Despite extensive studies on nicotinic acetylcholine receptors (nAChRs) and homologues, details of acetylcholine binding are not completely resolved. Here, we report the crystal structure of acetylcholine bound to the receptor homologue acetylcholine binding protein from Lymnaea stagnalis. This is the first structure of acetylcholine in a binding pocket containing all five aromatic residues conserved in all mammalian nAChRs. The ligand-protein interactions are characterized by contacts to the aromatic box formed primarily by residues on the principal side of the intersubunit binding interface (residues Tyr89, Trp143 and Tyr185). Besides these interactions on the principal side, we observe a cation-π interaction between acetylcholine and Trp53 on the complementary side and a water-mediated hydrogen bond from acetylcholine to backbone atoms of Leu102 and Met114, both of importance for anchoring acetylcholine to the complementary side. To further study the role of Trp53, we mutated the corresponding tryptophan in the two different acetylcholine-binding interfaces of the widespread α4β2 nAChR, i.e. the interfaces α4(+)β2(-) and α4(+)α4(-). Mutation to alanine (W82A on the β2 subunit or W88A on the α4 subunit) significantly altered the response to acetylcholine measured by oocyte voltage-clamp electrophysiology in both interfaces. This shows that the conserved tryptophan residue is important for the effects of ACh at α4β2 nAChRs, as also indicated by the crystal structure. The results add important details to the understanding of how this neurotransmitter exerts its action and improves the foundation for rational drug design targeting these receptors.

Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT) taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO) mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-β-erythroidine, and CP-601932 (a partial agonist of the α3β4* nAChR), inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT)-cAMP and loading taste cells with [Ca2+]i by topical lingual application of ionomycin + CaCl2. In contrast, CT responses to nicotine were enhanced when TRC [Ca2+]i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol.

Excess sugar consumption has been shown to contribute directly to weight gain, thus contributing to the growing worldwide obesity epidemic. Interestingly, increased sugar consumption has been shown to repeatedly elevate dopamine levels in the nucleus accumbens (NAc), in the mesolimbic reward pathway of the brain similar to many drugs of abuse. We report that varenicline, an FDA-approved nicotinic acetylcholine receptor (nAChR) partial agonist that modulates dopamine in the mesolimbic reward pathway of the brain, significantly reduces sucrose consumption, especially in a long-term consumption paradigm. Similar results were observed with other nAChR drugs, namely mecamylamine and cytisine. Furthermore, we show that long-term sucrose consumption increases α4β2 * and decreases α6β2* nAChRs in the nucleus accumbens, a key brain region associated with reward. Taken together, our results suggest that nAChR drugs such as varenicline may represent a novel treatment strategy for reducing sugar consumption.

Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca(2+)-dependent Cl(-) channels, since menthol inhibition remained unchanged by intracellular injection of the Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free bathing solution containing Ba(2+). Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [(125)I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca(2+) transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner.

Acetylcholine is one of the most important endogenous neurotransmitters in a range of organisms spanning different animal phyla. Within pituitary gland it acts as autocrine and paracrine signal. In a current study we assessed expression profile of the different subunits of nicotinic as well as muscarinic acetylcholine receptors in RC-4BC cells, which are derived from rat pituitary gland tumor. Our findings indicate that β2, δ, and M2 subunits are expressed by the cells with the lowest Ct values compared to other tested subunits. The detected Ct values were 26.6±0.16, 27.95±0.5, and 28.8±0.25 for β2, δ, and M2 subunits, respectively.

Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex "collides" with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to G(q/11), while even-numbered receptors prefer coupling to G(i/o). We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the α-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [(3)H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M(1) and M(3) receptors also pre-couple with non-preferential G(i/o) G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype and the class of G-protein.

In recent years a non-neuronal cholinergic system has been described in immune cells, which is often usually activated during the course of inflammatory processes. To date, it is known that Acetylcholine (ACh), a neurotransmitter extensively expressed in the airways, not only induces bronchoconstriction, but also promotes a set of changes usually associated with the induction of allergic/Th2 responses. We have previously demonstrated that ACh polarizes human dendritic cells (DC) toward a Th2-promoting profile through the activation of muscarinic acetylcholine receptors (mAChR). Here, we showed that ACh promotes the acquisition of an inflammatory profile by murine DC, with the increased MHC II IAd expression and production of two cytokines strongly associated with inflammatory infiltrate and tissue damage, namely TNF-α and MCP-1, which was prevented by blocking mAChR. Moreover, we showed that ACh induces the up-regulation of M3 mAChR expression and the blocking of this receptor with tiotropium bromide prevents the increase of MHC II IAd expression and TNF-α production induced by ACh on DC, suggesting that M3 is the main receptor involved in ACh-induced activation of DC. Then, using a short-term experimental murine model of ovalbumin-induced lung inflammation, we revealed that the intranasal administration of ACh-treated DC, at early stages of the inflammatory response, might be able to exacerbate the recruitment of inflammatory mononuclear cells, promoting profound structural changes in the lung parenchyma characteristic of chronic inflammation and evidenced by elevated systemic levels of inflammatory marker, TNF-α. These results suggest a potential role for ACh in the modulation of immune mechanisms underlying pulmonary inflammatory processes.

In a majority of patients with myasthenia gravis (MG), anti-acetylcholine receptor (AChR) antibodies target postsynaptic AChR clusters and thus compromise the membrane integrity of neuromuscular junctions (NMJs) and lead to muscle weakness. Antibody-induced endocytosis of AChRs in the postsynaptic membrane represents the initial step in the pathogenesis of MG; however, the molecular mechanisms underlying AChR endocytosis remain largely unknown. Here, we developed an approach to mimic the pathogenic antibodies for inducing the crosslinking and internalization of AChRs from the postsynaptic membrane. Using biotin-α-bungarotoxin and quantum dot (QD)-streptavidin, cell-surface and internalized AChRs could be readily distinguished by comparing the size, fluorescence intensity, trajectory, and subcellular localization of the QD signals. QD-induced AChR endocytosis was mediated by clathrin-dependent and caveolin-independent mechanisms, and the trafficking of internalized AChRs in the early endosomes required the integrity of microtubule structures. Furthermore, activation of the agrin/MuSK (muscle-specific kinase) signaling pathway strongly suppressed QD-induced internalization of AChRs. Lastly, QD-induced AChR crosslinking potentiated the dispersal of aneural AChR clusters upon synaptic induction. Taken together, our results identify a novel approach to study the mechanisms of AChR trafficking upon receptor crosslinking and endocytosis, and demonstrate that agrin-MuSK signaling pathways protect against crosslinking-induced endocytosis of AChRs.

Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4β2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4β2)2β2- and LS-(α4β2)2α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)β2 subunit binding interfaces. However LS-(α4β2)2α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4β2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4β2)2β2-nAChR exhibit a single conductance state, whereas LS-(α4β2)2α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4β2)2α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site's role in mediating LS-(α4β2)2α4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(α4β2)2α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)β2 subunit interfaces, rather than independent induction of high conductance channel openings.

How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified neuron R15 of the marine mollusk Aplysia californica. R15 is a bursting neuron in the abdominal ganglia of the central nervous system and is implicated in reproduction, water balance, and heart function. Exposure to acetylcholine (ACh) causes an increase in R15 burst firing. Whole-cell recordings of R15 in the intact ganglia dissected from mature and old Aplysia showed specific changes in burst firing and properties of action potentials induced by ACh. We found that while there were no significant changes in resting membrane potential and latency in response to ACh, the burst number and burst duration is altered during aging. The action potential waveform analysis showed that unlike mature neurons, the duration of depolarization and the repolarization amplitude and duration did not change in old neurons in response to ACh. Furthermore, single neuron quantitative analysis of acetylcholine receptors (AChRs) suggested alteration of expression of specific AChRs in R15 neurons during aging. These results suggest a defect in cholinergic transmission during aging of the R15 neuron.

To what extent do cholesterol-rich lipid platforms modulate the supramolecular organization of the nicotinic acetylcholine receptor (AChR)? To address this question, the dynamics of AChR particles at high density and its cholesterol dependence at the surface of mammalian cells were studied by combining total internal reflection fluorescence microscopy and single-particle tracking. AChR particles tagged with a monovalent ligand, fluorescent α-bungarotoxin (αBTX), exhibited two mobile pools: i) a highly mobile one undergoing simple Brownian motion (16%) and ii) one with restricted motion (∼50%), the rest being relatively immobile (∼44%). Depletion of membrane cholesterol by methyl-α-cyclodextrin increased the fraction of the first pool to 22% and 33% after 15 and 40 min, respectively; the pool undergoing restricted motion diminished from 50% to 44% and 37%, respectively. Monoclonal antibody binding results in AChR crosslinking-internalization after 2 h; here, antibody binding immobilized within minutes ∼20% of the totally mobile AChR. This proportion dramatically increased upon cholesterol depletion, especially during the initial 10 min (83.3%). Thus, antibody crosslinking and cholesterol depletion exhibited a mutually synergistic effect, increasing the average lifetime of cell-surface AChRs∼10 s to ∼20 s. The instantaneous (microscopic) diffusion coefficient D2-4 of the AChR obtained from the MSD analysis diminished from ∼0.001 µm2 s(-1) to ∼0.0001-0.00033 µm2 s(-1) upon cholesterol depletion, ∼30% of all particles falling into the stationary mode. Thus, muscle-type AChR exhibits heterogeneous motional regimes at the cell surface, modulated by the combination of intrinsic (its supramolecular organization) and extrinsic (membrane cholesterol content) factors.

The behavioral effects of nicotine and other nicotinic agonists are mediated by AChRs in the brain. The relative contribution of acute activation versus chronic desensitization of AChRs is unknown. Sustained "smoldering activation" occurs over a range of agonist concentrations at which activated and desensitized AChRs are present in equilibrium. We used a fluorescent dye sensitive to changes in membrane potential to examine the effects of acute activation and chronic desensitization by nicotinic AChR agonists on cell lines expressing human α4β2, α3β4 and α7 AChRs. We examined the effects of acute and prolonged application of nicotine and the partial agonists varenicline, cytisine and sazetidine-A on these AChRs. The range of concentrations over which nicotine causes smoldering activation of α4β2 AChRs was centered at 0.13 µM, a level found in smokers. However, nicotine produced smoldering activation of α3β4 and α7 AChRs at concentrations well above levels found in smokers. The α4β2 expressing cell line contains a mixture of two stoichiometries, namely (α4β2)2β2 and (α4β2)2α4. The (α4β2)2β2 stoichiometry is more sensitive to activation by nicotine. Sazetidine-A activates and desensitizes only this stoichiometry. Varenicline, cytisine and sazetidine-A were partial agonists on this mixture of α4β2 AChRs, but full agonists on α3β4 and α7 AChRs. It has been reported that cytisine and varenicline are most efficacious on the (α4β2)2α4 stoichiometry. In this study, we distinguish the dual effects of activation and desensitization of AChRs by these nicotinic agonists and define the range of concentrations over which smoldering activation can be sustained.

The ability to eliminate undesired cells by apoptosis is a key mechanism to maintain organismal health and homeostasis. Failure to clear apoptotic cells efficiently can cause autoimmune diseases in mammals. Genetic studies in Caenorhabditis elegans have greatly helped to decipher the regulation of apoptotic cell clearance. In this study, we show that the loss of levamisole-sensitive acetylcholine receptor, but not of a typical neuronal acetylcholine receptor causes a reduction in the number of persistent cell corpses in worms suffering from an engulfment deficiency. This reduction is not caused by impaired or delayed cell death but rather by a partial restoration of the cell clearance capacity. Mutants in acetylcholine turn-over elicit a similar phenotype, implying that acetylcholine signaling is the process responsible for these observations. Surprisingly, tissue specific RNAi suggests that UNC-38, a major component of the levamisole-sensitive receptor, functions in the dying germ cell to influence engulfment efficiency. Animals with loss of acetylcholine receptor exhibit a higher fraction of cell corpses positive for the "eat-me" signal phosphatidylserine. Our results suggest that modulation by ion channels of ion flow across plasma membrane in dying cells can influence the dynamics of phosphatidylserine exposure and thus clearance efficiency.

Heart rate variability is an important risk factor for cardiovascular disease and all-cause mortality. The acetylcholine pathway plays a key role in explaining heart rate variability in humans. We assessed whether 443 genotyped and imputed common genetic variants in eight key genes (CHAT, SLC18A3, SLC5A7, CHRNB4, CHRNA3, CHRNA, CHRM2 and ACHE) of the acetylcholine pathway were associated with variation in an established measure of heart rate variability reflecting parasympathetic control of the heart rhythm, the root mean square of successive differences (RMSSD) of normal RR intervals. The association was studied in a two stage design in individuals of European descent. First, analyses were performed in a discovery sample of four cohorts (n = 3429, discovery stage). Second, findings were replicated in three independent cohorts (n = 3311, replication stage), and finally the two stages were combined in a meta-analysis (n = 6740). RMSSD data were obtained under resting conditions. After correction for multiple testing, none of the SNPs showed an association with RMSSD. In conclusion, no common genetic variants for heart rate variability were identified in the largest and most comprehensive candidate gene study on the acetylcholine pathway to date. Future gene finding efforts for RMSSD may want to focus on hypothesis free approaches such as the genome-wide association study.

The formation of acetylcholine receptor (AChR) cluster is a key event during the development of the neuromuscular junction. It is induced through the activation of muscle-specific kinase (MuSK) by the heparan-sulfate proteoglycan agrin released from the motor axon. On the other hand, DC electric field, a non-neuronal stimulus, is also highly effective in causing AChRs to cluster along the cathode-facing edge of muscle cells.

Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by further experiments.

Animals change their behavior and metabolism in response to external stimuli. cAMP response element binding protein (CREB) is a signal-activated transcription factor that enables the coupling of extracellular signals and gene expression to induce adaptive changes. Biogenic amine neurotransmitters regulate CREB and such regulation is important for long-term changes in various nervous system functions, including learning and drug addiction. In Caenorhabditis elegans, the amine neurotransmitter octopamine activates a CREB homolog, CRH-1, in cholinergic SIA neurons, whereas dopamine suppresses CREB activation by inhibiting octopamine signaling in response to food stimuli. However, the physiological role of this activation is unknown. In this study, the effect of dopamine, octopamine, and CREB on acetylcholine signaling was analyzed using the acetylcholinesterase inhibitor aldicarb. Mutants with decreased dopamine signaling exhibited reduced acetylcholine signaling, and octopamine and CREB functioned downstream of dopamine in this regulation. This study demonstrates that the regulation of CREB by amine neurotransmitters modulates acetylcholine release from the neurons of C. elegans.

Acetylcholine (ACh) plays a crucial role in physiological responses of both the central and the peripheral nervous system. Moreover, ACh was described as an anti-inflammatory mediator involved in the suppression of exacerbated innate response and cytokine release in various organs. However, the specific contributions of endogenous release ACh for inflammatory responses in the lung are not well understood. To address this question we have used mice with reduced levels of the vesicular acetylcholine transporter (VAChT), a protein required for ACh storage in secretory vesicles. VAChT deficiency induced airway inflammation with enhanced TNF-α and IL-4 content, but not IL-6, IL-13 and IL-10 quantified by ELISA. Mice with decreased levels of VAChT presented increased collagen and elastic fibers deposition in airway walls which was consistent with an increase in inflammatory cells positive to MMP-9 and TIMP-1 in the lung. In vivo lung function evaluation showed airway hyperresponsiveness to methacholine in mutant mice. The expression of nuclear factor-kappa B (p65-NF-kB) in lung of VAChT-deficient mice were higher than in wild-type mice, whereas a decreased expression of janus-kinase 2 (JAK2) was observed in the lung of mutant animals. Our findings show the first evidence that cholinergic deficiency impaired lung function and produce local inflammation. Our data supports the notion that cholinergic system modulates airway inflammation by modulation of JAK2 and NF-kB pathway. We proposed that intact cholinergic pathway is necessary to maintain the lung homeostasis.

Despite the large number of studies available on nicotinic acetylcholine receptors, a complete account of the mechanistic aspects of their gating transition in response to ligand binding still remains elusive. As a first step toward dissecting the transition mechanism by accelerated sampling techniques, we study the ligand-induced conformational changes of the acetylcholine binding protein (AChBP), a widely accepted model for the full receptor extracellular domain. Using unbiased Molecular Dynamics (MD) and Temperature Accelerated Molecular Dynamics (TAMD) simulations we investigate the AChBP transition between the apo and the agonist-bound state. In long standard MD simulations, both conformations of the native protein are stable, while the agonist-bound structure evolves toward the apo one if the orientation of few key sidechains in the orthosteric cavity is modified. Conversely, TAMD simulations initiated from the native conformations are able to produce the spontaneous transition. With respect to the modified conformations, TAMD accelerates the transition by at least a factor 10. The analysis of some specific residue-residue interactions points out that the transition mechanism is based on the disruption/formation of few key hydrogen bonds. Finally, while early events of ligand dissociation are observed already in standard MD, TAMD accelerates the ligand detachment and, at the highest TAMD effective temperature, it is able to produce a complete dissociation path in one AChBP subunit.

The nicotinic acetylcholine receptor (nAChR) is an excitatory pentameric ligand-gated ion channel (pLGIC), homologous to the inhibitory γ-aminobutyric acid (GABA) type A receptor targeted by pharmaceuticals and endogenous sedatives. Activation of the GABAA receptor by the neurosteroid allopregnanolone can be inhibited competitively by thyroid hormone (L-3,3',5-triiodothyronine, or T3), but modulation of nAChR by T3 or neurosteroids has not been investigated. Here we show that allopregnanolone inhibits the nAChR from Torpedo californica at micromolar concentrations, as do T3 and the anionic neurosteroid pregnenolone sulfate (PS). We test for the role of protein and ligand charge in mediated receptor inhibition by varying pH in a narrow range around physiological pH. We find that both T3 and PS become less potent with increasing pH, with remarkably similar trends in IC50 when T3 is neutral at pH < 7.3. After deprotonation of T3 (but no additional deprotonation of PS) at pH 7.3, T3 loses potency more slowly with increasing pH than PS. We interpret this result as indicating the negative charge is not required for inhibition but does increase activity. Finally, we show that both T3 and PS affect nAChR channel desensitization, which may implicate a binding site homologous to one that was recently indicated for accelerated desensitization of the GABAA receptor by PS.