Introduction

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The steroids 35P (allopregnan-3-ol-20-one) and 35P (pregnanolone) are potent anesthetics (Phillipps, 1974). A large amount of data supports the hypothesis that the anesthetic actions of these steroids are correlated with their enhancement of -aminobutyric acid (GABA)_A receptor-mediated neuronal inhibition (Lambert et al., 1995). The number of binding sites for these steroids and their location on GABA_A receptors are not known. Moreover, whether the steroids share a common site is not known. Molecular-modeling studies suggest that the anesthetic activities of these steroids result from binding at a common binding site (Purdy et al., 1990; Han et al., 1996). However, binding studies for steroid-induced noncompetitive displacement of t-butylbicyclophosphorothionate (TBPS) from the picrotoxin-binding site on GABA_A receptors indicate that, although these steroids may share a common binding site, multiple binding sites are detectable for some steroids (Morrow et al., 1990; Hawkinson et al., 1994a,b, 1998).

Because 35P and 35P are molecules that each contain eight chiral centers (C-3, C-5, C-8, C-9, C-10, C-13, C-14, and C-17), their stereoselective modulation of GABA_A receptor function can be studied by inverting each, some, or all of the chiral centers in the molecules. Inversion of one center in a molecule containing multiple chiral centers gives compounds called epimers. Epimers are a subset of compounds called diastereomers. Diastereomers are compounds with multiple chiral centers that differ in configuration at one or some, but not all, chiral centers. Studies of the C-3 (inversion of the 3-hydroxyl group) and C-17 (inversion of the acetyl group) epimers of 35P and 35P have been very informative in establishing the structure-activity relationships for anesthetic steroids (Phillipps, 1974). No doubt, studies of additional diastereomers of 35P and 35P will further define these structure-activity relationships. However, because diastereomers have different physical properties due to the relative positions of some atoms in each diastereomer being different, the membrane-perturbing effects as well as the direct effects of these steroids on GABA_A receptor function will be different. Thus, C-3 epimers (3-OH) of 35P and 35P not only fail to potentiate GABA effects at GABA_A receptors but also interact differently with membrane phospholipids (Makriyannis et al., 1991).

To avoid the membrane-perturbing effects caused by studying anesthetic steroid diastereomers having different physical properties, we have studied anesthetic steroid enantiomers (Fig. 1). Enantiomers are the stereoisomers of optically active compounds that are mirror images of each other (all chiral centers have the opposite absolute configuration). Enantiomers have identical physical properties because the relative positions of all atoms in each enantiomer are identical. For example, any group having an axial configuration in one enantiomer also has an axial configuration in the other enantiomer.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Structures of the four pairs of anesthetic steroid enantiomers studied.

Enantioselectivity of 35P-induced GABA_A receptor modulation and anesthesia in Xenopus laevis tadpoles and mice has been reported previously (Wittmer et al., 1996). Herein, we report the corresponding actions of the 35P enantiomers. Additionally, new information for the displacement of TBPS binding by both pairs of enantiomers is reported. Some comparative data for the corresponding enantiomers of the 17-carbonitrile analogs of 35P and 35P also are presented. The new data show that enantioselectivity [comparison of 35P/ent-35P with 35P/ent-35P) and diastereoselectivity (comparison of 35P/35P with ent-35P/ent-35P) for modulation of GABA_A receptor function by these anesthetic steroids are different. The enantioselective actions of 35P are significantly greater than the enantioselective actions of 35P. The diastereoselective actions of the 35P/35P steroid pair are not significant and those of the ent-35P/ent-35P pair are significantly different.

	Materials and Methods

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Chemicals. The ent-35P, ent-35P, 35ACN [(3,5,17)-3-hydroxyandrostane-17-carbonitrile], ent-35ACN, and 35ACN [(3,5,17)-3-hydroxyandrostane-17-carbonitrile] were prepared and characterized as described previously (Hu et al., 1993, 1997; Han et al., 1996; Nilsson et al., 1998). The ent-35ACN was prepared from ent-(3,5)-3-hydroxyandrostan-17-one with the methods described for the preparation of 35ACN (Han et al., 1996). The infrared, ¹H NMR, and ¹³C NMR spectra of the 35ACN and ent-35ACN were identical. The 35P and 35P were purchased from either Sigma Chemical Co. (St. Louis, MO) or Steraloids (Newport, RI). The [³⁵S]TBPS was purchased from NEN Life Science Products (Boston, MA) and TBPS was purchased from Research Biochemicals International (Natick, MA).

[³⁵S]TBPS Binding. Rat brain cortical membranes were prepared with minor modifications of the method previously reported (Hawkinson et al., 1994a). Briefly, frozen rat cerebral cortices (Pel-freez, Rogers, AK) were thawed and homogenized in 10 volumes of ice-cold 0.32 M sucrose with a glass/Teflon pestle. The homogenate was centrifuged at 1500g for 10 min at 4°C. The resultant supernatant was centrifuged at 10,000g for 30 min at 4°C. The pellet (P2) from this centrifugation was resuspended in 200 mM NaCl, 50 mM potassium phosphate buffer, pH 7.4, and centrifuged at 10,000g for 20 min at 4°C. This washing procedure was done three times, and then pellets were resuspended in buffer (~4 ml/brain) with a glass/Teflon pestle. The membrane suspension was aliquoted, frozen in liquid nitrogen, and stored at 80°C before use. [³⁵S]TBPS binding assays were done according to the procedure described previously (Hawkinson et al., 1994a) with modifications. Briefly, aliquots of membrane solution (0.5 mg/ml final protein concentration in assay) were incubated with 5 µM GABA, 2 nM [³⁵S]TBPS (45-120 Ci/mmol), and 5 µl-aliquots of steroid in dimethyl sulfoxide (DMSO) solution (final assay concentrations ranged from 1 nM to 10 µM), and brought to a final volume of 1 ml with 200 mM NaCl, 50 mM potassium phosphate buffer, pH 7.4. Control binding was defined as binding observed in the presence of 0.5% DMSO and the absence of steroid. Nonspecific binding was defined as binding observed in the presence of 200 µM picrotoxin and ranged from 6.1 to 14.3% of total binding. Assay tubes were incubated for 2 h at room temperature. A Brandel (Gaithersburg, MD) cell harvester was used for filtration of the assay tubes through Whatman/GF/C glass filter paper. Filter paper was rinsed with 4 ml of ice-cold buffer three times. Radioactivity bound to the filters was read by liquid scintillation counter and data were fit with Sigma Plot version 3.0 to the Hill equation
where R_max is the maximal effect, [conc] is steroid concentration, IC₅₀ is the half-maximal inhibitor concentration, and n is the Hill coefficient. Each data point was determined in triplicate and two or three full concentration-response curves were generated for each steroid.

Electrophysiological Recording. Hippocampal neurons were cultured from 1- to 2-day-old Sprague-Dawley albino rats with methods described previously (Thio et al., 1991). After 4 to 7 days in culture, neurons were voltage clamped at 60 mV with whole-cell patch-clamp recording techniques. The extracellular recording solution contained 140 mM NaCl, 4 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 10 mM glucose, pH 7.3. Recording pipettes were filled with a solution containing 140 mM CsCl, 4 mM NaCl, 0.5 mM CaCl₂, 4 mM MgCl₂, and 10 mM HEPES, pH 7.3. GABA and steroids were applied for 500 ms with a pressure (20 psi of air) ejection drug delivery system with patch pipettes positioned ~5 µm from the recorded neuron. This system allowed reliable drug application while minimizing exposure of neurons to steroids. The concentrations reported are those in the pipette and are ~2- to 3-fold higher than the concentrations bathing the cell. Steroids were prepared in a DMSO stock at 10 to 100 mM and diluted so that the final concentration of DMSO was <0.5%. DMSO at this concentration had no effect on GABA responses. Concentration-response data were fit to an equation of the form
where R_max is the maximum effect, [conc] is the steroid concentration, EC₅₀ is the half-maximal effective concentration, and n is the Hill coefficient.

Tadpole Loss of Righting Reflex (LRR) Anesthesia Assay. Tadpole LRR was measured as described previously (Wittmer et al., 1996). Briefly, groups of 10 early prelimb-bud stage Xenopus laevis tadpoles (Nasco, Fort Atkinson, WI) were placed in 100 ml of oxygenated Ringer's stock solution containing various concentrations of compound. Compounds were added from a 10 mM DMSO stock (final concentration of DMSO in test solutions was 0.1%). After equilibrating at room temperature for 3 h, tadpoles were evaluated with the LRR behavioral endpoint. LRR was defined as failure of the tadpole to right itself within 5 s after being flipped by a smooth glass rod. In all cases, the tadpoles regained their righting reflex when placed in fresh oxygenated Ringer's solution. Control beakers containing up to 0.6% DMSO produced no LRR in tadpoles. Concentration-response curves were fit with Sigma Plot version 3.0 to the Hill equation
where R_max is the maximum effect, [conc] is the steroid concentration, EC₅₀ is the half-maximal effective concentration, and n is the Hill coefficient.

Mouse Anesthesia Assay. BALB/c mice (Sprague-Dawley; Harlan Breeders, Indianapolis, IN) weighing 20 to 30 g each were placed under a heat lamp for 1 to 2 min. Compounds were injected i.v. through a tail vein with various doses of either 35P or ent-35P in an 8% ethanol, 16% Cremaphor EL (Sigma Chemical Co.) solution at a rate of 50 to 250 µl/5 to 10 s. Sleep time was measured from the moment mice displayed LRR until they were able to right themselves. All mice recovered fully without observable neurological deficits. Control solutions without steroids were administered with no observable neurobehavioral effect.

	Results

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[³⁵S]TBPS Binding. Anesthetic steroids are known to be noncompetitive displacers of [³⁵S]TBPS from the picrotoxin-binding site of GABA_A receptors (Majewska et al., 1986). In this study, the enantioselectivity for [³⁵S]TBPS displacement by four pairs of anesthetic steroid enantiomers was examined (Fig. 2). For the two 5-reduced steroid enantiomer pairs, 35P/ent-35P and 35ACN/ent-35ACN, the natural enantiomers were each ~4-fold more potent displacers of [³⁵S]TBPS. For the two 5-reduced steroid enantiomer pairs, 35P/ent-35P and 35ACN/ent-35ACN, the natural enantiomers were the more potent displacers by factors of 26 and 80, respectively. Thus, enantioselectivity for [³⁵S]TBPS displacement was found for all enantiomer pairs, but the degree of enantioselectivity was much higher for the 5-reduced steroids.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Inhibition of [35S]TBPS binding to rat brain cortical membranes by steroid enantiomers. The enantiomers of 35ACN and 35P show a larger degree of enantioselectivity than do the enantiomers of 35ACN and 35P. The figure shows a representative concentration-response curve for each steroid. The IC50 ± S.E. values reported are from the fit of data points from two or three experiments. A, the 35ACN () and ent-35ACN () show an ~4-fold difference in IC50 (72 ± 8 versus 292 ± 21 nM) for inhibition of [35S]TBPS binding. B, the 35P () and ent-35P () enantiomer pair also show an ~4-fold difference in IC50 (71 ± 18 versus 276 ± 34 nM) for inhibition of [35S]TBPS binding. C, an 80-fold difference in IC50 (50 ± 5 versus 4020 ± 310 nM) for inhibition of [35S]TBPS binding is demonstrated between 35ACN () and ent-35ACN (). D, an ~26-fold difference in IC50 (74 ± 7 versus 1910 ± 270 nM) is seen between 35P () and ent-35P ().

Steroid Effects on GABA_A Currents. The enantioselectivity found for modulation of GABA_A receptor function in cultured rat hippocampal neurons by the enantiomer pairs is shown in Figs. 3 and 4. At a steroid concentration of 10 µM, there was no enantioselectivity for potentiation of 2 µM GABA-mediated currents by the 35P/ent-35P pair (Fig. 3). In contrast, an ~4-fold enantioselectivity for the degree of potentiation under the same experimental conditions was found for the 35P/ent-35P pair (Fig. 3). This last result is in close agreement with results previously reported from a more extensive electrophysiological evaluation of GABA_A receptor modulation in these cells by the 35P/ent-35P pair (Wittmer et al., 1996).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   A, traces show the enhancement of GABA-mediated currents (2 µM; ~EC10) by 10 µM 35P and ent-35P in hippocampal neurons recorded at a holding potential of 60 mV. GABA, either alone or in the presence of the steroids, was applied for 500 ms. B, average potentiation of 2 µM GABA currents by 10 µM enantiomers of 35P (right columns). For comparison, the effects of 10 µM 35P enantiomers on currents gated by 2 µM GABA are shown (left columns). Results represent the mean ± S.E. of four to six cells per experimental condition.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Concentration-response curves for the potentiation of currents gated by 1 µM (~EC5) GABA (A) and for direct chloride-channel gating in the absence of GABA (B) by 35P () and ent-35P (). A, points represent the percentage of increase ± S.E. in GABA response produced by the steroid for three to five cells per concentration. The solid lines represent the best fit of the logistic concentration-response equation. For 35P, the maximal response, Hill coefficient, and EC50 were 1725 ± 111%, 1.0 ± 0.3 µM, and 1.2 ± 0.4 µM, respectively, whereas for ent-35P these values were 1737 ± 1%, 1.4 ± 0.1 µM, and 3.6 ± 0.1 µM, respectively. B, responses to the steroids were normalized with respect to the response obtained at 10 µM steroid and represent data (points are the mean ± S.E.) from four or five cells per concentration. The responses to 10 and 100 µM 35P were 316 ± 64 pA and 1234 ± 224 pA, respectively (n = 4 each). The responses to 10 and 100 µM ent-35P were 346 ± 53 pA and 2010 ± 254 pA, respectively, (n = 4 each). The solid lines represent best fits with maximal responses of 1429 ± 1 and 1337 ± 1%, Hill coefficients of 0.8 ± 0.01 and 1.0 ± 0.01, and EC50 values of 272 ± 1 and 125 ± 1 µM for 35P and ent-35P, respectively. For the best fits, values ± S.E. given are from the fit of the data points.

Tadpole LRR. The concentration-response relationships for the loss of tadpole LRR were determined for the 35ACN/ent-35ACN and 35P/ent-35P pairs. Little, if any, enantioselectivity was observed for these enantiomer pairs (Fig. 5). The results contrast with the significant degrees of enantioselectivity found previously (Wittmer et al., 1996) for tadpole LRR with the 35ACN/ent-35ACN and 35P/ent-35P pairs (10- and 2.8-fold, respectively).

View larger version (12K): [in this window] [in a new window]   Fig. 5.   35ACN and 35P enantiomers cause tadpole LRR but do not act enantioselectively. Points on the tadpole concentration-response curves represent mean response of 10 to 40 animals, scored quantally. A, concentration-response curves for LRR in tadpoles showing the effects of 35ACN () versus ent-35ACN (). The EC50 values ± S.E. of the fit data were 80 ± 13 and 115 ± 19 nM, respectively. B, concentration-response curves for LRR in tadpoles showing the effects of 35P () versus ent-35P (). The EC50 values ± S.E. of the fit data were 61 ± 4 and 58 ± 18 nM, respectively.

Mouse LRR. When injected into mice, both 35P and ent-35P produced anesthesia (as indicated by LRR) in a dose-dependent manner (Fig. 6). The slopes of the lines from a regression analysis of the dose-response data for this enantiomer pair differ by a factor of 2. The 35ACN/ent-35ACN pair was not tested for anesthetic activity in mice because adequate amounts of ent-35ACN were not available. These 35P/ent-35P results for mouse anesthesia are different from those obtained previously for the 35ACN/ent-35ACN pair in this bioassay (Wittmer et al., 1996). In that study, the lowest dose of 35ACN shown to cause LRR was 4 mg/kg; whereas ent-35ACN did not cause LRR at a dose as high as 80 mg/kg.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Intravenous injection of 35P () or its enantiomer ent-35P () each produced dose-dependent LRR (sleep times) in mice. Points ± S.E. on mouse dose-response curves represent the average sleep time for three or four animals and were fit to a straight line.

	Discussion

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The results from this and our previous enantioselectivity study (Wittmer et al., 1996) provide new information about the enantioselective and diastereoselective interactions of anesthetic steroids with GABA_A receptors. Enantioselective interactions can be examined by comparing the results obtained with the 35/ent-35 and 35/ent-35 steroid pairs. Diastereoselective interactions can be examined by comparing the results obtained for the 35/35 and ent-35/ent-35 steroid pairs because only the chiral center at C-5 is different in each of these steroid pairs.

As summarized in Table 1, all pairs of anesthetic steroid enantiomers studied in this and our previous study (Wittmer et al., 1996) exhibit some degree of enantioselectivity in electrophysiology, [³⁵S]TBPS binding, and when performed, the mouse anesthesia tests. Significant enantioselectivity was not found in the tadpole LRR experiments for the 5-reduced enantiomer pairs. Notably, the degree of enantioselectivity is uniformly greater across the various assays for the steroids in the 5-reduced series. The consistency of these results is remarkable considering that different species and preparations were used in the bioassays. Of course, differences in species and preparation also limit interpretation of the results. For example, the enantioselectivity for anesthetic steroid effects on TBPS binding may differ for different GABA_A receptor subtypes. Additonal studies with different subtypes of GABA_A receptors are needed to address this possibility.

In the electrophysiological experiments, the enantioselectivity is manifested in different ways for the 5- and 5-series of compounds. For potentiation of GABA-mediated currents, enantioselectivity is detected as a difference in maximal response for the steroids in the 5-reduced series, whereas it is detected as a difference in the EC₅₀ values for the 35P/ent-35P enantiomer pair in the 5-reduced series. Based on prior studies (Wittmer et al., 1996; Zorumski et al., 1998), there are likely to be significant differences in the potencies of the 5-reduced enantiomers for potentiating GABA responses, but poor solubility at concentrations 100 µM limits the ability to generate accurate concentration-response data for the ent-steroids. There is also a marked enantioselectivity difference in the gating of GABA_A receptors by the two series of steroids. The ent-steroids in the 5-reduced series do not gate these ion channels at concentrations up to 100 µM, whereas ent-35P was found to gate the ion channels at least as well as 35P over the concentration range of 1 to 100 µM.

Comparing the IC₅₀ values from the [³⁵S]TBPS binding experiments and the EC₅₀ values from tadpole LRR and electrophysiological experiments shows that the effects of the compounds occur in the same concentration range throughout these bioassays. However, the enantioselectivity for [³⁵S]TBPS displacement by the 35ACN and 35P enantiomer pairs is much greater than the enantioselectivity found for the anesthetic effects of these compounds in the tadpole LRR test. Additionally, whereas a small degree of enantioselectivity is found for the 5-reduced steroids in the [³⁵S]TBPS-binding experiments, significant enantioselectivity for these compounds was not found in the tadpole LRR test. The reasons for the failure of the tadpole LRR results to more closely correlate with those of the [³⁵S]TBPS binding experiment are not clear, although the complexity of factors involved in behavioral responses is likely to contribute.

Overall the results show that 35-, 35- and ent-35-steroids potently modulate GABA_A receptors, whereas ent-35-steroids do not effectively modulate these receptors. Enantioselectivity for GABA_A receptor modulation/anesthesia by anesthetic steroids in the 5- and 5-reduced steroid series is different (5 > 5). Because of this enantioselectivity difference, the diastereoselective interactions of the ent-35/ent-35 and 35/35 steroid pairs are also different (ent-pairs > natural pairs). These stereoselectivity results raise interesting questions. Do these stereoselectivity differences imply different mechanisms (direct versus indirect) for modulation of GABA_A receptor function and the correlated anesthetic actions of the two series of anesthetic steroids? Do these stereoselectivity differences suggest different binding sites for the two series of anesthetic steroids on GABA_A receptors? These questions are addressed in the following discussion.

Our studies of the enantioselectivity of anesthetic steroid effects on GABA_A receptor function were initiated as a way to distinguish between direct (a steroid-binding site on the receptor) and indirect (membrane perturbation) effects of these compounds on receptor function. We define direct effects as those arising from molecular interactions involving the steroid and amino acids of the receptor. We define indirect effects as those arising from molecular interactions involving the steroid and other nonprotein membrane constituents such as cholesterol and phospholipids. The degree to which enantioselectivity can be used to make the distinction between the two types of interactions is dependent on how large a difference is expected for the direct and indirect actions of the steroid on receptor function.

Binding interactions of ligands with receptors are expected to be highly enantioselective because receptor proteins are made of only L-amino acids. For substrates binding to enzymes, where both the initial binding and then a subsequent chemical reaction must occur, the expected result is complete enantioselectivity (i.e., the enzymatic transformation is expected to be enantiospecific). However, there are exceptions to the expected results for these types of interactions. For example, the binding of nornicotine to nicotinic acetylcholine receptors is not enantioselective. Both (+)- and ()-nornicotine displace ()-[³H]nicotine with equal potency and efficacy (Zhang and Nordberg, 1993; Badio and Daly, 1994; Abreo et al., 1996).

Many examples of esterases that demonstrate varying degrees of enantioselectivity for the hydrolysis of racemic esters are reported in the literature. Indeed, the effective separation of racemic esters by enzymes (one ester enantiomer is preferentially hydrolyzed and readily separated from the unreacted ester enantiomer as the corresponding alcohol enantiomer) as a method for the resolution of the optically active components may require the screening of different esterases to identify the most enantioselective esterase for a particular separation (Chen et al., 1982 and references therein). The implication of these examples for this study is that the varying degrees of enantioselectivity found for the anesthetic steroids are all consistent with a direct steroid-receptor interaction. Indeed, even if enantioselectivity had not been observed in this study, the possibility of a direct steroid-receptor interaction could not be unequivocally ruled out.

Based on information currently available, no enantioselectivity would be expected for the indirect modulation of GABA_A receptor function by anesthetic steroid enantiomers. This conclusion is based on the following reasoning and supporting evidence. Because enantiomers have identical physical properties, they alter the physical properties of a membrane (e.g., fluidity) in an identical manner unless the physical properties of the membrane are already dependent on preexisting enantiospecific interactions between membrane constituents. Most obviously, because the cholesterol and phospholipids found in cell membranes occur in enantiomerically pure form, interactions between these molecules could fulfill the preexisting enantiospecificity requirement. The extent to which the different anesthetic steroid enantiomers altered these preexisting enantiospecific cholesterol-phospholipid interactions would then determine the anesthetic steroid enantioselectivity for indirect modulation of channel function.

Is there any evidence that enantiospecific cholesterol-phospholipid interactions are determinants of membrane physical properties? Because of the difficulty involved in addressing this question (the unnatural enantiomers of cholesterol and/or a phospholipid are required for the studies), few experiments have been conducted to examine this question. However, the few available experiments (lipid monolayer and NMR studies) provide no evidence that enantiospecific cholesterol-phospholipid interactions have any effect on the physical properties of the membrane (Ghosh et al., 1971; Arnett and Gold, 1982). Thus, we have referred previously to the membrane as a nonchiral environment and we have concluded that no enantioselectivity for anesthetic steroid action is expected if the effects of these compounds are indirectly caused by membrane perturbation (Wittmer et al., 1996). Realizing that this conclusion is based on very limited experimental data, we recently reported a new method for preparing the unnatural enantiomer of cholesterol (Kumar and Covey, 1999) and we plan to address the biological significance of enantiospecific interactions between cholesterol and different classes of phospholipids in future studies. Because, in this study, we find some degree of enantioselectivity for both series of anesthetic steroids, we find no compelling reasons to conclude that either series of anesthetic steroids modulates GABA_A receptor function by an indirect mechanism.

Whether the different degrees of enantioselectivity imply different receptor-binding sites for the two series of anesthetic steroids is the final question to address. Although there are data from previous studies suggesting the existence of more than one class of binding sites for some steroids on GABA_A receptors (Morrow et al., 1990; Hawkinson et al., 1994a,b, 1998), the enantioselectivity results we have obtained thus far present no new evidence for this phenomenon. Previous data from studies of the diastereoselective modulation of GABA_A receptor function by 35P and 35P have been reasonably explained by a common binding-site hypothesis developed from molecular modeling studies (Purdy et al., 1990; Han et al., 1996). The results from this and the previous enantioselectivity study of 5-reduced anesthetic steroids (Wittmer et al., 1996) establish new criteria that must be satisfied by molecular models of a common binding site for 5- and 5-reduced anesthetic steroids. Molecular models of a common binding site for both classes of anesthetic steroids also must be able to explain the greater enantioselectivity of 5-reduced anesthetic steroids and the greater diastereoselectivity observed for the ent-35P/ent-35P steroid pair versus the 35P/35P steroid pair.