The Journal of Neuroscience, January 15, 1997, 17(2):625–634 Bicuculline and Gabazine Are Allosteric Inhibitors of Channel Opening of the GABAA Receptor Shinya Ueno,1 John Bracamontes,1 Chuck Zorumski,2 David S. Weiss,3 and Joe Henry Steinbach1 Departments of 1Anesthesiology and 2Psychiatry, Washington University School of Medicine, St. Louis, Missouri 63110, and 3University of Alabama at Birmingham, Neurobiology Research Center and Department of Physiology and Biophysics, Birmingham, Alabama 35294-0021 Anesthetic drugs are known to interact with GABAA receptors, bicuculline only partially blocked responses to pentobarbital. both to potentiate the effects of low concentrations of GABA and These observations indicate that the blockers do not compete to directly gate open the ion channel in the absence of GABA; with alphaxalone or pentobarbital for a single class of sites on the however, the site(s) involved in direct gating by these drugs is not GABAA receptor. Finally, at receptors containing a1b2(Y157S)g2L known. We have studied the ability of alphaxalone (an anesthetic subunits, both bicuculline and gabazine showed weak agonist steroid) and pentobarbital (an anesthetic barbiturate) to directly activity and actually potentiated responses to alphaxalone. These activate recombinant GABAA receptors containing the a1, b2, and observations indicate that the blocking drugs can produce allo- g2L subunits. Steroid gating was not affected when either of two steric changes in GABAA receptors, at least those containing this mutated b2 subunits [b2(Y157S) and b2(Y205S)] are incorporated mutated b2 subunit. We conclude that the sites for binding ste- into the receptors, although these subunits greatly reduce the roids and barbiturates do not overlap with the GABA-binding site. affinity of GABA binding. These observations indicate that steroid Furthermore, neither gabazine nor bicuculline competes for bind- binding and subsequent channel gating do not require these ing at the steroid or barbiturate sites. The data are consistent with particular residues, as already shown for barbiturates. Bicuculline a model in which both gabazine and bicuculline act as allosteric or gabazine (two competitive antagonists of GABA binding) re- inhibitors of channel opening for the GABAA receptor after binding duced the currents elicited by alphaxalone and pentobarbital from to the GABA-binding site. wild-type GABAA receptors; however, gabazine produced only a Key words: GABAA receptor; GABA; neurosteroids; bicucul- partial block of responses to pentobarbital or alphaxalone, and line; inverse agonist; anesthetics; allosteric inhibitor GABA activates a ligand-gated ion channel (the GABAA recep- the GABAA receptor that are involved in direct gating by anes- tor), which underlies most rapid inhibition in the brain. Various thetics, and we have initiated studies of channel activation by other compounds also bind to the GABAA receptor and can gate alphaxalone (an anesthetic steroid analog) and pentobarbital (an the channel or modulate channel function (Macdonald and Olsen, anesthetic barbiturate). 1993). In particular, steroids and barbiturates are each able to Accordingly, we have examined the ability of alphaxalone to directly gate the GABAA receptor channel (in the absence of gate mutated GABAA receptors, and we found that residues that GABA), and they can also enhance the activation produced by are important in determining the binding affinity of GABA do not low concentrations of GABA. It is not known whether the same affect activation by steroids. We also examined the actions of sites are involved in producing direct gating and in potentiating blocking drugs and found that neither bicuculline nor gabazine the effects of GABA. For the sites involved in potentiation, are competitive inhibitors of currents gated by alphaxalone or however, the steroid-binding site and the barbiturate-binding site pentobarbital. Finally, both gabazine and bicuculline act as weak are distinct from each other and are also distinct from the GABA- agonists for GABAA receptors containing the b2(Y157S) mutated binding site (Macdonald and Olsen, 1993). Because the charac- subunit. These data indicate that steroids and barbiturates do not terized sites for steroid and barbiturate binding differ from the bind to the GABA-binding site when they directly gate the chan- GABA-binding site, it is puzzling that a competitive antagonist of nel of the GABAA receptor. Furthermore, the data support the GABA binding, bicuculline, is also a potent blocker of channel idea that bicuculline and gabazine act as negative allosteric mod- gating by steroids (Barker et al., 1987) or pentobarbital (Nicoll ulators of function of GABA receptors. and Wojtowicz, 1980). We are interested in defining the sites on A Received Aug. 15, 1996; revised Oct. 31, 1996; accepted Nov. 15, 1996. MATERIALS AND METHODS This research was supported by National Institutes of Health (NIH) Grant PO1 All chemicals were from Sigma (St. Louis, MO) unless specified other- GM47969 to J.H.S. and C.Z., NIH Grants AA09212 and NS35291 to D.S.W., wise. Gabazine (SR-95531) and alphaxalone were obtained from Re- National Institute of Mental Health Research Scientist Development Award search Biochemicals International (Natick, MA). MH00964 to C.Z., and funds from the Anesthesiology Department, Washington A complementary DNA construct for the rat a1 subunit of the GABA University School of Medicine. We thank A. Tobin for the a1 subunit cDNA. receptor was provided by Dr. A. Tobin (University of California Los Correspondence should be addressed to Joe Henry Steinbach, Department of Anesthesiology, Washington University School of Medicine, 660 South Euclid, St. Angeles). The rat g2L and b2 subunits and the point mutants b2(Y205S) Louis, MO 63110. and b2(Y157S) have been described (Amin and Weiss, 1993). GABA Dr. Ueno’s present address: Division of Pharmacology, National Institute of receptor subunit cDNAs were transferred to the eucaryotic expression Health Sciences, 1-18-1 Kamiyoga, Tokyo 158, Japan. vector pcDNA3 (Invitrogen, San Diego, CA), for expression in QT6 cells. Copyright q 1997 Society for Neuroscience 0270-6474/97/170625-10$05.00/0 Direct sequencing of the mutated b2 subunits confirmed that the con- 626 J. Neurosci., January 15, 1997, 17(2):625–634 Ueno et al. • Binding and Gating by Steroids structs contained the appropriate base changes (Sequenase version 2 kit; Amersham, Arlington Heights, IL). Quail fibroblasts (QT6 cells; initially provided by Dr. J. Merlie, Wash- ington University) were maintained in Medium 199 (Earle’s salts) con- taining 5% fetal bovine serum (Hyclone, Logan, UT), 10% tryptose phosphate broth (Life Technologies, Grand Island, NY), 1% dimethyl- sulfoxide (DMSO), and penicillin (100 U/ml) plus streptomycin (100 mg/ml) in a humidified atmosphere containing 5% CO2. Calcium phos- phate precipitation was used to transfect QT6 cells (Chen and Okayama, 1987), with the additional step of an initial wash to remove tryptose phosphate broth (Phillips et al., 1991). QT6 cells were used for expression because of anomalous results obtained when subunits were expressed in HEK293 cells (Ueno et al., 1996b). Cells that expressed a high level of protein from exogenous cDNA were identified using the bead-labeling technique described by Jurman et al. (1994). We inserted a flag epitope tag into the N-terminal region of the a1 subunit (Ueno et al., 1996b). Starting with the N terminus of the predicted mature peptide, the predicted sequence of the resulting peptide is YGQPSQDELKDYKDDDDKLKDNTT, where the introduced resi- dues are shown underlined. This construct was identified on the surface of intact cells using a mouse monoclonal antibody to the FLAG epitope (M2, Eastman Kodak Scientific Imaging Systems, New Haven, CT), which had been adsorbed to beads with covalently attached goat anti-mouse IgG antibody (Dynal, Great Neck, NY). Control experiments indicated that the tag had no functional effects on receptors incorporating the tagged a1 subunit (Ueno et al., 1996b). Recordings were made using standard whole-cell methods (Hamill et al., 1981) 24–72 hr. after transfection. All experiments were performed at room temperature (21–238C), and drugs were dissolved in external solu- tion. In all cases, data were obtained from isolated single cells. Experi- Figure 1. Activation of GABAA receptors containing b2orb2(Y205S) ments were performed in two laboratories. In the Steinbach laboratory subunits. Each panel shows concentration–response curves for an agonist: (Ueno et al., 1996a), drugs were applied with a polyethylene “Y tube.” GABA (A), pentobarbital (PENT, B), alphaxalone (ALPH, C), and The bath was perfused continuously with normal external solution from a DHP-OH (D). In each panel, the open symbols show responses from QT6 separate perfusion line, and solution was removed from the bath with a cells transfected with a1b2g2L subunits, whereas filled symbols show Leiden aspirator (Medical Systems, Greenvale, NY). In the Zorumski responses from cells transfected with a1b2(Y205S)g2L subunits. GABA laboratory (Hu et al., 1993), drugs were applied by pressure ejection from produced no gating of receptors containing the mutated subunit (points at “puffer” pipettes positioned within 5 mm of the patch-clamped cell, using 100 and 1000 mM GABA in A). For the other agonists tested, the data from a 500 msec pulse of air pressure (10–20 psi) to the back of the pipette. receptors containing wild-type or mutated subunits were indistinguishable. The data shown