetine treatment is mediated through this metabolic product.