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NEUROPHARMACOLOGY

Neuroactive Steroids and Human Recombinant 1 GABA Receptors

Departments of Anesthesiology (W.L., X.J., J.H.S.) and Molecular Biology and Pharmacology (D.F.C.), Washington University School of Medicine, St. Louis, Missouri

Received June 15, 2007; accepted July 16, 2007.

	Abstract

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The -aminobutyric acid type C (GABA_C) receptor is structurally related to the GABA_A receptors, yet quite distinct physiologically and pharmacologically. Neuroactive steroids are known to be potent and efficacious modulators of the GABA_A receptor, but they are less well characterized in their actions on the GABA_C receptor. We have examined the actions of neuroactive steroids and analogs on 1 (GABA_C) receptors expressed in Xenopus laevis oocytes, with two goals in mind. First, we tested a larger number of endogenous steroids, to determine whether particularly potent steroids could be found. Second, we examined the structure-activity relationship for steroid actions, and some mechanistic features, to determine the possible numbers of steroid binding sites and mechanisms of action. In total, 41 compounds were examined. Estradiols are inhibitors, essentially equipotent with picrotoxinin. No endogenous steroid tested was highly efficacious at potentiation. The results of the structure-activity studies and the effects of two mutations to the second transmembrane region of the 1 GABA_C receptor indicate that there are several mechanisms by which steroids can inhibit the receptor. Surprisingly, estradiol shares some features with picrotoxin. Inhibition by negatively charged compounds was not sensitive to membrane potential, and inhibition by all compounds tested was reduced at higher concentrations of GABA. The data indicate that the binding sites mediating potentiation and inhibition differ from each other and that there are several (three or more) mechanisms for producing inhibition.

There were two primary motivations for these studies. The first motivation was to determine whether some endogenous steroids have higher potency or efficacy than those examined to date. Because steroids are so effective in actions on the GABA_A receptor, it seemed possible that a particular steroid might demonstrate a physiologically relevant selectivity for the GABA_C receptor. The GABA_C receptor plays a less well defined role in the function of the nervous system (Zhang et al., 2001). It has a clear role in the retina (Lukasiewicz et al., 2004), and studies of genetically modified mice suggest that it may play a role in processing of nociception in the spinal cord (Zheng et al., 2003). Activation of GABA_C receptors by synaptically released GABA has been shown to occur in the rat hippocampus (Alakuijala et al., 2006), and a role for GABA_C receptors in short-term memory has been demonstrated in young chickens (Gibbs and Johnston, 2005). Furthermore, there is accumulating evidence that subunits may coassemble with GABA_A receptor subunits both in vitro (Pan et al., 2000; Pan and Qian, 2005) and in vivo (Milligan et al., 2004). There is also increasing interest in developing drugs targeting the GABA_C receptor (Johnston et al., 2003). Hence, identifying possible endogenous modulators of receptors containing the 1 subunit is of general interest.

The second motivation was to obtain additional information for comparisons of steroid actions on the GABA_A and GABA_C receptor. The GABA_A receptors are the major type of receptors that underlie the physiologically and clinically relevant effects of neurosteroids, and they are the most extensively studied type (Belelli et al., 2006). The GABA_C receptor has been less studied, but it shows both similarities to and differences from the GABA_A receptor. In particular, for pairs of steroids such as pregnanolone and allopregnanolone (which differ only in terms of the stereochemistry at the C5-position), both members potentiate GABA_A receptors, whereas the 5 steroid (pregnanolone) inhibits and the 5 steroid (allopregnanolone) potentiates GABA_C receptors (Morris et al., 1999). Because the GABA_A and GABA_C receptors show sequence similarities and hence may have some similarity in steroid binding sites, it is of interest to explore the structure-activity relationship for potentiation and inhibition of the GABA_C receptor by steroids.

View larger version (43K): [in this window] [in a new window]   Fig. 1. Data are summarized for the effects of groups of structurally related steroids and analogs. The first row of the figure shows the steroid backbone; four rings are identified by letters inside the rings. The three positions (3, 5, and 17), which were studied most extensively, are indicated with arrows. The compounds studied are then shown in the following rows, separated into groups based on structures. The basic structure for members in a group is shown in the first column; it is the structure for the first compound listed in the second column (the "exemplar"). The tables in the second column summarize data for the compounds named (for full names, see Table 1), as the response to 200 nM GABA in the presence of 10 µM drug normalized to the response of the same oocyte to 200 nM GABA alone (mean ± S.D.; number of oocytes in parentheses). P1 gives the probability of a significant difference to no effect (paired t test versus a ratio of 1), whereas P2 gives the probability of a significant difference to the exemplar drug in a group (examplar denoted by EX; multiple comparisons with Dunnett's post hoc correction). N.S., P > 0.05; *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

	Materials and Methods

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A large number of steroids and analogs were tested (Table 1). Commercially available steroids were purchased from Steraloids (Newport, RI). Other steroids were synthesized by published methods (Table 1). Stock solutions of steroids were made at a concentration of 10 mM in DMSO, and then they were diluted to the final concentration in bath solution containing 200 nM GABA. At the highest concentration of DMSO used (0.2%), DMSO had no effect on responses (also see Morris et al., 1999; Li et al., 2006b).

Responses were recorded 2 to 5 days after injecting the oocytes, at a holding potential of –50 mV (unless otherwise noted) using a two-electrode oocyte clamp (Warner Instruments, Hamden, CT). Both voltage and current electrodes were patch-clamp electrodes filled with 3 M KCl, and they had resistances of 0.5 to 1 MOhm. The bath solution contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, and 10 mM HEPES, pH adjusted to 7.5 with NaOH. The bath had a volume of approximately 0.1 ml, and it was perfused at approximately 7 ml/min. Bath solution was perfused between all test applications. Solutions were switched by hand, using Teflon rotary valves (Rheodyne, Rohnert Park, CA). Solutions were applied using glass reservoirs, fluorocarbon valves, and fluorocarbon or metal tubing, to reduce adsorption. Most studies were performed using 200 nM GABA, a concentration producing a response approximately 5% of the maximal response (Li et al., 2006b). In each case, GABA alone was applied, then GABA + steroid, then GABA alone, and the effect was calculated by taking the ratio of the response to GABA + steroid to the mean response for the bracketing control applications. Applications were separated by 3 to 5 min. In studies of mutated receptors, an initial concentration-response curve was obtained for GABA, and the concentration that produced approximately 5% of maximal response was used (see Results).

Data were acquired using a Labmaster analog-to-digital converter (Molecular Devices, Sunnyvale, CA). Digitized traces were analyzed using pClamp software (Molecular Devices), and reduced data were analyzed with Excel (Microsoft, Redmond, WA) and SigmaPlot (Systat Software, Inc., San Jose, CA). Data values are presented in the text and shown in the figures as mean ± S.D. (N cells). The significance of effect for a given compound was assessed using a two-tailed Student's t test for paired observations for a difference to a ratio of 1. To compare effects of multiple drugs a one-way analysis of variance with a Dunnett's post hoc correction (SYSTAT; Systat Software, Inc.) was used to compare effect ratios.

GABA concentration-response relationships were obtained by interspersing responses to a control concentration (1 µM) and normalizing responses for that egg to the control. The relationships were analyzed using the following equation: relative , where maximum is the maximal relative response, EC₅₀ is the concentration producing half-maximal response, and n is the Hill coefficient.

Inhibition curves were analyzed using the following equation: relative , where Y0 is the relative response at a saturating drug concentration, IC₅₀ is the concentration producing half-maximal inhibition, and n is the Hill coefficient. In both cases, the equations were fit to all the individual data points pooled from all eggs with that drug.

	Results

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Structure-Activity Relationship for Steroids and Analogs
The structure-activity relationships were evaluated using a drug concentration of 10 µM, for effects on the response to 200 nM GABA. This concentration of GABA is about the EC₅ concentration (Li et al., 2006b), and it was chosen because it has been shown that both potentiation and inhibition by steroids are maximal at a low concentration of agonist (see below; Morris et al., 1999). We also note that it has been proposed that GABA_C receptors produce a relatively tonic response to low concentrations of GABA; thus, a low concentration may be most appropriate for exploring physiologically relevant actions. A single concentration of steroid was adopted for comparisons among multiple drugs. However, it means that a difference in effect could be produced by a change in potency, efficacy, or both.

We use a standard set of abbreviations for the steroids used (full names and abbreviations are given in Table 1). For example, the pair allopregnanolone and pregnanolone are abbreviated as 3517P and 3517P (e.g., the 3-hydroxyl group is in the orientation; the 5-hydrogen is or , respectively; and the 17-methylketone is ).

The overall results for 41 compounds are summarized in Fig. 1 (with representative structures), and selected comparisons are presented below. Structures for all steroids and analogs are provided in the Supplemental Material.

Comparison of 5- and 5-Reduced Compounds. We compared pairs of drugs with identical structures except for the reduction at the C5-position (Fig. 2). In general, if drugs in a pair had activity, the 5-reduced compound potentiated and the 5-reduced compound inhibited. These results are consistent with previous studies (Morris et al., 1999; Goutman and Calvo, 2004). The major exception is the pair of sulfated steroids 3SO₄517P and 3SO₄517P, for which these C5 diastereomers inhibited equally. This observation was surprising enough that we confirmed it with new supplies of both steroids, and we also performed a full concentration-effect study (see below). It suggests that inhibition by the sulfated steroids may differ from inhibition by unsulfated steroids.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Comparisons of pairs of steroids with 5 and 5 stereochemistry. The figure shows the mean ± S.D. for the ratio of the response to 200 nM GABA in the presence of 10 µM steroid to the response in the absence of steroid. (For values, numbers of oocytes and probability of significant differences, see Fig. 1.) In general, steroids with 5 stereochemistry potentiate (if they have any effect), whereas those with 5 stereochemistry inhibit. A notable exception is the pair of 3-sulfated steroids, which both inhibit responses.

View larger version (8K): [in this window] [in a new window]   Fig. 3. Comparisons of pairs of steroids with 3 and 3 stereochemistry. Steroids with 3 stereochemistry are active as potentiators or inhibitors, whereas those with 3 stereochemistry are inactive. This difference is true even for sulfated steroids. Data are presented as in Fig. 2.

Structure of the Substituent at the 17-Position. We examined a number of drugs that were identical at the 3- and 5-positions but that differed in terms of the group attached at the 17 position, to determine whether the sites mediating potentiation and inhibition could be distinguished based on this structural feature (Fig. 4). Overall, we found that the two types of effector sites showed similar structure-activity relationships, at the resolution afforded by our methods. That is, drugs with carbonitrile, methylketone, and hydroxymethylketone groups in the 17-position all were able to produce potentiation or inhibition. Ketone and hydroxyl groups resulted in compounds that were essentially inactive (Fig. 1). These observations suggest similarities between the sites mediating potentiation by 5-reduced steroids and inhibition by 5-reduced steroids.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Comparisons of steroids with 5 (top group) or 5 (bottom group) stereochemistry, depending on the type of group at position 17. In general, if a substituent produces a compound that is a potentiator when the structure is 5, the paired compound with 5 structure is an inhibitor. The 17 hydroxyl and keto steroids are relatively ineffective at either potentiation or block. Data are presented as in Fig. 2.

Inhibition by Estradiols. We have already noted that both 17-Est and 17-Est inhibit responses of the 1 GABA_C receptor. We also examined the requirement for a free 3-OH group, and we found that methylation (3MeO17-Est) significantly reduces inhibition (Fig. 1). The ability of estrogens to potentiate responses of the human neuronal nicotinic 42 receptor shows similar structural requirements, including the lack of selectivity between 17- and 17-Est (Paradiso et al., 2001), so we tested an additional compound (17-ethynyl17-Est), which potentiates the nicotinic receptor. This drug showed reduced inhibitory effects on the 1 GABA_C receptor compared with 17-Est (Fig. 1).

Sulfated and Carboxylated Steroids. Both 3SO₄517P and 3SO₄517P inhibit responses (Fig. 2), whereas 3SO₄517P and 3SO₄517P do not (Fig. 2). In addition, 3SO₄517P and 3SO₄517K are only weak blockers (Fig. 1). We also tested the effects of adding a sulfate to either the 3- or 17-position of 17-Est; both sulfated estradiols were significantly less active than 17-Est (Fig. 1). This result suggests that the sites mediating inhibition by sulfated steroids and by estradiols are distinct.

To examine the role of a different anionic group at the 3-position, we tested a carboxylic acid derivative (3COOH517P). This proved to be a very effective blocker (Fig. 2). Because the sulfated steroids inhibited whether the structure was 5- or 5-reduced, we also examined 3COOH517P. This compound was inactive (Fig. 2). Accordingly, the carboxylated and the sulfated compounds have a different structure-activity relationship.

Comparisons of Enantiomer Pairs. Previous studies of the 1 GABA_C receptor have shown that the enantiomer of 3517P(ent-3517P) is inactive (rather than potentiating) and that ent-3517P actually potentiates rather than blocking currents (Li et al., 2006b; see Fig. 1). We also compared 17-estradiol and ent-17-estradiol; again, the unnatural enantiomer is inactive [17-Est: 0.44 ± 0.12 (11); ent-17-Est: 0.92 ± 0.10 (6)]. This suggests that there is a specific site involved, although it can, apparently, recognize both 17- and 17-estradiol. The enantiomer pairs 3SO₄517P and ent-3SO₄517P and 3SO₄517K and ent-3SO₄517K are only weakly active (Fig. 1), but they are consistent in the sense that if the natural enantiomer has a discernible effect, the unnatural enantiomer has a reduced, absent or reversed effect. Accordingly, it seems most likely that all of the effects are mediated by interactions between the steroid and the 1 GABA_C receptor (Li et al., 2006b).

Studies of the Mechanism of Steroid Action
Concentration Effect Studies. We first determined the consequences of testing a drug at 10 µM and eliciting responses with different concentrations of GABA (Fig. 5). The goal was to determine whether there is an indication that a drug acts preferentially on the resting or activated states of the receptor. The potentiation produced by 3517CN and the inhibition produced by 17-Est or 3SO₄517P are maximal at a low concentration of GABA, and they are reduced at a high (saturating) concentration of GABA. We have also confirmed that the inhibition by 3517P is reduced at higher concentrations of GABA (Goutman and Calvo, 2004); 1 µM3517P reduced the response to 200 nM GABA to 0.65 ± 0.14 (N = 27), whereas it only reduced the response to 10 µM GABA to 0.96 ± 0.06 (N = 14). Similar observations have been made for potentiation by 3517POH and inhibition by 3517POH (Morris et al., 1999). In general, it is expected that there will be reduced potentiation when a high concentration of agonist is used, since many mechanisms for potentiation will be ineffective when a maximal response is elicited. In contrast, there is no reason to think that any of these inhibiting drugs act as competitive inhibitors of GABA binding. The fact that inhibition is reduced at higher [GABA] suggests that the inhibitors preferentially bind to resting states of the receptor.

View larger version (12K): [in this window] [in a new window]   Fig. 5. The effect of 10 µM compound is shown, as a function of the GABA concentration used to elicit the response (open triangles, 3517CN; closed circles, 17-Est; and closed triangles, 3SO4517P). Note that both potentiation and inhibition are reduced at higher GABA concentration. Data are mean ± S.D. for two or three cells (error bars often concealed by symbols). The lines simply connect the points.

We also examined the inhibition produced by various concentrations of inhibitors on responses elicited by 200 nM GABA. The goal was to determine whether inhibition was partial or complete, over the accessible range of steroid concentrations. As shown in Fig. 6, 3COOH517P is the most potent inhibitory steroid. The diastereomers of estradiol are about equipotent with picrotoxinin and with each other. The sulfated steroids also are essentially equipotent with each other, although less potent than picrotoxinin. These compounds produced full inhibition. However, 3517P produced only a partial block (Fig. 6). Morris et al. (1999) have tested 3517P and other 5-reduced steroids (including 3517POH), and they also found that they did not produce full block but showed only partial block at maximal inhibition (also see Goutman and Calvo, 2004). These observations suggest that 35P and other 5-reduced steroids are allosteric inhibitors. These results also indicate that the screening protocol (a drug concentration of 10 µM and an EC₅ concentration of GABA) is appropriate for enhancing the magnitude of any effects.

View larger version (9K): [in this window] [in a new window]   Fig. 6. Inhibition curves are shown for seven compounds. Data were fit with the Hill equation (lines through the data show fits), and fitted parameters are given as the best fitting value ± the standard error of the fit estimated during the fitting procedure (see Materials and Methods). A, data are shown for 17-Est (closed circles; fit values are Y0 = 0.06 ± 0.15, Hill coefficient =–1.29 ± 0.31, IC50 = 6.50 ± 1.93 µM), 17-Est (open circles; Y0 = 0.03 ± 0.45, Hill coefficient =–0.73 ± 0.35, IC50 = 4.92 ± 7.21 µM), and picrotoxinin (closed triangles; Y0 = 0.00 ± 0.09, Hill coefficient =–0.85 ± 0.15, IC50 = 2.68 ± 0.69 µM). B, data are shown for 3SO4517P (closed circles; Y0 = 0.00 ± 0.18, Hill coefficient =–1.17 ± 0.31, IC50 = 5.98 ± 2.07 µM), 3SO4517P (closed triangles; Y0 = 0.06 ± 0.10, Hill coefficient =–1.36 ± 0.28, IC50 = 4.45 ± 0.78 µM), and 3COOH517P (open circles; Y0 = 0.00 ± 0.04, Hill coefficient =–1.06 ± 0.12, IC50 = 1.02 ± 0.11 µM). C, data for 3517P (Y0 = 0.25 ± 0.14, Hill coefficient =–0.67 ± 0.22, IC50 = 1.30 ± 0.98 µM).

Consequences of Mutations in the Second Membrane-Spanning Helix. We then explored the consequences of mutations in the 1 subunit. We selected two residues in the predicted second membrane-spanning region (TM2) for mutation, based on the likelihood that they would produce changes in the actions of inhibitory drugs. We evaluated consequences for the effects of 10 drugs, including three potentiating steroids, the corresponding 5 inhibitory steroids, and representative sulfated inhibitory steroids, carboxylated inhibitory steroids, and 17-estradiol. The first mutation is to the residue in the second position of TM2, TM2 P2'S (1 P294S numbered according to the first residue in the predicted mature subunit). Residues homologous to this have been identified as important for inhibition in several studies. Initially, it was found to be a critical residue for conferring resistance to dieldrin in an insect GABA receptor (rdl TM2 P2'S; Ffrench-Constant et al., 1993). Subsequently, it was identified as a determinant for block of glycine receptors by cyanotriphenylborate (Rundström et al., 1994) and lactones (glycine 3 TM2 G2'A, Steinbach et al., 2000). Finally, mutation of this residue in the 1 subunit of the GABA_A receptor reduces block by pregnenolone sulfate (1 TM2 V2'S; Akk et al., 2001), by a benz[e]indene (Li et al., 2006a) and by 3-hydroxysteroids and sulfated steroids (Wang et al., 2002). This mutation does not affect block by picrotoxinin of responses of 1 GABA_C receptors to low concentrations of GABA (Wang et al., 1995). However, we note that mutations to 1 TM2 P2' do affect activation by agonists, including changes in the efficacy of partial agonists (Carland et al., 2004).

This mutation shifted the concentration response curve for GABA to higher concentrations (data not shown; EC₅₀ 3.4 ± 0.2 µM; Carland et al., 2004). Accordingly, we used 1 µM GABA to elicit responses to test the actions of steroid.

The consequences of this mutation on the actions of drugs are shown in Table 2. The ability of 5-reduced steroids to inhibit the response is diminished to such an extent that it did not differ from no effect (Table 2). However, the actions of a sulfated steroid, a carboxylated steroid, 17-estradiol, and picrotoxinin are less affected. Potentiation is not significantly affected. The results suggest that the 5-reduced steroids form a group that is distinct from the other inhibitors. The fact that potentiation is not strongly affected also suggests that the incomplete inhibition by 3517P is not the result of combined inhibition plus potentiation, with differing EC₅₀ values.

View this table: [in this window] [in a new window]   TABLE 2 Consequences of the 1 P2'S mutation on the effects of steroids and analogs The first column gives the drug abbreviation; the compounds are arranged in groups: 5-reduced steroids, sulfated or carboxylated steroids, 17-Est and picrotoxinin, and 5-reduced steroids. The second column summarizes effects on wild-type receptors (relative response: mean ± S.D.; number of eggs in parentheses) and gives the probability that the relative response differs from 1 (paired t test). The third column gives data for the effect on receptors containing the 1 P2'S mutation; the first probability gives the probability that the relative response differs from 1, whereas the second probability gives the probability that the relative response of the mutated receptor differs from that of wild type (unpaired two-tailed t test).

Consequences of a Mutation to TM2 T6'. The second mutation was TM2 T6'F(1 T298F). This residue was first implicated in block by picrotoxin in studies of the subunit of the glycine receptor (Pribilla et al., 1992), and it was later shown to be critical for picrotoxin block of the GABA_A receptor (Gurley et al., 1995) and the 1 GABA_C receptor (Zhang et al., 1995). This mutation did not shift the activation curve for GABA (data not shown; EC₅₀ = 0.6 ± 0.2 µM); thus, 200 nM GABA was used to elicit responses.

As expected, this mutation essentially removes block by picrotoxinin (Table 3). However, the consequences of this mutation on the steady-state effects of steroids were startling (Table 3). First, it removes block by 17-Est, suggesting similarities between the actions of estradiol and picrotoxinin. Second, it increases block by several drugs, including converting 3517CN into a relatively strong blocker.

View this table: [in this window] [in a new window]   TABLE 3 Consequences of the 1 T6'F mutation on the effects of steroids and analogs Data are presented as in Table 2. However, in this case for each drug the relative responses of wild type, T6'F, and T6'F tail were compared using a one-way analysis of variance with Bonferroni post hoc correction. The third probability listed in the column for T6'F tail gives the probability that the tail ratio differs from the steady-state ratio.

To understand these effects, we examined the time course of the responses. As shown in Fig. 7, a large "tail" forms when drug and GABA are removed, most markedly for 3COOH517P, 3517CN, and 3517CN. Tails are not apparent with 17-Est, picrotoxin, 3517POH, or 3517P, and they are less marked for other compounds. This tail is ascribed to a rapid relief of inhibition. If recovery from inhibition occurs more rapidly than deactivation of the receptor, then as receptors unblock the response actually increases when drug is removed. Often, the type of block producing these tails is an "open channel" block, but this specific mechanism is not required. It simply has to be the case that recovery from block is more rapid than deactivation. Recordings from Xenopus oocytes are not suitable for detailed kinetic analysis, so we did not attempt to correct for the time courses of the multiple processes occurring. Instead, we simply reanalyzed the traces, but we measured the amplitude of the tail current (Fig. 8; Table 3). When this is done, it seems that this mutation does not affect potentiation. It does remove block by 17-Est and picrotoxinin. It does not strongly affect block by 5-reduced steroids, except in the case of 3517POH. Finally, it partially removes block by sulfated and carboxylated steroids. Again, 17-Est and picrotoxinin fall in a group that is distinct from the other types of compounds examined.

View larger version (8K): [in this window] [in a new window]   Fig. 7. Responses of 1T6'F receptors to 200 nM GABA (dashed lines) and to 200 nM GABA + 10 µM steroid (solid lines), aligned to show the tail response. The control and test responses are sequential applications to one egg, whereas each pair was obtained from a different egg. The top row shows 5-reduced steroids (3517P, 3517CN, and 3517POH), the second row shows 3SO4517P and 3COOH517P, the third row shows 17-Est and picrotoxinin, and the bottom row shows 5-reduced steroids (3517P, 3517CN, and 3517POH). Most compounds produce tails, of different amplitudes, although 17-Est, picrotoxinin, 3517P, and 3517POH show no apparent tail. Each pair of responses was normalized to the peak control response for that pair. The dashed horizontal line shows the 0 current level, whereas the heavy bar above the dashed line shows the end of the drug application. Scale bar, 60 s.

View larger version (5K): [in this window] [in a new window]   Fig. 8. Superimposed responses of the same egg to 200 nM GABA (dashed line) and to 200 nM GABA + 10 µM3517CN. The vertical line shows the time at which the relative tail response was calculated, as the ratio between the response to GABA + 3517CN to that of GABA alone. The figure is presented as in Fig. 7.

	Discussion

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These results have extended the analysis of steroid actions at the 1 GABA_C receptor in several ways. As a result of our studies of enantiomer pairs (also see Li et al., 2006b), it seems that these steroids interact with specific chiral sites, most probably on the 1 GABA_C receptor itself. Because the comparisons are relatively extensive, we start by considering potentiation by 5-reduced steroids and inhibition by 5-reduced steroids (Morris et al., 1999).

Potentiation by 5 Compounds and Inhibition by 5 Compounds. Previous work has shown that potentiation and inhibition seem to be independent processes. When 3517P (10 µM) was applied in the presence of 10 µM 3517P, the amount of block was indistinguishable from the block in the absence of 3517P (Li et al., 2006b). This observation indicates that the two types of drug do not compete for the same site. However, the present data show similarities in the structural requirements for steroid action. For both inhibition and potentiation, drugs containing a 3-hydroxy group are inactive, as are drugs containing a 17-carbonitrile. Furthermore, the types of the substituent at the 17-position that support potentiation or inhibition are broadly similar, with hydroxy or keto groups resulting in relatively weak compounds for either action. Thus, the sites mediating the two effects may be similar in structure, although distinct. Neither of the mutations in the TM2 region that we examined had a significant effect on the ability of 5-reduced steroids to potentiate, suggesting that the mechanisms for potentiation and inhibition are distinct. We discuss other studies of mutated receptors below.

5-Reduced steroids interact preferentially with inactive receptors, because the inhibition is reduced at higher concentrations of GABA, and it is likely to be allosteric, because the maximal inhibition can be less than complete (also see Morris et al., 1999; Goutman and Calvo, 2004). Somewhat surprisingly, a mutation (TM2 P2'S) homologous to a mutation that has been shown to reduce the ability of sulfated steroids to block the GABA_A receptor has the largest effect on inhibition by 5-reduced steroids. It has previously been argued that this residue is unlikely to form part of a binding site for 3SO₄517P (pregnenolone sulfate) on the GABA_A receptor (Akk et al., 2001). We note that an earlier study found that a different mutation at this site (TM2 P2'A) did not affect inhibition by 3517P (Morris and Amin, 2004).

A comparison to the steroid structure-activity relationship for actions on the GABA_A receptor is more complex, for several reasons. First, 5 steroids potentiate the GABA_A receptor. The consequences of a 3 substituent orientation are also somewhat different. For the 1 GABA_C receptor, this orientation removes both potentiation or block (at least for the concentrations tested), whereas for the GABA_A receptor 3 steroids (e.g., 3517P) block weakly by a mechanism similar to that of 3SO₄517P (Wang et al., 2002). However, the lack of potentiation by steroids with 3 substituents is in agreement with our observations on the 1 GABA_C receptor. The requirement for the 17 configuration for potentiation is found for the GABA_A receptor (Phillipps, 1975), although it does not seem to have been examined for inhibitory steroids. Therefore, overall, there are some strong similarities between the structural requirements for steroid actions at the GABA_A and 1 GABA_C receptors. The major difference is in the effects of 5-reduced steroids, which potentiate GABA_A and block 1 GABA_C receptors. It should be noted that there are some indications that 5- and 5-reduced steroids may have nonidentical binding sites on the GABA_A receptor (Gee et al., 1988; Mennerick et al., 2004), so the possibility exists that the sites may be similar on both types of receptor but transduction mechanisms differ. It is also known that potentiating steroids interact with more than one site on the GABA_A receptor (Akk et al., 2004); the studies of 1 GABA_C receptors were not performed with techniques that could address this issue.

We note that many steroids and analogs inhibit the rodent nicotinic 42 receptor (Sabey et al., 1999; Paradiso et al., 2000). The structure-activity relationship for this inhibition suggests that the site involved is distinct from any of the sites we have characterized on the 1 GABA_C receptor.

Inhibition by Sulfated or Carboxylated Compounds. Turning to inhibition of 1 GABA_C receptors, several observations were noted under Results that suggest that there is more than one mechanism for producing inhibition of the 1 GABA_C receptor. Inhibition could be produced by 5-reduced steroids, by sulfated or carboxylated steroids, and by estrogens. In comparing sulfated to 5-reduced steroids, both the 5- and 5-reduced sulfates inhibited. This is a clear difference to the inhibition by unsulfated steroids, for which the 5 configuration is required. Inhibition by sulfated steroids is reduced at higher GABA concentrations, suggesting that they interact preferentially with the inactive receptor. It does not seem to involve binding in which the charge on the steroid interacts with the membrane field, because the inhibition is voltage-independent. Somewhat surprisingly, a mutation homologous to a mutation that has been shown to reduce the ability of sulfated steroids to block the GABA_A receptor has no effect on block by 3SO₄517P, whereas it reduced inhibition by the uncharged 5 steroids tested. Overall, it seems likely that the receptor-steroid interactions that mediate inhibition by sulfated steroids (and possibly the mechanism as well) differ from those mediating inhibition by nonsulfated 5 steroids.

Many aspects of the effects of sulfated steroids differ between the 1 GABA_C and GABA_A receptors. One aspect is that the 3-sulfated steroids do not block the 1 GABA_C receptor, whereas they do block the GABA_A receptor (Mennerick et al., 2001). Block of GABA_A receptors by 3SO₄517P is voltage-dependent, and it is enhanced by higher concentrations of GABA (Mennerick et al., 2001), possibly reflecting a form of open-channel block. Finally, block is reduced by a mutation, 1V2'S (Wang et al., 2002), that did not strongly affect block of the 1 GABA_C receptor. 3SO₄517P differs from other sulfated steroids in that the block of the GABA_A receptor is not voltage-dependent (Akk et al., 2001; Eisenman et al., 2003). The mechanism by which pregnenolone sulfate acts on GABA_A receptors is not known, although it has been proposed that it enhances desensitization (Shen et al., 2000; Akk et al., 2001).

A 3-carboxysteroid (3COOH517P) produces the largest inhibition of any of the drugs we tested at 10 µM. In common with the sulfated steroids, inhibition does not show voltage dependence. In clear distinction to the sulfated steroids, 3COOH517P had little effect at either block or potentiation. This may mean that this compound shares some aspects of inhibition with other 5-reduced compounds. However, the effects of two mutations showed a rather different pattern than for uncharged 5-reduced steroids. This analog potentiates GABA_A receptors when applied at 10 µM concentrations, but it acts as a voltage-dependent blocker at higher concentrations (Mennerick et al., 2001), and its mechanism of action is not clear.

Inhibition by Estradiols. The most surprising observation with natural steroids is the block by 17-estradiol, which is an effective inhibitor. 17-Estradiol is relatively inactive on the GABA_A receptor (Akk et al., 2007). It potentiates the human nicotinic 42 receptor (Paradiso et al., 2001), whereas it inhibits the rat isoform (Paradiso et al., 2000). At 1 GABA_C receptors, estradiols are also unusual in that the 17 hydroxy group can be oriented in either the 17 or 17 configuration, with equal effect. The saturated androstanediols are largely inactive, irrespective of stereochemistry, indicating the importance of the unsaturated A ring for this action. Adding a sulfate group to either the 3- or 17-position of 17-Est reduces activity, suggesting a difference to inhibition by sulfated steroids. To our surprise, inhibition by 17-Est is removed completely by a mutation, which also removes inhibition by picrotoxinin. Inhibition by estradiols does not fit well with the structural requirements for inhibition by either 5-reduced steroids or sulfated steroids, and it is likely to reflect an additional site.

Other Studies of the Effects of Mutations to the 1 GABA_C receptor on Steroid Actions. An extensive study has been made of the consequences of mutations to a residue in the TM2 region of the 1 GABA_C receptor, I307, which is near the extracellular end of the helix (Morris and Amin, 2004). This residue is homologous to a residue that has been implicated in several drug effects on the GABA_A receptor. Initially, it was reported that in the subunits the nature of this residue is critical for the action of etomidate (Belelli et al., 1997). Subsequent work has also indicated the importance of this residue, in the subunit, in the actions of volatile anesthetics and alcohols (Mascia et al., 2000; Jenkins et al., 2001). Finally, the mutation 1 I307S makes the receptors sensitive to potentiation by pentobarbital (Belelli et al., 1999). It has been proposed that this residue forms part of a pocket behind the TM2 membrane-spanning helix, into which a drug may insert itself to produce effects (Mascia et al., 2000; Jenkins et al., 2001). A large series of substitutions have been made of 1 I307, and the consequences for actions of two inhibitory steroids (3517P and 5-dihydroprogesterone or 3K517P) and one potentiating steroid (3517P) were determined (Morris and Amin, 2004). The pattern of results is complicated and difficult to interpret. In brief, inhibitory steroids, in general, could be converted to show either mixed effects (inhibition at lower concentrations and potentiation at higher concentrations) or only potentiating effects. A more limited set of mutations was tested on the potentiating steroid, but, in general, potentiation was enhanced. The initial interpretation was that neuroactive steroids had their actions by effects in the lipid surrounding the 1 GABA_C receptor, which were sensed by this residue and transduced into functional changes (Morris and Amin, 2004). This initial interpretation is made much less likely by the strong enantioselectivity of many steroid actions (see above; Li et al., 2006b), but the pattern of observations is difficult to explain by a unifying theory. However, we note that in our more limited examination of the consequences of mutations we have obtained evidence that a point mutation may have complicated effects. In the TM2 T6'F mutation, we were fortunate that the appearance of a novel form of inhibition was signaled by the tail currents.

Summary. In terms of the first objective, examination of a greater number of endogenous steroids, two major observations can be made. First, estradiols are much more effective inhibitors of the 1 GABA_C receptor than of GABA_A receptors. Second, the 3 configuration is required for all other steroid actions examined. In terms of the second objective, to clarify the structure-activity relationship for the actions of steroids on the GABA_C receptor and compare it with that for the GABA_A receptor, the results of this initial survey of the structure-activity relationship for steroid actions on the 1 receptor suggest multiple sites for interaction between steroids and the 1 receptor. The sites mediating potentiation by 5- and inhibition by 5-reduced steroids have similar qualitative requirements, and some similarities to those for potentiation of GABA_A receptors. In contrast, the sites mediating inhibition by sulfated steroids and by estradiols seem to differ from those sites, from each other, and from sites characterized on other members of the nicotinic receptor family.

	Acknowledgements

 
We thank Gustav Akk and John Bracamontes for advice and constructive criticism, and Gustav Akk, Peter Lukasiewicz, and Steve Mennerick for comments on the manuscript.

	Footnotes

 
This work was supported by National Institutes of Health Grant P01 GM 47969 (to D.F.C. and J.H.S.). J.H.S. is the Shelden Professor of Anesthesiology.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.127365.

ABBREVIATIONS: DMSO, dimethyl sulfoxide; TM, transmembrane.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Joe Henry Steinbach, Department of Anesthesiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO. E-mail: jhs{at}wustl.edu

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