Vol. 4, 759-765,
May, 1990, Copyright
0 1990 by Cell Press
Neurosteroids Act on Recombinant Human GABAA Receptors Giulia Puia: MariaRita Santi: Stefano Vicini: Dolan B. Pritchett,+ Robert H. Purdy,* Steven M. Paul,5 Peter H. Seeburg,+ and Erminio Costa* * Fidia-Georgetown Institute for the Georgetown University Washington D. C. 20007 +Center for Molecular Biology University of Heidelberg, INF 282 D6900 Heidelberg Federal Republic of Germany *Department of Organic Chemistry Southwest Foundation for Biomedical San Antonio, Texas 78284 JClinical Neuroscience Branch National Institute of Mental Health Bethesda, Maryland 20892
Summary The endogenous steroid metabolites 3aJldihydroxy-5apregnan-20-one and 3a-hydroxyda-pregnan-20-one potentiate GABA-activated Cl- currents recorded from a human cell line transfected with the (3,, a,&, and alPly, combinations of human CABA* receptor subunits. These steroids are active at nanomolar concentrations in potentiating GABA-activated Cl- currents and directly elicit bicuculline-sensitive Cl- currents when applied at micromolar concentrations. The potentiating and direct actions of both steroids were expressed with every combination of subunits tested. However, an examination of single-channel currents recorded from outside-out patches excised from these transfected cells suggests that despite the common minimal structural requirements for expressing steroid and barbiturate actions, the mechanism of CABA* receptor modulation by these pregnane steroids may differ from that of barbiturates. Introduction The 3a-hydroxy ring A-reduced metabolites of progesterone and deoxycorticosterone have been shown to interact potently with the GABAA receptor-coupied Cl- ion channel (Majewska et al., 1986; Gee et al., 1988; Harrison et al., 1987; Morrow et al., 1987; Peters et al., 1988). For example, 3a-hydroxy-5a-pregnan-20-one (allopregnanolone or 3a-OH-DHP) and 3a,2ldihydroxy-5a-pregnan-20-one (allotetrahydrodeoxycorticosterone or THDOC) compete with the high affinity binding of [%]i-bufylbicyclo-phosphorothionate (TPBS) to GABAA receptor-associated Cl- channels; and their apparent affinities are greatly enhanced in the presence of CABA (Gee et al., 1988). Both steroids potentiate GABA-activated Cl- currents recorded in rat hippocampal neurons in primary cul-
ture (Majewska et al., 1986; Harrison et al., 1987) as well as CABAA receptor-mediated 36CI- ion uptake in rat cerebral cortical synaptoneurosomes (Morrow et al., 1987, 1990). Behavioral studies have demonstrated that THDOC, like benzodiazepines and barbiturates, possesses anticonflict and hypnotic activity upon parenteral administration to rats (Crawley et al., 1986; Mendelson et al., 1987). These data suggest that one or both of these naturally occurring steroid metabolites may function in the regulation of various physiological states, including sleep and anxiety. Recently, on the basis of receptor binding and electrophysiological studies, it has been suggested that the GABA receptor-active pregnane steroids and barbiturates act at different sites on the GABAA receptor complex (Callathan et al., 1987; Gee et al., 1988). Moreover, the presence of multiple steroid binding sites has been proposed on the basis of the shallow and complex (biphasic) concentration-response curves observed for their potentiation of GABAA receptor-mediated 36CIuptake (Morrow et al., 1990). The availability of cDNA clones for various subunits of the GABAA receptor complex (Schofield et al., 1987; Pritchett et al.,l989) has prompted studies of various molecular forms of GABA* receptors by transient expression of these cDNAs in human embryonic kidney cells (293 cell line; Pritchett et al., 1988). Characterization of these GABAA receptors, assembled from different subunits, has revealed the minimal structural requirements for the actions of various compounds previously known to affect this receptor. While GABA, bicuculline, and barbiturates act on receptors formed from only a or fi subunits (Pritchett et al., 1988; Levitan et al., 1988), the coexpression of these subunits with they subunit is required for the positive or negative allosteric modulation of GABAevoked Cl- currents through the GABAJbenzodiazepine receptor (Pritchett et al.,l989). In the present study we have investigated the effects of 3a-OH-DHP and THDOC on GABA* receptor-mediated Cl- ion conductance using various molecular forms of recombinantly expressed GABAA receptors. We have observed that 3a-OH-DHP is more efficacious than flunitrazepam in potentiating GABA responses on the GABAA receptor assembly that includes aI, B1, and y2 subunits. Moreover, while the potentiation of GABA responses by these steroids was observed with all subunit combinations examined, potentiation of GABA by the benzodiazepine was not observed in receptors lacking the y2 subunit, as previously reported (Pritchett et al., 1988). Finally, a comparison of the effects of these steroids with those of pentobarbital, on single-channel currents, suggests that while both increase GABA-gated Clchannel activity, they apparently do so by different mechanisms. Thus, pregnane steroids appear to constitute
a unique class of endogenous with intrinsic efficacy equal benzodiazepines.
GABA* receptor to or greater than
ligands that of
Results Steroid Hormones Potentiate GABA-Activated Cl- Currents in Transfected Cells GABA activates inward Cl- currents in cultured human cells transfected with various combinations of cDNAs encoding the a,, P,, and y2 GABAA receptor subunits (Figure 1). These currents revert at 0 mV with the ionic condition chosen. CABA fails to activate CIcurrents in nontransfected cells. At nanomolar concentrations, the endogenous steroid metabolites 3aOH-DHP and THDOC potentiate GABA-activated CIcurrents in voltage-clamped cells expressing different subunits and their combinations (Figure 1). The potency of the two steroids is similar (Figure 2), and neither appears to act preferentially on a given GABAA receptor assembly (Figure 2), including the homooligomeric fi, combination. In contrast to flunitrazepam, the potentiation of GABA-activated Cl- conductance by 3a-OH-DHP over a wide range of concentrations
Figure 2. Dose-Dependent Steroids of GA&Activated Transfected with Different tions
Each data point is the average dishes (n = 121.
c Pi MDOC
Figure 7. Inward Cl- Currents Induced by CABA lontophoretic Application (30 ms, 100 nA) on a,&y,, a& or B1 Receptor Subunits in Transfected Mammalian Cells THDOC (100 nM) potentiates these Cl- currents with any of the CABA receptor subunits tested, even with the homooligomeric B, combination. The cells were held at -50 mV
Potentiation Induced by Pregnane Cl- Currents in Mammalian Cells CABA Receptor Subunit Combinaof 3 cells
appears to be biphasic. This may be due to the ability of steroids to activate Cl- channels directly at high concentrations (see Figure 4) and to augment GABA actions at lower concentrations (Figure 2). Alternatively, these steroids may interact with multiple binding sites to augment GABA-activated Cl- ion conductance. This biphasic action of the pregnane steroids in potentiating GABA-activated Cl- currents is also observed with homooligomeric fi, subunit-containing GABAA receptors. This latter receptor fails to be modulated by benzodiazepines, as shown previously (Pritchett et al., 1989). The coexpression of al, Pl, and y2 GABAA receptor subunits results in receptors that are sensitive to the augmenting actions of benzodiazepines. In Figure 3, a direct comparison between the effects of various concentrations of 3a-OH-DHP and flunitrazepam on GABA-activated Clcurrents is illustrated. 3a-OHDHP is more efficacious than flunitrazepam, whereas the inactive enantiomer 3P-OH-DHP (3B-hydroxy-5apregnan-20-one) fails to enhance CABA-activated Clcurrents, except to a small degree at micromolar concentrations, supporting a previous report on the stereoselective action of these steroids.
io cLM !
Vh -40 I
Figure 3. A Comparison between the Dose-Dependent Potentiation Induced by3a-OH-DHP, 3b-OH-DHP, and Flunitrazepam on the CABA-Evoked Cl- Currents in Cells Transfected with the c&yz Receptor Subunits Each data
Steroid Hormones Activate Cl- Currents in Transfected in the Absence of CABA
At higher (micromolar) concentrations, 3a-OH-DHP and THDOC alone activate inward Cl- currents in transfected cultured cells (Figure4). These inward currents revert at a holding potential of 0 mV and are sensitive to bicuculline methiodide (Figure 4A), indicating that they are due to the direct activation of the GABAA receptor Cl- channel complex. This finding suggests that there are at least two sites of action of the steroids, each having a different threshold. We tested 3a-OH-DHP and THDOC at concentrations between IO-’ and 10m5 M on cells expressing different combination of al, PI, and y2 receptor subunits and observed activation of Cl- currents only at steroid concentrations of lO-‘j M or higher. Figure 4B summarizes the results obtained at steroid concentrations of IO-5 M on various molecular forms of GABAA receptor.
THDOC IO C~
The presence of electrical coupling in transfected 293 cells has been inferred from the finding that GABA responses are often greater in cells adjacent to those being recorded from (Pritchett et al., 1988). This feature facilitates finding cells that transiently express GABAA receptor subunits. The patch-clamp, single-channel recording technique (Hamill et al., 1981) was used in the outside-out configuration, with symmetrical Cl- concentrations on both sides of the membrane patch and the Cl- equilibrium potential close to 0 mV. The GABA-activated, inward single-channel current reverted at 0 mV and displayed a conductance of 30 pS for the al&y2 combination and 20 pS for either the [3, or the a& receptor assembly. This is in agreement with a previous report by Verdoorn et al. (1989,
al/31 Figure Cells
(A) 3a-OH-DHP (IO PM) elicits inward currents in cells transfected with a&y, being antagonized by bicuculline methiodide (BMI, IO PM). (B) A comparison between the direct action of 3a-OH-DHP (‘IO PM) and THDOC (IO PM) on different GABA receptor subunits combination (IO cells per data point).
Sot. Neurosci., abstract). In patches derived from the a&y, subunit combination, channel openings having lower conductance states were also observed, possibly because of the simultaneous expression in the same patch of channels with and without the y2 subunit (Verdoorn et al., 1989, Sot. Neurosci., abstract). In the present study, we discarded patches containing more than 5% of these low conductance openings. In Figure 5A, the channel activity of an outside-out membrane patch excised from cells expressing the a&y, subunits combination is shown. 3a-OH-DHP or pentobarbital in combination with GABA was pressure-applied from micropipettes (5-10 pm tip diameter) placed in proximity to the membrane patch. Both the steroid and the pentobarbital increased the GABA-gated Cl- channel activity (Figure 5A), being the product of the number of active channels and the channel open probability, but apparently by different mechanisms, as can be seen in Figure 5B. Fluctuation analysis of the channel current records (Figure 58; see also Table I) reveals that the power spectra obtained in these conditions are best fitted by two lorentzian components. The slow component of these power spectra is a reflection of the length of
Figure 5. Single-Channel Currents Recorded in Outside-Out Patches Excised from u,&y, Transfected Cells (A) Channel currents evoked by GABA alone (1 PM, left) or in combination with 3a-OH-DHP (100 nM, center) or pentobarbitol (50 PM, right). (B) Power spectra derived from fluctuation analysis of CABA-activated channel currents in outside-out patches records represented in part in (A).
channel opening bursts and is similar for channels activated by GABA alone or by the association of GABA with 3a-OH-DHP. Conversely, the slow time constant derived from spectra obtained with GABA combined with pentobarbital is significantly increased (Figure 5B), indicating an increase in the duration of channel opening bursts, as previously reported (Study and Barker, 1981). Since the corner frequency corresponding to the slow time constant of the spectra is in the plateau region of the experimental data, incorrect estimates of these data can occur. Moreover, a slower sampling rate will produce errors in the estimate of the fast time constant. Thus our results on the increase of the slow time constant induced by barbiturates suggest, but do not prove, that there is a difference in the mechanisms of enhancement by barbiturates compared with neurosteroids. When the outside-out patches were excised from transfected cells expressing a#, subunit, 3a-OH-DHP or pentobarbital, in combination with GABA, also increased
CABA-gated Cl- channel activity (Figure 6A). Fluctuation analysis of the channel current records (Figure 68; see also Table 1) also reveals that the power spectra obtained are best fitted by two lorentzian components. The slow component of these spectra, however, contributes minimally to the total amplitude. Cells expressing the a& subunit combination may contain channels with different kinetic properties. However, we cannot rule out the presence of channel openings that occur faster than the resolution of our measurement. Nevertheless, 3a-OH-DHP fails to affect these time constants, while pentobarbital significantly increases them (Table I), further suggesting a difference in the mechanisms of action of neurosteroids and barbiturates. Discussion Molecular tor subunits
cloning of cDNAs encoding (Schofield et al., 1987;
Table 1. Power Spectrum Time Constants Derived From Fluctuation Analysis of GABA-Activated Cl- Channel Currents from Reconstituted Receptors in the Presence of Pentobarbital and 3a-OH-DHP
a#772 CABA + 3a-OH-DHP + PB
1.1 f 0.6 1.2 + 0.8 1.8 f 0.9
11.5 & 1.8 12.7 f 2.6 28.1 f 3.5
23k13 20&9 32+15
5 6 3
1.3 f 0.9 1.4 + 0.7 4.2 f 1.2
10.5 & 1.5 11.6 + 1.6 18.9 + 3.7
5+2 4*3 12+3
6 5 3
a& GABA + 3a-OH-DHP f PB rr and rr are fast and cent slow components pentobarbital.
slow time constants in ms; %S is the perof the power spectra, +SD of n cells. PB,
1989) has facilitated a more complete characterization of the minimal structural requirements forthe actions of GABA, bicuculline, picrotoxin, barbiturates, and benzodiazepines at GABAA receptors (Pritchett et al.,
1988, 1989; Levitan et al., 1988). Coexpression of a or 8 subunits with ay subunit is required for the positive or negative allosteric modulation of GABA-evoked Cl- currents by either benzodiazepines or b-carboline derivatives (Pritchett et al., 1989), while barbiturates can act on homooligomeric a or 8 receptors (Pritchett et al., 1988). In the present study we have demonstrated that at low (nanomolar) concentrations, two endogenous steroid metabolites, 3a-OH-DHP and THDOC, produce a potentiation of GABA-evoked Cl- currents in cells transfected with GABA, receptor subunit cDNAs. At higher (micromolar) concentrations, these steroids have a direct action on GABA* receptor-mediated Cl- conductance. It is noteworthy that the efficacy of these steroids for facilitating GABA-evoked Cl- currents is greater than that of the relatively potent benzodiazepine flunitrazepam, using the a&y2 molecular configuration of the CABAA receptor. The concentration-response curves for 3a-OHDHP-mediated augmentation of CABA-activated Cl-
4.4 rf ’ 7s 20.1 %S 10 0.1; *
(A) Channel currents evoked by GABA alone (1 nM, right). (8) Power spectra derived from fluctuation analysis part in (A).
left) or in combination of GABA-activated
Excised with channel
Transfected (100 nM, in outside-out
ms -Y:( . ms “;. . .. ‘., \ o k. ___
Cells center) patch
or pentobarbital records
currents are biphasic in each molecular configuration of GABAA receptors investigated. This finding is reminiscent of the biphasic concentration-response curves for steroid and barbiturates in the facilitation of GABAA receptor-mediated 36CI- uptake studied in synaptoneurosomes (Morrow et al., 1987, 1990). We also observed the reported stereoselectivity for the facilitatory and biphasic action of 3a-OH-DHP on GABA*-activated Cl- currents (Morrow et al., 1990). Taken together, these data suggest the presence of two distinct steroid binding sites on the GABAA receptor complex. One of these binding sites may be responsible for facilitation of GABA action, its efficacy remaining unchanged whether or not the y2 subunit is expressed. The other binding site may be responsible for the direct chan’nel opening properties of these steroids observed in the absence of GABA, since these responses are inhibited by bicuculline, an isosteric GABAA receptor antagonist. Since the direct action of these steroids was tested in the absence of GABA, we infer that these steroids may elicit this action in the vicinity of the GABA recognition site. The same site may mediate the direct action of these steroids iv stimulating s6CIuptake in subcellular brain preparation observed at higher steroid concentrations (Majewska et al., 1986; Callachan et al., 1987). However, other possibilities may also be operative. In cultured murine neurons, GABA activates a single Cl- channel with a main conductance state of 30 pS and with sequential closed and open states typical of bursting behavior (Hamill et al., 1983; Bormann and Clapham 1985; Vicini et al., 1987; MacDonald et al., 1989). In cultured neurons, barbiturates increase the open probability of GABA-activated, single-channel currents, promoting bursts of channel openings (Study and Barker, 1981; Mathers, 1985; Mienville and Vicini, 1989; MacDonald et al., 1989). Similarly, pregnanolone (3a-hydroxy-5P-pregnan-2(rone) potently increases the burst durations of GABA-activated, single-channel currents recorded from bovine chromaffin cells (Challachan et al., 1987). GABA activates mostly 30 pS CIchannels in outside-out patches excised from the 293 cells expressing the alPly, subunit combinations of the GABA* receptor (Verdoorn et al., 1989, Sot. Neurosci., abstract). The absence of the y2 subunit results in the expression of channels with mainly a 20 pS conductance state (Verdoorn et al., 1989, Sot. Neurosci., abstract). In the present study, we failed to observe an increase of burst duration produced by 3a-OH-DHP on GABA-activated Cl- channel currents recorded from outside-out patches excised from cells expressing both a,& and al&y, subunits, in contrast with the apparent action of pentobarbital on these same patches. Thus, despite the technical limitations of our measurements, it appears that the actions of 3a-OHDHP on GABA-gated Cl- channel may be primarily due to an increase in channel opening frequency, which is similar to that previously reported for benzodiazepines (Study and Barker, 1981; Vicini et al.,
1987; Rogers et al., 1989, Sot. Neurosci., abstract). This would therefore indicate that the mechanisms of action of these endogenous steroids and barbiturates are quite different, despite their common minimal structural requirements and the similarity in the shape of their concentration-response curves. This observation may relate to the reported differences between the steroid and barbiturates recognition sites (Turner et al., 1989) on the GABAA receptor complex. Furthermore, unlike benzodiazepines, barbiturates induce REM sleep suppression in rodents, whereas THDOC has minimal to no effects on REM sleep and produces an overall hypnotic effect (Mendelson et al., 1987). Taken together with previous data (Majewska et al., 1986; Gee et al., 1988; Harrison et al., 1987; Morrow et al., 1987,199O; Peters et al., 1988), our findings support the hypothesis that these endogenous 3a-hydroxy ring A-reduced steroid metabolites interact with GABA* receptors at unique recognition site and affect channel activity in a manner different from both benzodiazepines and barbiturates. Given the high affinity and potent efficacy of these steroids, their role as modulators of GABAA receptor-mediated Cl- ion conductance should prove to be of significant physiological interest. Experimental
Cultures from Kidney Embryonic Cell Line and cDNA Transfection Transformed human embryonic kidney cells 293 (ATCC #CRL 1573)weregrown in minimum essential medium (MEM; GIBCO) supplemented with 10% fetal bovine serum, 100 U/ml penicillin (GIBCO), and 100 U/ml streptomycin (GIBCO) in a 6% CC& incubator. Exponentially growing ceils were ttypsinized and seeded at 2 x IO5 cells per 35 mm dish in 2 ml of growth medium. The transfection was performed using the calcium phosphate precipitation technique (Chen and Okayama, 1987). The cloned human GABAA receptor a,, PI, and y2 subunit cDNAs (Pritchett et al., 1989; Schofield et al., 1987), inserted singly or together into a eukaryotic expression vector, pCIS2 (Pritchett et al., 1988), were used to perform the transfection. The cells were incubated in the presence (3 pg per 35 mm dish) of one or two supercoiled plasmids for 12-16 hr at 37OC under 3% CO>. The medium was removed, and the cells were rinsed twice with growth medium, refed, and incubated in the same medium for 24 hr at 37°C under 6% COz before electrophysiological studies. Whole-Cell Recordings Employing the single-electrode, voltage-clamp technique in the whole-cell configuration (Hamill et al., 1981), CABA-activated Cl- currents were studied from transfected cells (24 hr after transfection). The recording pipette contained 145 mM CsCI, 1 mM MgCi*, 11 mM EGTA, and IO mM HEPES-CsOH (pH 7.2). Cells were bathed in 145 mM NaCI, 5 mM KCI, 2 mM CaCI,, 5 mM HEPES-NaOH (pH 74). Osmolarity was adjusted to 325 mOsm with sucrose. CABA (0.5 M in H,O, pH 4 with HCI) was applied by iontophoresis using 30 ms pulses of positive current. Using CABA iontophoretic currents in the 50-100 nA range, inward currents were generated in neurons in such a way as to obtain a peak amplitude of 150-200 PA. Steroids, prepared as described by Purdy et al. (1990), were dissolved in dimethyl sulfoxide (DMSO, 0.01% final dilution) and applied by pressure (2-4 psi) in the proximity of the cell body with micropipettes of 5-10 pm diameter. Applying DMSO (0.01% in medium) failed to modify CABA responses. To avoid unconlrolled drug leakage,
this pipette was kept outside the recording bath before pressure injection and brought in proximity to the recorded cell just before drug application. Steroids were applied for 5 s between two GABA pulses delivered every 10 s. All of the concentrations reported in the figures are apparent concentrations. The concentration in the drug application pipette set an upper limit. Dilution at the cell can occur, although dye application experiments reveal an efficacious microperfusion of the cell. Current traces were amplified by a patch-clamp amplifier (EPC-7 List Electronics), filtered at 1500 Hz (g-pole low-pass Bessel, Frequency Devices), and recorded on a chart recorder (Gould 2600s) for off-line analysis. Single-Channel Recordings The patch-clamp, single-channel recording technique (Hamill et al., 1981) was used in the outside-out configuration. Cultures were bathed in the same medium as used for whole-cell recordings. The patch pipette contained the same high Cs+ intracellular solution. In a few patches, the presence of spontaneous, long duration, inward single-channel currents was observed. We discarded these membrane patches and analyzed only those patches in which exogenous GABA application directly elicited channel current activity. Steroids and pentobarbital, alone or in combination with GABA, weredissolved at the reported concentrations in bath medium containing 0.01% DMSO and were pressure-applied by micropipettes (5-10 Km tip diameter) placed in proximity of the membrane patch. Data Analysis In the noise analysis experiments, power spectra were obtained from single-channel records at a sampling frequency of 1 kHz after filtering with a low-pass Butterworth filter (Frequency Devices) with corner frequency at 0.45 kHz. An iterative, nonlinear least-squares regression routine fitted the experimental spectra with double lorentzian curves. The time constants (T values) were derived from each corner frequency. More detailed description of the methods used for noise analysis can be found elsewhere (Vicini and Schuetze, 1985). Received
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