Introduction

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GABA-gated ion channels in the mammalian central nervous system can be classified into GABA_A and GABA_C receptors according to their pharmacological properties (Woodward et al., 1992; Feigenspan et al., 1993; Qian and Dowling, 1993; Macdonald and Olsen, 1994; Johnston, 1996; Lukasiewicz, 1996). GABA_A receptors can be modulated by benzodiazepines, barbiturates, and neurosteroids and can be blocked by bicuculline, whereas GABA_C receptors are insensitive to all of these compounds. These two types of GABA receptors are also distinct in their activation characteristics. For example, GABA_C receptors activate and deactivate more slowly than GABA_A receptors and show minimal desensitization (Polenzani et al., 1991; Feigenspan et al., 1993; Qian and Dowling, 1993, 1994, 1995; Lukasiewicz et al., 1994). These differences between the two types of receptors arise from their different subunit compositions. Recombinant receptors can reconstitute most of the pharmacological and physiological properties of GABA_A receptors (Pritchett et al., 1989; Sigel et al., 1990; Verdoorn et al., 1990), whereas recombinant homomeric 1 GABA receptors have similar properties to native GABA_C receptors (Cutting et al., 1991; Polenzani et al., 1991; Feigenspan et al., 1993; Amin and Weiss, 1994, 1996).

The exogenously expressed 1 homomeric GABA_C receptor can be activated by several GABA agonists including cis-4-aminocrotonic acid (CACA), trans-4-aminocrotonic acid (TACA), muscimol, imidazole-4-acetic acid (I4AA), and isoguvacine (Woodward et al., 1992; Kusama et al., 1993). These agonists differ in their sensitivities (EC₅₀, or concentration of agonist required for half-maximal activation) and efficacies (maximal current). It is unclear whether these differences arise from distinct binding affinities and/or distinct gating kinetics.

In this study, using the intact single oocyte binding technique (Chang and Weiss, 1999) in conjunction with the two-electrode voltage clamp, we have determined the apparent binding affinities (K_i) as well as agonist sensitivities (EC₅₀) and potencies (I_max) for TACA, GABA, muscimol, I4AA, CACA, and isoguvacine on 1 homomeric GABA_C receptors. The results showed that the apparent affinities of these agonists fell into two groups. The high affinity group was comprised of agonists with longer distances between the nitrogen atom of the amino or imidazole group and the carbon atom of the carboxyl or isoxazole group. This study represents the first direct comparison of agonist affinities, efficacies, and potencies on intact, nondesensitized ligand-activated ion channels.

	Materials and Methods

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cDNA and cRNA Preparation. The cDNA of the human 1 GABA receptor subunit was cloned into the pALTER-1 vector (Promega, Madison, WI) as previously described (Amin et al., 1994). A silent mutation to remove the EcoRI site within the gene was made by site-directed mutagenesis (Promega) using the following mutagenic oligonucleotide: 5' CTC ATT CAG GAG TTC CAC ACC ACC 3'. The gene was then subcloned into the pGEMHE high expression vector (Liman et al., 1992) between EcoRI sites in the T7 orientation. The cDNA was linearized with the NheI restriction enzyme, and capped cRNA was transcribed by T7 RNA polymerase using standard in vitro transcription procedures. The yield and integrity of the cRNA were examined on a 1% agarose gel.

Oocyte Preparation and cRNA Injection. Female Xenopus laevis (Xenopus I, Ann Arbor, MI) were anesthetized by 0.2% MS-222 (3-aminobenzoic acid ethyl ester, methanesulfonate salt). The ovarian lobes were surgically removed from the frog and placed in calcium-free oocyte Ringers-2 (OR2) incubation solution consisting of 92.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 1 mM Na₂HPO₄, and 5 mM HEPES; 50 U/ml penicillin, and 50 µg/ml streptomycin, pH 7.5. The lobes were cut into small pieces and digested with 0.3% collagenase A (Boehringer Mannheim Biochemicals, Indianapolis, IN) with constant stirring at room temperature for 1.5 to 2 h. The dispersed oocytes were thoroughly rinsed with the above solution plus 1 mM CaCl₂. The stage VI oocytes were selected and the follicular layer (if still present) was manually removed with fine forceps. The oocytes were incubated at 18°C.

Electrophysiology. One to 2 days after injection, the oocyte expressing 1 GABA receptors was voltage-clamped at 70 mV. The agonist-induced currents were in the range of 100 to 1000 nA, except the I4AA-induced current, which had a maximum of 30 to 100 nA. The dose-response relationships were determined by measuring the current induced by a range of agonist concentrations. The EC₅₀ and Hill coefficient of the dose-response relationship was determined by fitting the data to the Hill equation in the following form:

(1)

where I is the current amplitude, I_max is the maximum current amplitude for that particular agonist ([A]), EC₅₀ is the agonist concentration that induces a 50% maximal response, and n is the Hill coefficient.

Single Oocyte Binding. Two to 3 days after injection, the expression level of the 1 GABA receptors in oocytes were examined by two-electrode voltage clamp at 70 mV. Oocytes with a current response (to 10 µM GABA) of more than 3000 nA were selected for the binding assay. Most of the oocytes tested had a maximum current amplitude of 4000 to 6000 nA. Details of the single oocyte binding have been previously described (Chang and Weiss, 1999). Briefly, the oocyte expressing 1 GABA receptors was held by gentle suction at the end of a sequencing gel loading pipette tip with a Pasteur bulb on the end. The tip, bulb, and attached oocyte were held by a small clamp attached to a base. In this way, the oocyte could be moved manually between the incubation, rinse, and dissociation solutions as follows. The oocyte was first incubated in 1 µM [³H]GABA (in 100 µl of OR2) for 30 s at room temperature, then rinsed (to remove unbound [³H]GABA) for 6 s in a 150 ml 0°C OR2 bath with constant stirring, and finally placed in 250 µl of OR2 at room temperature for 60 s to let the bound [³H]GABA dissociate. The 250 µl of OR2 containing the dissociated [³H]GABA was thoroughly mixed with 4 ml of scintillation fluid, and the radioactivity of each sample (cpm) was determined in a liquid scintillation counter. For the measurement of the binding affinities of each GABA receptor agonist, the unlabeled GABA receptor agonist was added to the incubation solutions at increasing concentrations. Due to the lack of desensitization of the 1 receptor, each oocyte could be examined over the entire range of agonist concentrations without the need for a recovery period between the test concentrations (Chang and Weiss, 1999). The IC₅₀ (concentration of agonist that decreases the [³H]GABA binding by 50%) for each GABA receptor agonist was determined by least-squares fit of the following relationship to the data:

(2)

where specific binding (B) is a function of the inhibitor concentration ([I]). B_max is the maximum binding of 1 µM GABA in the absence of unlabeled agonist and n is the slope factor.

(3)

	Results

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EC₅₀ Values and Maximum Currents for Different Agonists on 1 Homomeric GABA Receptors. Figure 1A are examples of currents induced by TACA, GABA, muscimol, I4AA, CACA, and isoguvacine in oocytes expressing 1 homomeric GABA_C receptors. The normalized average dose-response relationships of these compounds are shown in Fig. 1B. The continuous lines are least-squares fits of eq. 1 to the data points. The resulting EC₅₀ values and Hill coefficients are provided in Table 1. Clearly, these compounds activated the receptor with distinct sensitivities of the order: TACA > GABA > muscimol > I4AA CACA > isoguvacine.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Dose-response relationships of TACA, GABA, muscimol, I4AA, CACA, and isoguvacine, and efficacies on 1 homomeric GABA receptor expressed in Xenopus oocytes. A, current traces induced by these compounds. B, average dose-response relationships of different agonists normalized to their own maxima. Continuous lines are least-squares fit of eq. 1 to the data points. The resulting EC50 values and Hill coefficients are in Table 1. C, average dose-response relationships of different agonists normalized to the maximum for GABA.

The Binding Affinities of the Agonists to 1 Homomeric GABA Receptors Fall into Two Groups. Using the single oocyte binding technique in a competition assay (Chang and Weiss, 1999), we determined the apparent binding affinities of the different agonists in intact oocytes. Figure 2 shows that nonlabeled GABA agonists could reduce the specific binding of 1 µM [³H]GABA to oocytes expressing 1 GABA receptors in a concentration-dependent manner. Note that the dose-inhibition relationships for TACA, GABA, and muscimol were very similar, whereas CACA and isoguvacine required much higher concentrations to displace the [³H]GABA. I4AA displayed the highest apparent affinity and will be considered separately in a later section. The continuous lines are least-squares fits of eq. 2 to the data yielding IC₅₀ values and Hill coefficients (Table 2) for [³H]GABA competition. The apparent binding affinities (K_i) calculated from the IC₅₀ values (Cheng and Prusoff, 1973) are presented in Table 2. Note that the binding affinities of these agonists can be divided into two groups: TACA, GABA, muscimol, and I4AA have relatively high apparent binding affinities, whereas CACA and isoguvacine have significantly lower apparent affinities.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Dose-dependent inhibition of the [3H]GABA (1 µM) binding to 1 GABA receptors by different GABA receptor agonists. Continuous lines are best fit of eq. 2 to the data points. Note that TACA, GABA, muscimol, and I4AA have similar IC50 values, whereas CACA and isoguvacine need significantly higher concentrations to inhibit [3H]GABA binding.

The Binding Affinities and EC₅₀ Values of the Agonists at 1 Homomeric GABA Receptors Are Correlated. The single oocyte binding technique allows us to measure binding under similar conditions as the electrophysiological recording, and in the same set of functional receptors. This has made it possible to directly correlate binding and channel activation. Figure 3 is a plot of the EC₅₀ values of these agonists as a function of their apparent dissociation constants (K_i). The continuous line is from a linear regression to all the data points excluding I4AA. The dashed line represents a theoretical exact correspondence between these two parameters. Note that all data points fall below the line. This difference may be due, at least in part, to the requirement that multiple, probably three (Amin and Weiss, 1996), agonist molecules must bind to open the GABA_C receptor.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Relationship between EC50 and Ki values. Continuous line is from a linear regression to all the data points excluding I4AA. The dashed line is the prediction assuming an equivalent EC50 and Ki. Note that all data points are below the line, indicating that the Ki values are lower than the EC50 values.

The IC₅₀ Values for I4AA Competition of GABA Binding and GABA-Mediated Activation Are Similar. I4AA exhibited the greatest discrepancy between the EC₅₀ and K_i values among the agonists we examined (Fig. 3). At the same time, I4AA displayed the lowest efficacy for activation; the maximal I4AA-induced current was 2% of the maximal GABA-induced current (Fig. 1C, Table 1). I4AA also exhibited the highest apparent affinity of any of the compounds tested (Fig. 2, Table 2). Therefore, electrophysiologically, if coapplied with GABA, I4AA should act like an antagonist. Figure 4 shows the dose-dependent inhibition of I4AA on the GABA-induced current (1 µM GABA) and [³H]GABA binding. The I4AA-mediated inhibition of the GABA-induced current (1 µM GABA) demonstrated an IC₅₀ value of 0.67 ± 0.10 µM (n = 5), which was similar to the IC₅₀ value measured in the competitive binding assay: 0.39 ± 0.02 µM (n = 5).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Comparison of the dose-dependent inhibition of the GABA-activated current and GABA binding by I4AA. A, current traces of I4AA inhibition of GABA-induced current. B, average dose-inhibition relationships of I4AA on GABA-induced current () and [3H]GABA binding (). For the current, the I4AA dose-dependent activation was subtracted from the data points.

	Discussion

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Activation Mechanism. The activation mechanism of ligand-gated ion channels includes both binding and gating steps. Investigation of the coupling between binding and gating requires both radioligand binding and electrophysiological recording techniques. We have developed a single intact oocyte binding technique that allows us to study binding and activation properties in functional receptors under identical experimental conditions. In addition, homomeric 1 GABA receptors do not desensitize, further simplifying the analysis of the coupling between binding and channel activation. Using the single oocyte binding technique to investigate the 1 GABA receptor, we have already provided fundamental insights into the activation mechanism of this receptor by GABA (Chang and Weiss, 1999). Here, we have extended the application of this technique to measuring the apparent binding affinity for other GABA agonists on the recombinant 1 GABA_C receptors in an attempt to directly correlate binding and activation of different agonists in functional, intact receptors.

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View larger version (14K): [in this window] [in a new window]   Fig. 5.   Relationship between the GABA-activated current (normalized to the maximum for GABA) and the experimentally observed EC50 values. The dashed lines represent the predicted relationship between the maximum current and the EC50 using the rate constants and three-bind-to-open kinetic model provided under Materials and Methods. The dashed lines were produced by holding all rate constants fixed and varying only the opening rate (). The shift along the abscissa for the two dashed lines results from the different forward binding rates. The predicted EC50 and maximum current for each set of parameters was determined by using a Q-matrix algorithm to generate a dose-response relationship, which was then fitted with eq. 1 (Colquhoun and Hawkes, 1981; Chang and Weiss, 1999). The dashed lines were generated by holding all rates fixed except for  (opening rate), which was varied to produce the relationship between maximum activatable current and EC50. The maximum for GABA is plotted as one, which is not the efficacy because the maximum open probability for GABA is 0.92 (Chang and Weiss, 1999).

Structure/Activity Relationship for the Agonists. A conformational analysis to explain the structure/activity relationships of these GABA agonists for 1 receptors has been published (Kusama et al., 1993; Chebib and Johnston, 2000). Using the nomenclature of Kusama et al. (1993), the high affinity agonists GABA, TACA, muscimol, and I4AA are in extended planar conformations when bound to 1 receptors (Fig. 6A). The lower affinity agonist CACA is in a folded planar conformation and isoguvacine is in a nearly planar conformation (Fig. 6B). For the un-ionized forms of the agonists shown in Fig. 6, the distances from the nitrogen atom in the amino or imidazole group (I4AA) to the carbon atom in the carboxyl or isoxazole group (muscimol) vary between 4.29 and 5.02 Å. For the high affinity agonists, the CN distance varies from 4.63 to 5.02 Å. For the low affinity agonists, the CN distance varies from 4.29 to 4.51 Å. Thus, the high affinity agonists have a larger CN distance than the low affinity agonists.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Three-dimensional structures of GABA agonists. The conformations of the structures shown were generated using the CS Chem 3D Pro molecular modeling software from CambridgeSoft Corp., Cambridge, MA. For clarity, hydrogen atoms are not shown. A, extended conformations of GABA, TACA, muscimol, and I4AA. The distance between the amino group nitrogen and the carboxyl group carbon in GABA (CN distance) is 5.02 Å. The distances between the corresponding atoms in TACA, muscimol, and I4AA are 4.90, 4.63, and 4.83 Å, respectively. B, folded conformation of CACA and isoguvacine conformation. The CN distances are 4.51 and 4.29 Å, respectively. C, superimposition of muscimol on TACA. The intermolecular distances between the superimposed nitrogen and carbon atoms of TACA (starting at N and ending with the carboxyl C) and the corresponding atoms in muscimol are 0.14, 0.11, 0.22, 0.25, and 0.13 Å, respectively. D, a superimposition of I4AA on TACA. The intermolecular distances, as defined in panel C, for the TACA/I4AA superimposition are 0.03, 0.09, 0.21, 0.18, and 0.03 Å, respectively. Part of the imazole ring of I4AA occupies a region of space that is unoccupied by TACA and muscimol. E, a superimposition of CACA and isoguvacine. The intermolecular distances, as defined in panel C, for the TACA/I4AA superimposition are 0.11, 1.69, 0.49, 1.35, and 0.11 Å, respectively.