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Accelerated Communication

Enantiomers of Neuroactive Steroids Support a Specific Interaction with the GABA-C Receptor as the Mechanism of Steroid Action

Departments of Anesthesiology (W.L., J.H.S.) and Molecular Biology and Pharmacology (D.F.C.), Washington University School of Medicine, St. Louis, Missouri; and Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine/Biochemistry, University of Helsinki, Finland (J.-M.A., P.K.J.K.)

Received January 23, 2006; accepted March 9, 2006

	Abstract

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Neuroactive steroids can either potentiate or inhibit a variety of membrane channels. Most studies have suggested that the effects are mediated by specific association of the steroid with the affected channel. However, a recent study of the 1 (GABA-C) receptor (Mol Pharmacol 66:56-69, 2004) concluded that the actions were consistent with an action of the steroid in the lipid bilayer to alter the lateral pressure profile in the membrane. The enantiomers of an optically active compound are expected to have identical physical properties, including interactions with hydrophobic portions of the cell membrane. We have used two pairs of enantiomers (pregnanolone and ent-pregnanolone, allopregnanolone and ent-allopregnanolone) and show that the ability to potentiate (allopregnanolone) or inhibit (pregnanolone) the 1 receptor is enantioselective. Therefore, these results strongly suggest that the actions of these neuroactive steroids are mediated by interactions with chiral regions of the target protein, rather than by a change in membrane properties (including lateral pressure).

A recent study (Morris and Amin, 2004) of the actions of pregnanolone [35P; (3,5)-3-hydroxypregnan-20-one] and allopregnanolone [35P; (3,5)-3-hydroxypregnan-20-one] on the 1 GABA-C receptor concluded that the actions of these neuroactive steroids were most consistent with the idea that the steroids altered the lateral pressure profile in the membrane (Cantor, 1997). 35P potentiates the receptor response to low concentrations of GABA, whereas 35P inhibits (Morris et al., 1999, Goutman and Calvo, 2004).

We examined the actions of the natural and unnatural forms of these steroids on the responses of 1 receptors (the unnatural form is indicated by the prefix ent-; for example, ent-35P). We confirmed the findings that 35P inhibits the response to low concentrations of GABA, whereas 35P potentiates the receptor response. In contrast, the enantiomers of the natural steroids are much less effective at either potentiating (35P) or inhibiting (35P) the activation of the 1 receptor.

We also examined the biophysical interactions of each enantiomer pair with natural and artificial membranes (J.-M. Alakoskela, D. F. Covey, and P. K. J. Kinnunen, submitted). 35P and ent-35P show no difference in effects on a variety of measures, including packing of the interior or headgroups, mobility of hydrocarbon chains, phase transitions, or association of the steroid with headgroup or interior region of the leaflet. Likewise, 35P and ent-35P show no differences in any measured parameter. The data indicate that the enantiomers have identical interactions with membranes for all the parameters examined. As expected, 35P and 35P are clearly distinct from each other in their biophysical effects on membranes. Therefore, although 35P and 35P clearly differ from each other in terms of effects on membrane properties, there is no indication that the two enantiomers of a given steroid (that is, 35P and ent-35P or 35P and ent-35P) do.

These data do not support the idea that steroids act on the 1 receptor by affecting properties of the lipid bilayer. Rather, they are more consistent with the idea that the steroids interact with specific sites, probably on the receptor protein, that recognize specific structural features of the steroid.

	Materials and Methods

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Experiments were conducted using two-electrode voltage clamp of Xenopus laevis oocytes, as described previously (Steinbach et al., 2000; Paradiso et al., 2001). A full-length construct of the human 1 receptor was provided by D. Weiss (University of Texas Health Center, San Antonio, TX; see Amin and Weiss, 1996), and transferred to pcDNA3 (Invitrogen, Carlsbad, CA). The construct was sequenced and found to correspond to the published sequence (Gen-Bank accession number M62400 [GenBank] ). cRNA was synthesized using mMessage mMachine T7 kit (Ambion, Austin, TX). Eight nanograms were injected into each oocyte in 23 nl. Oocytes were incubated for 40 to 60 h at 18°C before being studied electrophysiologically.

35P and 35P were purchased from Sigma (St. Louis, MO), and enantiomers were prepared as described previously (Hu et al., 1997; Nilsson et al., 1998). Steroids were dissolved in DMSO at a concentration of 10 mM, then diluted to the appropriate final concentration in recording saline. The maximal final concentration used was 20 µM to avoid problems with steroid solubility. Solutions were applied using glass reservoirs and fluorocarbon or metal tubing to reduce adsorption.

As reported previously (Morris et al., 1999; Goutman and Calvo, 2004), the effects of steroids reversed very slowly. Because we were concerned about time-dependent changes in control responses during the protracted washes, most experiments were performed by paired applications. At first, 2 applications of GABA alone (200 nM) were applied to measure control responses. Then GABA (200 nM) was applied with 10 µM steroid (either the native or unnatural form). A second control application was then made, followed by GABA with the other member of the enantiomeric pair. A final control application was then made. Responses were measured from the peak (average current for an approximately 5-s interval centered at the peak) to the baseline (average current for an approximately 5-s interval before the response). The effect of steroid was measured by the relative response in the presence of steroid to the control response preceding the application of steroid. All applications were separated by at least 3 min. The order of application (natural or unnatural) was switched between eggs. Subsequent analysis of the effects of the steroids on responses showed that there was no effect of order of application—that is, the relative potentiation of 35P was the same irrespective of whether it was applied before or after ent-35P. The effects of the enantiomeric forms were then compared for each oocyte. Steroids were tested on six separate sets of oocytes injected with 1 cRNA. The maximal concentration of DMSO used was 0.2%. When this concentration of DMSO was applied (without steroid), it had no effect on responses to 200 nM GABA (relative response 0.97 ± 0.05; three oocytes tested). All results are reported as mean ± S.D. (number of observations).

	Results

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The responses of the injected oocytes to GABA alone were similar to those reported previously (Amin and Weiss, 1996; Morris et al., 1999; Goutman and Calvo, 2004). The concentration-response relationship is described by the Hill equation with an EC₅₀ value of 700 ± 70 nM and a Hill coefficient of 2.0 ± 0.2 (mean ± S.D. of fits to curves from five oocytes). A GABA concentration of 200 nM was chosen for studies of steroids because it corresponded to the concentration required to elicit a response of approximately 10% of the maximum response (Morris and Amin, 2004). (The predicted response to 200 nM is 6.4% of maximum.)

The essential results are shown in Fig. 1. This figure shows the responses of an oocyte expressing the 1 receptor to 200 nM GABA, GABA plus 10 µM 35P, and GABA plus 10 µM ent-35P (Fig. 1A). Note that 35P blocks the response, whereas ent-35P seems to potentiate. Similar data are shown for responses of a different oocyte to 10 µM35P and ent-35P (Fig. 1D). In this case, the enantiomer has no effect, whereas the natural form potentiates. The concentration of 10 µM steroid was chosen because it has been reported to be maximally effective at inhibiting and close to maximal at potentiating responses (Morris et al., 1999; see also Fig. 2). In all, paired responses were obtained from eight oocytes for 35P and ent-35P, and nine oocytes for 35P and ent-35P. In every oocyte tested, ent-35P potentiated less than 35P whereas ent-35P inhibited less than 35P (and, actually, ent-35P potentiated responses). If we assume that the natural and unnatural forms are equally effective and potent, then this result would be obtained by chance in less than 0.4% of sets of eight pairs and 0.2% for nine pairs. Therefore, we conclude that the natural and unnatural forms are not equivalent in actions on the 1 receptor. Likewise, in five pairs of oocytes tested with 1 µM steroids and five other pairs tested with 20 µM steroids, the enantiomer always produced less potentiation (35P) or less inhibition (35P), which would happen in fewer than 4% of pairs if the enantiomer was equivalent to the natural steroid.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Responses of oocytes to GABA and enantiomer pairs. Top left (A), response of an oocyte to 200 nM GABA (solid line), then to 200 nM GABA plus 10 µM ent-35P (dashed line), then to 200 nM GABA plus 10 µM 35P (dot-dashed line). Note that the natural enantiomer inhibits the response, whereas the unnatural form potentiates. Top right (D), shows the responses of a different oocyte to 200 nM GABA, then to 200 nM GABA plus 10 µM ent-35P (dashed line), then to 200 nM GABA plus 10 µM 35P (dot-dashed line). In this case, the natural form potentiates whereas the unnatural form has no effect. B and C, structures of 35P as a flat structure and in a three-dimensional view; E and F, similar structures for 35P.

View larger version (9K): [in this window] [in a new window]   Fig. 2. Summary of effects of steroids on responses to 200 nM GABA. Left, summary of the effects of 35P (filled symbols) and its enantiomer (open symbols) on the response to 200 nM GABA. An asterisk indicates that the relative response is significantly different from 1 (P < 0.05, t test). The relative responses to 35P and ent-35P are significantly different from each other at each concentration tested. The data were obtained from five oocytes (1 µM), nine oocytes (10 µM), and five oocytes (20 µM). Right, summary of similar data for the effects of 35P (filled symbols) and its enantiomer (open symbols). The relative responses to 35P and ent-35P are significantly different from each other at all concentration tested. The data were obtained from five oocytes (1 µM), eight oocytes (10 µM) and five oocytes (20 µM).

The paired comparisons were used because of concerns about slow reversibility of steroid effects (see Materials and Methods). Therefore, the quantitative values for potentiation and inhibition are somewhat less critical for testing the hypothesis. However, the differences are clearly apparent in the parametric data. The concentration-effect relationships for the natural and unnatural forms of the steroids are shown in Fig. 2. The potentiation curve for 35P is very similar to that reported earlier (Morris et al., 1999), whereas ent-35P shows essentially no effect over the same concentration range. The inhibition curve for 35P is also very similar to that reported earlier (Morris et al., 1999). However, ent-35P shows a potentiating, rather than inhibiting, action at higher concentrations. For neither steroid pair does the unnatural form show similar effects to the natural form.

In studies of neuroactive steroids at other transmittergated channels, the inhibiting and potentiating effects seem to be independent (e.g., Akk et al., 2001; Paradiso et al., 2001). Therefore, we performed some experiments to indicate whether 35P and 35P seem to have independent (that is, multiplicative) actions on the 1 receptor. To do this, we applied 200 nM GABA plus 10 µM 35P for approximately 100 s, then switched to 200 nM GABA plus 10 µM35P plus 10 µM 35P. As shown in Fig. 3, a rapid inhibition occurs. The amount of inhibition was assessed by comparing the response at the maximal inhibition to the response immediately before the blocker was applied. The amount of inhibition for the potentiated response (response reduced to 0.38 ± 0.04, results from three oocytes) is indistinguishable from that obtained for responses to GABA alone (0.39 ± 0.08, n = 9 oocytes; Fig. 2). The response just before the application of 35P was potentiated to an average of 1.47 (±0.08, n = 3) times the control response to 200 nM GABA, measured at the same time in the application, which is similar to the data shown in Fig. 2 (1.41 ± 0.17, n = 8). It is difficult to evaluate the predictions of the lateral pressure mechanism when a mixture of steroids is applied. However, the data suggest that the potentiating effects of 35P and the inhibiting effects of 35P have independent mechanisms, and the net effect is produced by the multiplication of the potentiation and inhibition. We also performed similar experiments using enantiomer pairs. 35P (10 µM) blocks responses to the application of 200 nM GABA plus 10 µM ent-35P (relative response in the presence of 35P was reduced to 0.36 ± 0.03) whereas 10 µM ent-35P has little effect on responses potentiated by 35P (relative response 0.91 ± 0.05).

View larger version (8K): [in this window] [in a new window]   Fig. 3. 35P inhibits responses potentiated by 35P. GABA (200 nM) plus 10 µM 35P was applied to an oocyte for approximately 100 s; then the solution was changed to 200 nM GABA plus 10 µM35P plus 10 µM 35P for approximately 50 s, and then the solution was switched back to 200 nM GABA plus 10 µM 35P for approximately 20 s. 35P shows a rapid inhibitory effect, which is only partially reversed. On average, the response in the presence of 35P is potentiated by 1.47-fold, whereas the addition of 35P reduces the potentiated response to 0.38 ± 0.04.

	Discussion

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In a full study (J.-M. Alakoskela, D. F. Covey, and P. K. J. Kinnunen, submitted) of the interactions of 35P, 35P, and their enantiomers with lipid membranes, the members of an enantiomer pair showed no difference in effects on a variety of measures, including packing of the interior or headgroups, mobility of hydrocarbon chains, hydration of the headgroup region, phase transitions, surface potential, and association of the steroid with headgroup or interior region of the leaflet. In contrast, the 5- and 5-reduced steroids differ in most of the measures, as expected. These data strongly suggest that the enantiomers of a given steroid interact identically with the membrane.

No technique can directly measure the lateral pressure profile, which can only be calculated or simulated. However, its integral moments, the first of which is related to the splay curvature elastic modulus and spontaneous curvature, and the second to the Gaussian curvature elastic modulus (Cantor, 1999), can both be calculated from simulations (Gullingsrud and Schulten, 2004) and can be measured as well. We did not directly measure these parameters. In addition, simulations of the behavior of cholesterol-containing bilayers suggest that the lateral pressure profile may be very complex, having an alternating array of very strong tension (contracting) and pressure (expanding) components (Patra, 2005). It is not possible to predict how a membrane protein, of irregular shape and presently unknown volume changes during gating, would interact with such a spatially complex pressure distribution. Hence, even the measurement of the first two integral moments could not confirm the pressure profiles to this level of detail. Therefore, the possibility exists that the members of an enantiomer pair could have different effects on the lateral pressure profile even though they have identical effects on all the parameters we measured.

Subject to that caveat, the present results indicate that these neurosteroids, and probably other steroids, act on the 1 receptor by interacting with a chiral site, probably on the receptor protein. It is somewhat surprising that ent-35P acts as a potentiator. If the potentiation and inhibition are mediated by interactions at specific sites, this observation suggests that ent-35P might have some efficacy at the site for potentiation. Indeed, previous studies of the GABA-A receptor have found that the enantiomers of 5-reduced steroids retain more ability to potentiate than do enantiomers of 5-reduced steroids (Covey et al., 2000). Our results also suggest that 35P and 35P have independent actions to (respectively) inhibit and potentiate responses of 1 receptors. It seems less likely that a mechanism mediated by effects on lateral pressure would show such a simple interaction as more steroid is incorporated into the membrane.

In any case, the strong enantioselectivity for both 35P and 35P suggests that an action in the membrane to change a membrane property, such as lateral pressure, is less likely than a direct interaction with a chiral site, probably on the 1 receptor.

	Acknowledgements

 
We thank John Bracamontes for advice on the molecular biology and Steven Mennerick and Charles F. Zorumski (Department of Psychiatry, Washington University School of Medicine, St. Louis, MO) for providing oocytes.

	Footnotes

 
This work was supported by National Institutes of Health grant P01-GM47969 (to D.F.C. and J.H.S.). J.H.S. is the Russell and Mary Shelden Professor of Anesthesiology.

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

doi:10.1124/mol.106.022863.

ABBREVIATIONS: 35P, allopregnanolone ((3,5)-3-hydroxypregnan-20-one); 35P, pregnanolone ((3,5)-3-hydroxypregnan-20-one); DMSO, dimethyl sulfoxide.

Address correspondence to: Joe Henry Steinbach, Department of Anesthesiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: jhs{at}wustl.edu

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