Anesthetic drugs are known to interact with GABA A receptors, both to potentiate the effects of low concentrations of GABA and to directly gate open the ion channel in the absence of GABA; however, the site(s) involved in direct gating by these drugs is not known. We have studied the ability of alphaxalone (an anesthetic steroid) and pentobarbital (an anesthetic barbiturate) to directly activate recombinant GABA A receptors containing the ␣1, ␤2, and ␥2L subunits. Steroid gating was not affected when either of two mutated ␤2 subunits [␤2(Y157S) and ␤2(Y205S)] are incorporated into the receptors, although these subunits greatly reduce the affinity of GABA binding. These observations indicate that steroid binding and subsequent channel gating do not require these particular residues, as already shown for barbiturates. Bicuculline or gabazine (two competitive antagonists of GABA binding) reduced the currents elicited by alphaxalone and pentobarbital from wild-type GABA A receptors; however, gabazine produced only a partial block of responses to pentobarbital or alphaxalone, and bicuculline only partially blocked responses to pentobarbital. These observations indicate that the blockers do not compete with alphaxalone or pentobarbital for a single class of sites on the GABA A receptor. Finally, at receptors containing ␣1␤2(Y157S)␥2L subunits, both bicuculline and gabazine showed weak agonist activity and actually potentiated responses to alphaxalone. These observations indicate that the blocking drugs can produce allosteric changes in GABA A receptors, at least those containing this mutated ␤2 subunit. We conclude that the sites for binding steroids and barbiturates do not overlap with the GABA-binding site. Furthermore, neither gabazine nor bicuculline competes for binding at the steroid or barbiturate sites. The data are consistent with a model in which both gabazine and bicuculline act as allosteric inhibitors of channel opening for the GABA A receptor after binding to the GABA-binding site.Key words: GABA A receptor; GABA; neurosteroids; bicuculline; inverse agonist; anesthetics; allosteric inhibitor GABA activates a ligand-gated ion channel (the GABA A receptor), which underlies most rapid inhibition in the brain. Various other compounds also bind to the GABA A receptor and can gate the channel or modulate channel function (Macdonald and Olsen, 1993). In particular, steroids and barbiturates are each able to directly gate the GABA A receptor channel (in the absence of GABA), and they can also enhance the activation produced by low concentrations of GABA. It is not known whether the same sites are involved in producing direct gating and in potentiating the effects of GABA. For the sites involved in potentiation, however, the steroid-binding site and the barbiturate-binding site are distinct from each other and are also distinct from the GABAbinding site (Macdonald and Olsen, 1993). Because the characterized sites for steroid and barbiturate binding differ from the GABA-binding site, it is puzzling that a competit...

1. Neurosteroids are produced in the brain, and can have rapid actions on membrane channels of neurons. Pregnenolone sulfate (PS) is a sulfated neurosteroid which reduces the responses of the y-aminobutyric acid A (GABA A ) receptor. We analysed the actions of PS on single-channel currents from recombinant GABA A receptors formed from a1, b2 and y2L subunits.2. Currents were elicited by a concentration of GABA eliciting a half-maximal response (50 µM) and a saturating concentration (1 mM). PS reduced the duration of clusters of single-channel activity at either concentration of GABA.3. PS had no discernable effect on rapid processes: no effects were apparent on channel opening and closing, nor on GABA affinity, and a rapidly recovering desensitised state was not affected. Instead, PS produced a slowly developing block which occurred at a similar rate for receptors with open or closed channels and with one or two bound GABA molecules.4. The rate of block was independent of membrane potential, implying that the charged sulfate moiety does not move through the membrane field.5. Change in a specific residue near the intracellular end of the channel lining portion of the a1 subunit had a major effect on the rate of block. Mutation of the residue a1 V256S reduced the rate of block by 30-fold. A mutation at the homologous position of the b2 subunit (b2 A252S) had no effect, nor did a complementary mutation in the y2L subunit (y2L S266A). It seems likely that this residue is involved in a conformational change underlying block by PS, instead of forming part of the binding site for PS.

Neuroactive steroids are among the most efficacious modulators of the mammalian GABA-A receptor. Previous work has proposed that receptor potentiation is mediated by steroid interactions with a site defined by the residues ␣1Asn407/Tyr410 in the M4 transmembrane domain and residue ␣1Gln241 in the M1 domain. We examined the role of residues in the ␣1 subunit M1 domain in the modulation of the rat ␣1␤2␥2L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the ␣1Q241W or ␣1Q241L mutations were insensitive to (3␣,5␣)-3-hydroxypregnan-20-one (3␣5␣P), albeit with different underlying mechanisms. The ␣1Q241S mutant was potentiated by 3␣5␣P, but the kinetic mode of potentiation was altered by the mutation. It is noteworthy that the ␣1Q241L mutation had no effect on channel potentiation by (3␣,5␣)-3-hydroxymethylpregnan-20-one, but mutation of the neighboring residue, ␣1Ser240, prevented channel modulation. A steroid lacking an H-bonding group on C3 (5␣-pregnan-20-one) potentiated the wild-type receptor but not the ␣1Q241L mutant. The findings are consistent with a model in which the ␣1Ser240 and ␣1Gln241 residues shape the surface to which steroid molecules bind.Potentiating neurosteroids are among the most efficacious modulators of the mammalian GABA-A receptor having potential applications as anxiolytics, anticonvulsants, sedatives, and anesthetics. Recent work has given significant insights into the functional and structural mechanisms of steroid actions. Potentiating steroids [e.g., (3␣,5␣)-3-hydroxypregnan-20-one (3␣5␣P) and (3␣,5␤)-3-hydroxypregnan-20-one (3␣5␤P)] act on the GABA-A receptor by modifying the channel open and closed times, leading to an increase in the open probability of the channel, enhanced macroscopic peak current, and a slower current decay when exposure to agonist is terminated. The putative steroid binding site is located in the membrane-spanning regions of the ␣ subunit of the receptor, extending from the ␣1Gln241 residue in the M1 membrane-spanning region to the residues ␣1Asn407 and ␣1Tyr410 in the M4 domain (Hosie et al., 2006). Mutations that reduce the H-bonding ability of these residues reduce receptor potentiation by both 5␣-and 5␤-reduced steroids. It was proposed that a common interaction site mediates the effects of the two classes of steroids, the ␣1Gln241 residue acting as an H-bond acceptor to the 3␣-hydroxyl group of the steroid molecule and the ␣1Asn407/Tyr410 residues interacting with the ketone group in the side chain on the D ring of steroids (Hosie et al., 2006). Subsequent studies showed that mutations that disrupt channel potentiation by steroids also affect modulation by a tricyclic benz[e]indene neurosteroid analog (Li et al., 2006), enantiomers of natural steroids (Li et al., 2007a), and the marine cembranoid eupalmerin acetate (Li et al., 2008), suggesting that the site may function as a common interaction site for a number of GABA-A receptor modul...

The effects of neuroactive steroids on the function of GABA A receptors were studied using cell-attached records of single channel activity recorded from HEK293 cells transfected with α1 β2 γ2L subunits. Activity was elicited with a half-maximal (50 µM) concentration of GABA. Two steroids were studied in detail: ACN ((3α,5α,17β)-3-hydroxyandrostane-17-carbonitrile) and B285 ((3α,5β,17β)-3-hydroxy-18-norandrostane-17-carbonitrile). ACN, 20 nM B285). As in the case for action on open times these data suggest that there are two recognition sites and two independent mechanisms, perhaps the sites and mechanisms associated with actions on open times. The presence of 1 µM ACN had no effect on the estimated channel opening rate or on the apparent affinity of the receptor for GABA. Mutation of the carboxy terminus of the γ2 subunit, but not the α1 or β2 subunits, abolished the ability of ACN to increase the duration of OT 3 but had no effect on the reduction of the rate of occurrence of the activation-related closed state. These observations are also consistent with the idea that there is more than one distinguishable steroid recognition site on the GABA A receptor.

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(−) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action.