Benzodiazepines act on GABA_A receptors via two distinct and separable mechanisms

Benzodiazepines (BZs) act on -aminobutyric acid type A (GABA_A) receptors such as ₁₂₂ through key residues within the N-terminal region of subunits, to render their sedative and anxiolytic actions. However, the molecular mechanisms underlying the BZs' other clinical actions are not known. Here we show that, with low concentrations of GABA, diazepam produces a biphasic potentiation for the ₁₂₂-receptor channel, with distinct components in the nanomolar and micromolar concentration ranges. Mutations at equivalent residues within the second transmembrane domains (TM₂) of , and subunits, proven important for the action of other anesthetics, abolish the micromolar, but not the nanomolar component. Converse mutation of the corresponding TM₂ residue and a TM₃ residue within ₁ subunits confers diazepam sensitivity on homo-oligomeric ₁-receptor channels that are otherwise insensitive to BZs. Thus, specific and distinct residues contribute to a previously unresolved component (micromolar) of diazepam action, indicating that diazepam can modulate the GABA_A-receptor channel through two separable mechanisms.

Sedative-hypnotic drugs such as diazepam (a benzodiazepine) can impose graded effects on the function of the central nervous system (CNS), resulting in a spectrum of clinical actions. This range of therapeutic actions progresses from sedation at low doses, to induction of anesthesia at significantly higher doses. BZs act at nanomolar concentrations to modulate chloride ion flux through GABA_A-receptor channels, thereby affecting synaptic transmission^{1, 2, 3}. The crucial amino acids responsible for this potent action of BZs in part have been determined, and are located within the N-terminal domains of and subunits^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16}. The importance of two subunit isoforms (₁ or ₂) in mediating sedative or anxiolytic action of diazepam, respectively, has been demonstrated using transgenic animals^{17, 18, 19, 20}.

In this study, we have demonstrated that diazepam, in the presence of low GABA concentrations, produces two distinct components of potentiation within the nanomolar (nM) and micromolar (M) concentration ranges for the ₁₂₂-receptor channel. In contrast to the nM component, the M component of diazepam action is independent of the ₂ subunit and is insensitive to the selective BZ antagonist flumazenil. Mutations at equivalent residues within the TM₂ of , and subunits, proven important for the action of other anesthetics, nullify the M component without significantly affecting the nM component of response to diazepam. Furthermore, the M action of diazepam can also be conferred upon the BZ-insensitive ₁-receptor channel by converse mutation of the corresponding TM₂ residue and a TM₃ residue. The physiological significance of the M action of diazepam is discussed in the light of the dynamics of benzodiazepine concentrations, the persistence of low concentrations of GABA and the tonic level of GABA_A receptor activity within the CNS.

Biphasic potentiation of ₁₂₂ by diazepam

Complementary RNA (cRNA) from rat wild-type ₁, ₂ and ₂ (_2s)²² subunits were co-injected into Xenopus laevis oocytes. Three days after injection, GABA-elicited currents were recorded in the absence and presence of a broad range of diazepam concentrations (2 to 150,000 nM) using the two-electrode voltage-clamp technique. Figure 1a and b depicts current traces and the concentration−response relationship for diazepam at EC₃, EC₈ and EC₁₆ (effective concentration giving rise to 3%, 8% and 16% of the maximal GABA current, respectively) GABA for the ₁₂₂-receptor channel. These data reveal two different patterns of response to diazepam, which are dependent on the GABA concentration. At all three GABA concentrations tested, diazepam produced a two- to three-fold potentiation of the GABA-evoked currents, with EC₅₀ values between 32 and 54 nM, and a Hill coefficient approaching unity (nM component, Table 1). On the other hand, at EC₃ and EC₈ GABA, M concentrations of diazepam (20 M and above) evoked a second component of potentiation, further increasing GABA-elicited currents at EC₃ from three-fold (the nM component) to approximately eight-fold. At EC₃ GABA, the M component of diazepam had an apparent EC₅₀ of 63 8.5 M and a notably greater Hill coefficient (3.04 0.37) than that of the nM component (Table 1). The extent of the M diazepam-induced potentiation was markedly dependent upon the GABA concentration; potentiation was diminished significantly at EC₈ GABA, and eventually abolished at EC₁₆ GABA.

Figure 1. Diazepam modulation of wild type 122- or 12-receptor channels. (a, b) The current traces and concentration−response relationship for diazepam at EC3 (3.2 M), EC8 (4.6 M) and EC16 (7.75 M) GABA for the 122-receptor channel. The presence of the diazepam M component of potentiation is highly dependent on GABA concentration. (c, d) The diazepam concentration− response relationship (10 to 150,000 nM) and current traces at EC3 GABA (0.2 M) for the 12-receptor channel. Diazepam at M concentrations (but not at nM concentrations) elicited a significant potentiation for the 12-receptor channel. Full Figure and legend (27K)

Table 1. Parameters obtained from fitting the Hill equation to GABA, diazepam and flurazepam data. Full Table

Monophasic potentiation of ₁₂ by diazepam

Co-expression of cRNA for ₁ and ₂ subunits also yielded GABA-gated receptor channels. The ₁₂-receptor channel can be distinguished from the ₁₂₂-receptor channel due to ₁₂'s unique pharmacological and kinetic characteristics. For example, the ₁₂-receptor channel is more sensitive to GABA in comparison to the ₁₂₂-receptor channel (Table 1). Figure 1c and d shows the diazepam concentration−response relationship (10 to 150,000 nM) at EC₃ GABA for the ₁₂-receptor channel. Diazepam elicited only a single component of potentiation within the M concentration range for the ₁₂-receptor channel, increasing the control EC₃ GABA currents by more than twelve-fold (Fig. 1c and d). Furthermore, the EC₅₀ and the Hill coefficient values for the M component of diazepam action were similar for both ₁₂ and ₁₂₂-receptor channels (at EC₃ GABA, Table 1). The lack of ₁₂-receptor channel sensitivity to diazepam within the nM concentration range has been documented previously, as the presence of the subunit is essential in conferring diazepam's nM action¹¹.

Zn²⁺ does not block the M action of diazepam

Zinc inhibits the GABA-evoked currents from ₁₂- but not from ₁₂₂-receptor channels^{23, 24} (Fig. 2). Zinc exhibited properties of an open channel blocker for ₁₂, inhibiting the GABA-evoked currents back to the baseline. In comparison, the GABA currents for ₁₂₂-receptor channels were reduced to 64 4% of the control in the presence of 10 M Zn²⁺, suggesting that a fraction of the total GABA current may have arisen from ₁₂-type-receptor channels. To confirm that the M potentiation of ₁₂₂ at EC₃ GABA was not due to the presence of ₁₂-receptor channels (see above), the ₁₂₂ diazepam concentration−response relationship at EC₃ GABA was determined in the presence of 10 M Zn²⁺ (Fig. 2b). In these experiments, the characteristics of ₁₂₂'s biphasic potentiation by diazepam were unaltered in the presence of Zn²⁺ (Table 1). For the nM and the M components, the EC₅₀/Hill coefficient values obtained in the presence of Zn²⁺ were 0.048 0.003/1.23 0.05 and 60.5 4.7/2.97 0.25, respectively. Moreover, the absolute magnitudes of potentiation for the two components were 3.44 0.26 for the nM and 4.34 0.26 for the M component of diazepam action. These values are similar to the values determined for the ₁₂₂-receptor channel in the absence of Zn²⁺ (Table 1). These data indicate that the significant potentiation observed at M concentration of diazepam for ₁₂₂ (at EC₃ GABA) does not arise from the concomitant presence of ₁₂-receptor channels.

Figure 2. Diazepam biphasic potentiation of the 122-receptor channel is unaltered in the presence of zinc. (a, b) Differential effect of Zn2+ (10 M) on the 12- versus 122-receptor channel. (a) The GABA-evoked currents (3.2 M) for the 12-receptor channel are completely blocked by 10 M Zn2+. (b) The current traces for the diazepam concentration− response relationship for the 122-receptor channel in the presence of Zn2+ (10 M). Although Zn2+ partially blocks the GABA-evoked currents for the 122-receptor channel (see current traces in b), it does not significantly affect the biphasic potentiation of the 122-receptor channel by diazepam at EC3 GABA. Full Figure and legend (27K)

The M action of diazepam is insensitive to flumazenil

Flumazenil is a selective antagonist for the high-affinity (nM) component of BZ action upon the GABA_A-receptor channel²⁵. For the ₁₂₂-receptor channel at EC₃ GABA, flumazenil at concentrations of 20 M (or 10 M, data not shown) abolished the nM, but not the M, component of diazepam action (Fig. 3). The overall potentiation at the M component was reduced from approximately 8 to 3.31 0.59. However, the EC₅₀ and the Hill coefficient values derived for the diazepam M component in the presence and absence of flumazenil were similar for the ₁₂₂-receptor channel at EC₃ GABA (Table 1). Concentrations of flumazenil up to 100 M were co-applied with 100 M diazepam (at EC₃ GABA) to test the effect of flumazenil at equivalent concentrations on M potentiation by diazepam. In the presence of such high concentrations of flumazenil, 100 M diazepam also produced a significant potentiation (data not shown). These experiments suggest that the nM and M actions of diazepam are mediated through distinct mechanisms.

Figure 3. Flumazenil abolishes the nM but not the M component of diazepam action upon the 122-receptor channel. Top, current traces for diazepam modulation of GABA currents in the presence of 20 M flumazenil. The nM but not the M component of diazepam action is eliminated by the co-application of flumazenil (bottom). Full Figure and legend (25K)

TM₂ mutations abolished the M action of diazepam

The diazepam concentration−response relationships for _{S267IN265IS280I} at EC₃, EC₈ and EC₁₆ GABA were next determined. At all three GABA concentrations tested, diazepam produced only a single component of potentiation within the nM concentration range (Fig. 4b). The EC₅₀ and Hill coefficient values derived for _{S267IN265IS280I} (32 to 55 nM and 1.1 to 0.8 nM, respectively; Table 1) were similar to the values determined for the nM component of wild-type ₁₂₂ (Table 1). The _{S267IN265IS280I}-receptor channel experiments further demonstrate that the nM and the M actions of diazepam are distinct and separable.

Figure 4. Diazepam modulation of the S267IN265IS280I-receptor channel. (a) Amino-acid sequence comparison for the TM2 domains of 1, 2, 2 and 1 subunits. The mutated residues are shown in boxes. (b) The current traces and the concentration−response relationship for diazepam, for the S267IN265IS280I-receptor channel at EC3 (0.09 M), EC8 (0.17 M) and EC16 (0.35 M) GABA. At all three GABA concentrations examined, diazepam produced a single component of potentiation occurring within the nM concentration range. The line is derived from the fit of the Hill equation to the data up to 5 M diazepam. Full Figure and legend (48K)

In comparison to the ₁₂₂,-receptor channel, the _{S267IN265IS280I}-receptor channel exhibited an approximately 40-fold higher sensitivity to GABA (Table 1). This difference in sensitivity indicates the importance of these TM₂ residues in the GABA-dependent modulation of the ₁₂₂-receptor channel.

Imparting diazepam sensitivity to ₁-receptor channels

₁-receptor channels are insensitive to BZs and other intravenous anesthetics²⁶ (excepting neuroactive steroids²⁷). This property of the homo-oligomeric ₁-receptor channel has been used to study the molecular basis of anesthetic action by conferring anesthetic sensitivity upon ₁. For example, the specific mutation of residue 307 within TM₂ or residue 328 within TM₃ of the ₁ subunit (Fig. 5a) can impart pentobarbital sensitivity upon the ₁-receptor channel^{28, 29}. Within the ₁ subunit, residues 307 and 328 were substituted, either singly or in combination, to their amino acid counterparts present within the ₁ (Ser, TM₂) and ₂ (Met, TM₃) subunits. Diazepam did not modulate the GABA-elicited currents at EC₄ in wild-type ₁-, _I307S- and _W328M- receptor channels (Fig. 5b). In contrast, doubly mutated ₁ became highly sensitive to diazepam (Fig. 5c and d). The EC₄ GABA currents for _I307S/W328M were markedly potentiated in the presence of diazepam, resulting in an increase of GABA currents up to 9-fold (20 M diazepam). Figure 5e and f show the potentiation of GABA currents by diazepam (10 M) at EC₁, EC₄, EC₅₀ and EC₉₉ GABA for ₁, _I307S, _W328M and _I307S/W328M mutants. For _I307S/W328M, diazepam potentiation was most prominent at GABA concentrations below the EC₅₀ value. The fractional potentiation by diazepam (10 M) was greatest at low GABA concentrations (EC₁ 5.40 0.50 and EC₄ 3.20 0.50), whereas at higher GABA concentrations (EC₉₉), little potentiation was observed. In comparison, ₁, _W328M and _I307S did not respond to diazepam at any of the GABA concentrations tested.

Figure 5. Imparting diazepam sensitivity to the 1-receptor channel by double mutation. (a) The position of the crucial amino acids within the putative second and third transmembrane domains. (b) GABA-evoked currents at EC4 from 1 (0.27 M GABA), W328M (0.35 M GABA) or I307S (0.11 M GABA) are insensitive to diazepam concentrations up to 30 M. (c, d) GABA currents (EC4 = 0.04 M) from the I307S/W328M are markedly potentiated by M concentrations of diazepam. (e, f) Poten-tiation of GABA currents by diazepam (10 M) at EC1, EC4, EC50 and EC99 GABA for 1, I307S, W328M and I307S/W328M mutants. The potentiation of the I307S/W328M-receptor channel is dependent on the GABA concentrations. 1, W328M and I307S do not respond to diazepam across the range of GABA concentrations tested (relative to their EC50 values). Full Figure and legend (35K)

The co-application of 20 M flumazenil and 20 M diazepam at EC₄ GABA did not significantly alter the diazepam-induced potentiation of the GABA currents elicited from _I307S/W328M (data not shown). These data indicate that the M action of diazepam on _I307S/W328M and its nM action on the ₁₂₂-receptor channel are intrinsically different.

Diazepam at higher concentrations is an agonist for _I307S/W328M (Fig. 6). The EC₅₀ value for the direct action of diazepam was 93 1.9 M associated with a high Hill coefficient (3.3 0.18, Table 1). Diazepam was highly efficacious relative to GABA, eliciting a relative maximum current of 0.8. In comparison, wild-type ₁ and singly mutated ₁ were not directly activated by diazepam at concentrations up to 300 M.

Figure 6. The diazepam concentration− response relationship, and diazepam's relative efficacy with respect to GABA in its direct activation of I307S/W328M. Diazepam is an agonist for the I307S/W328M-receptor channel. (a) Current traces showing the direct activation of I307S/W328M by 30 to 300 M diazepam. (b) Concentration− response relationship for diazepam. Diazepam activation of the I307S/W328M-receptor channel appears to be highly cooperative (high Hill coefficient, Table 1). (c) Diazepam was highly effective relative to GABA (current magnitude at 300 M diazepam divided by current magnitude at 10 M GABA; EC50 GABA for I307S/W328M, 0.095 0.003 M) for direct activation of the I307S/W328M-receptor channel. In comparison, diazepam alone, at concentrations up to 300 M, did not evoke a significant response in either wild-type or singly mutated I. Full Figure and legend (19K)

Differential sensitivity of _I307S/W328M to different BZs

The structure−function relationships of different BZs were assessed using the _I307S/W328M model. Figure 7 shows the current traces and the relative efficacies of diazepam, midazolam, flunitrazepam and flurazepam in potentiating GABA responses (EC₄) of the _I307S/W328M-receptor channel. Diazepam was the most efficacious among the BZs tested. Nevertheless, midazolam and flunitrazepam also elicited a marked potentiation, increasing GABA currents by approximately 0.5-fold and 3-fold at 5 and 20 M concentrations, respectively. In contrast, flurazepam at concentrations up to 20 M did not significantly alter the GABA-evoked currents from the _I307S/W328M-receptor channel.

Figure 7. The relative effectiveness of diazepam, midazolam, flunitrazepam and flurazepam in potentiating the GABA responses (EC4) of the I307S/W328M-receptor channel. Diazepam is the most effective among the benzodiazepines tested. In comparison, flurazepam up to 20 M concentration did not potentiate the GABA-evoked currents for the I307S/W328M-receptor channel. Full Figure and legend (15K)

At EC₃ GABA, the flurazepam concentration−response relationship was determined for the wild-type ₁₂₂-receptor channel to examine whether flurazepam could also produce a biphasic potentiation of GABA currents similar to that observed for diazepam (Fig. 8). In contrast to diazepam, flurazepam elicited only a single component of potentiation with an EC₅₀ of 501 96 nM and a Hill coefficient of 0.89 0.06 (Table 1). Thus, at M concentration, flurazepam neither produced a M component of potentiation for ₁₂₂, nor augmented the GABA-evoked current from the _I307S/W328M-receptor channel. Diazepam and midazolam, which possess a M action upon _I307S/W328M, are used routinely to induce and maintain anesthesia on their own, but flurazepam is not²¹.

Figure 8. Flurazepam elicits only a single component of potentiation within the nM concentration range. Note that the flurazepam induces inhibition at M concentrations. The line is derived from the fit of the Hill equation to the data up to 20 M flurazepam. Full Figure and legend (20K)

However, at higher concentrations (20 M and above), flurazepam produced a significant inhibition of the current (Fig. 8) similar to the inhibitory effect observed at equivalent concentrations of diazepam for _{S267IN265IS280I} (Fig. 4) and wild-type ₁₂₂ (between 5 and 20 M, Fig. 1). Previous studies have shown that impairing the high-affinity (nM) component of diazepam action within the ₁₂₂ by mutation of a single residue within the N-terminal domain of the subunit causes the M component to become more prominent⁵. On the other hand, eliminating the diazepam M component (or its absence in the case of flurazepam) seems to reveal an inhibitory phase (which is otherwise masked by M concentrations of diazepam; Figs. 4 and 8, respectively). The co-existence of an inhibitory phase associated with the nM component, as well as the apparent absence of an inhibitory phase following the abolition of the nM component⁵, suggests a close association between the diazepam inhibitory action and the nM component. BZs can enhance the desensitization of GABA currents^{30, 31}. It is thus possible that the inhibitory action observed here is a reflection of this induced desensitization upon the GABA current.

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The nM and the M components of diazepam action within the ₁₂₂-receptor channel have intrinsic differences. First, the nM component of diazepam action depends on the presence of the subunit. However, both ₁₂- and ₁₂₂-receptor channels are sensitive to M concentrations of diazepam. Second, for the ₁₂₂-receptor channel, flumazenil inhibits the nM but not the M component of potentiation by diazepam. Third, there is a difference of three orders of magnitude in potency between the two components. Fourth, mutation of specific TM₂ residues within the ₁, ₂ and ₂ subunits abolishes the M but not the nM effect of diazepam. Fifth, the action of diazepam in eliciting the M component of both ₁₂₂ and ₁₂ receptors appears highly cooperative as inferred from the Hill coefficient. Diazepam's direct action upon the _I307S/W328M-receptor channel and pentobarbital's direct action on _W328M (ref. 28) also have a high Hill coefficient (Table 1), whereas the nM component persistently displays a low cooperativity (Hill coefficient 1). Sixth, there is a correlation between lipid solubility and the extent of the M potentiation for the BZs tested. For example, diazepam, which has the highest octanol:buffer partition coefficient³² among the BZs tested, has the greatest efficacy for the _I307S/W328M-receptor channel. In comparison, no such relationship has been shown for the nM component. These differences provide strong evidence that the nM and the M component of diazepam action are mediated via distinct and separable mechanisms.

The aforementioned transmembrane residues (_S267 and _M286 and the equivalent residues within ₁) are centrally important for a number of anesthetic compounds^{28, 29, 33, 34, 35, 36, 37}. Also, mutation of the equivalent residue (_N265) within human ₂ or ₃ subunits (to a Ser) of is sufficient to impair the action of loreclezole, a broad-spectrum anticonvulsant that acts at M concentrations³⁸. It is intriguing that so many structurally diverse and hydrophobic compounds mediate their action through these common residues.

The prevalent activation scheme for GABA_A receptors postulates two open states³⁹. In this scheme, one open state is derived from receptor channels occupied by a single GABA, whereas the other open state arises from doubly bound receptor channel. From the simple law of mass action, the proportion of the singlybound form of ₁₂₂-receptor channels may predominate at EC₃ and EC₈ GABA. Thus, the M component of potentiation by diazepam could be the result of selective enhancement of currents from ₁₂₂-receptor channels, which are present in a mono-liganded state.

It has been suggested that the incremental CNS effects of benzodiazepines, from sedation to the induction of anesthesia, are all consequences of the relative occupancy of the benzodiazepine nM receptor. We postulate that the more marked actions of benzodiazepine upon the CNS, such as induction of anesthesia, could be the result of its M action upon GABA_A-receptor channels. Three observations suggest this view. First, the nM action of a benzodiazepine such as diazepam is dependent upon the isoform of the subunit expressed as well as the presence of the subunit, and therefore, its nM activity is limited to only certain subtypes of GABA_A receptors. On the other hand, diazepam's M action seems less specific (since both ₁₂- and ₁₂₂-receptor channels are sensitive to M concentrations of diazepam) and thus may occur at all GABA_A receptors. Second, diazepam produces the second component of potentiation only at low concentrations of GABA. Within the CNS, a low extracellular concentration of GABA has been documented^{40, 41}. In addition, a tonic level of functional GABA_A channel activity in a number of CNS regions has been shown^{42, 43}. Third, concentrations of benzodiazepines can reach M levels within the plasma⁴⁴, and given their high lipid solubility, their concentrations can reach even higher levels within the CNS⁴⁵. Finally, the additional chloride flux due to the M effect upon GABA_A-receptor channels can be predicted to dramatically increase diazepam's overall CNS depressant action. Therefore, under circumstances in which a large dose of diazepam is administered, the marked and ubiquitous action of diazepam at M concentrations may be central in producing a state of general CNS depression, such as the induction of anesthesia.

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Oligonucleotide-mediated site-directed mutagenesis was done according to the manufacturer's protocol (Altered Sites, Promega, Madison, Wisconsin). Successful mutagenesis was verified by DNA sequencing. The cDNAs were linearized with HpaI (for ₁) or SspI (for ₁,₂, or ₂ subunits) leaving a 3' tail several hundred base pairs long. These additional 3' sequences may increase cRNA stability within the oocyte. The cRNA was transcribed from the linearized cDNAs by standard in vitro transcription procedures (Megascript, Ambion, Austin, Texas).

Xenopus laevis (Xenopus I, Ann Arbor, Michigan) were anesthetized by hypothermia, and oocytes were surgically removed and placed in oocyte Ringer (OR₂) that consisted of 82.5 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM Na₂HPO₄, 50 U/ml penicillin, 50 g/ml streptomycin, pH 7.5. Oocytes were dispersed and then incubated in OR₂ without Ca²⁺ plus 0.3% collagenase A (Boehringer Mannheim, Indianapolis, Indiana) for approximately 1 h. After isolation, the oocytes were thoroughly rinsed with OR₂, and stage VI oocytes were separated and maintained at 18°C.

Micropipettes for injecting cRNA were made on a Sutter P87 puller (Sutter Instrument Company, Novato, California), and the tips were cut with microscissors. The cRNA in diethylpyrocarbonate-treated water was drawn up into the micropipette with negative pressure and then injected into the oocytes by applying positive pressure using a Picospritzer II (General Valve Corporation, Fairfield, New Jersey). The oocytes were incubated in OR₂ at 18°C for three days before the experiment. To ensure quality and to estimate the quantity of the cRNA, set dilutions of cRNA from mutants were electrophoresed on a 1% formaldehyde-containing agarose gel. The amount of cRNA was judged and matched by interpolation of lanes containing different dilutions of the corresponding cRNA. In addition, for nearly all mutants, two independent isolates were characterized and tested. The ₁, ₂ and ₂ subunits were co-injected into Xenopus laevis oocytes in the ratio of 1:1:1.8 to ensure the incorporation of ₂ subunits.

Three days after injection, oocytes were placed on a mesh machined in a small perfusing volume chamber (75 l), with a t_1/2 and clearance time of approximately 3 and 10 seconds, respectively. The chamber had an inlet in the top and an outlet in the bottom, which allowed continuous and rapid perfusion. Twenty separate reservoirs (100-ml glass containers) were connected to four six-way valves and the outlet of each of these six-way valves (the sixth position was continuous with the reservoir containing the control solution) was connected to one four-way valve. The outlet of the four-way valve led to the chamber. In this way, up to 20 different solutions could be bath-applied to an individual oocyte. Switching between the different solutions was controlled manually. The oocyte was continuously perfused with recording OR₂ (without antibiotics and the 1 mM Na₂HPO₄, which was replaced with 1 mM NaCl) and switched to the test solution containing drug.

Recording microelectrodes were made with a Narishige PP-83 puller (Tokyo, Japan) and filled with 3 M KCl. Electrodes with resistances of 0.6−1.6 M were used. A two-electrode voltage-clamp amplifier (Turbo TEC-05 npi, Adams and List, Westbury, New York) was used to record currents in response to the application of drugs. In all cases, membrane potential was clamped to -70 mV. Data were visualized on a Gould TA240 chart recorder (Valley View, Ohio) during the experiment and stored online using Pulse Fit or on a VCR using Instrutech 10b (Port Washington, New York) for subsequent off-line analysis.

The EC₅₀ and Hill coefficients were estimated by fitting the concentration−response relationships to the Hill equation according to the following formula (Sigma plot, or software provided by D.S. Weiss). Alternatively, the data containing two components of potentiation were fitted with a sum of two Hill equations.

The fractional potentiation was calculated using the following equation:

The GABA concentration−response relationships were shifted to lower concentrations (two-fold) in oocytes that had high levels of expression. For this reason, oocytes used to collect the data were mostly limited to those that had less than 1 A in maximal response to GABA. The EC values (for example, EC₄) were first determined from the averaged concentration− response relationship. The obtained values were then empirically tested for each experiment by comparing the elicited GABA response at this concentration and the maximal response evoked by a GABA concentration equivalent to 20 EC₅₀. The values were then adjusted and retested accordingly until a true EC value was determined. The low EC values (for example, EC₁) often had a standard error of 30% relative to expected values.

All BZs were purchased from Sigma, except for diazepam, which was purchased from Sigma or BIOMOL (Plymouth Meeting, Pennsylvania). Stock solutions of BZs at 100 mM were made in DMSO. Test solutions containing drugs were made by adding the BZ stock solutions to rapidly stirred recording OR₂ or OR₂ containing GABA. The presence of the vehicle solution DMSO at the maximum tested concentration (0.2%) did not alter the GABA-induced current from ₁, ₁₂₂ or _{S267IN265IS280I}-receptor channels.

All data presented are the mean standard error (s.e.m.).

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