Intrasubunit and Intersubunit Steroid Binding Sites Independently and Additively Mediate α1ß2γ2L GABAA Receptor Potentiation by the Endogenous Neurosteroid Allopregnanolone.

Prior work employing functional analysis, photolabeling, and X-ray crystallography have identified three distinct binding sites for potentiating steroids in the heteromeric GABAA receptor. The sites are located in the membrane-spanning domains of the receptor at the ß-α subunit interface (site I) and within the α (site II) and ß subunits (site III). Here, we have investigated the effects of mutations to these sites on potentiation of the rat α1ß2γ2L GABAA receptor by the endogenous neurosteroid allopregnanolone (3α5αP). The mutations were introduced alone or in combination to probe the additivity of effects. We show that the effects of amino acid substitutions in sites I and II are energetically additive, indicating independence of the actions of the two steroid binding sites. In site III, none of the mutations tested reduced potentiation by 3α5αP, nor did a mutation in site III modify the effects of mutations in sites I or II. We infer that the binding sites for 3α5αP act independently. The independence of steroid action at each site is supported by photolabeling data showing that mutations in either site I or site II selectively change steroid orientation in the mutated site without affecting labeling at the unmutated site. The findings are discussed in the context of linking energetic additivity to empirical changes in receptor function and ligand binding. SIGNIFICANCE STATEMENT: Prior work has identified three distinct binding sites for potentiating steroids in the heteromeric γ-aminobutyric acid type A receptor. This study shows that the sites act independently and additively in the presence of the steroid allopregnanolone and provide estimates of energetic contributions made by steroid binding to each site.

Enhancement of Muscimol Binding and Gating by Allosteric Modulators of the GABAA Receptor: Relating Occupancy to State Functions.

Muscimol is a psychoactive isoxazole derived from the mushroom Amanita muscaria and a potent orthosteric agonist of the GABAA receptor. The binding of [3H]muscimol has been used to evaluate the distribution of GABAA receptors in the brain, and studies of modulation of [3H]muscimol binding by allosteric GABAergic modulators such as barbiturates and steroid anesthetics have provided insight into the modes of action of these drugs on the GABAA receptor. It has, however, not been feasible to directly apply interaction parameters derived from functional studies to describe the binding of muscimol to the receptor. Here, we employed the Monod-Wyman-Changeux concerted transition model to analyze muscimol binding isotherms. We show that the binding isotherms from recombinant α1ß3 GABAA receptors can be qualitatively predicted using electrophysiological data pertaining to properties of receptor activation and desensitization in the presence of muscimol. The model predicts enhancement of [3H]muscimol binding in the presence of the steroids allopregnanolone and pregnenolone sulfate, although the steroids interact with distinct sites and either enhance (allopregnanolone) or reduce (pregnenolone sulfate) receptor function. We infer that the concerted transition model can be used to link radioligand binding and electrophysiological data. SIGNIFICANCE STATEMENT: The study employs a three-state resting-active-desensitized model to link radioligand binding and electrophysiological data. We show that the binding isotherms can be qualitatively predicted using parameters estimated in electrophysiological experiments and that the model accurately predicts the enhancement of [3H]muscimol binding in the presence of the potentiating steroid allopregnanolone and the inhibitory steroid pregnenolone sulfate.

Volatile anesthetic sevoflurane pretreatment alleviates hypoxia-induced potentiation of excitatory inputs to striatal medium spiny neurons of mice.

Sevoflurane, a commonly used anesthetic in surgery, has drawn attention because of its preconditioning effects in hypoxic conditions. To investigate the preconditioning effects in the striatum, a common site for ischemic stroke, we collected whole-cell current-clamp recordings from striatal medium spiny neurons. In our in vitro brain slice experiments, deprivation of oxygen and glucose depolarized the striatal neurons to subthreshold potentials, and the pre-administration of sevoflurane (4%, 15 min) prolonged the time to depolarization. Furthermore, transient hypoxia induced the potentiation of excitatory postsynaptic potentials, which play a part in post-ischemic excitotoxicity. Glibenclamide, a KATP channel inhibitor, reversed the prolonged time to depolarization and the prevention of the pathological potentiation of excitatory responses, indicating that the short exposure to sevoflurane likely participates in neuroprotection against hypoxia via activation of KATP channels. A monocarboxylate transporter blocker, 4-CIN, also depolarized striatal neurons. Interestingly, the blockade of monocarboxylate transporters that supply lactate to neurons caused the pathological potentiation, even in the presence of enough oxygen and glucose. In this case, sevoflurane could not prevent the pathological potentiation, suggesting the involvement of monocarboxylate transporters in the sevoflurane-mediated effects. These results indicate that sevoflurane protects striatal neurons from hypoxic damage and alleviates the pathological potentiation. Under these conditions, sevoflurane may become an effective intervention for patients undergoing surgery.

Prevalence of chronic postsurgical pain after thoracotomy and total knee arthroplasty: a retrospective multicenter study in Japan (Japanese Study Group of Subacute Postoperative Pain).

We performed a multicenter observational study to assess the prevalence and risk factors of persistent pain after lung cancer surgery and total knee arthroplasty (TKA) in the Japanese population. After receiving Ethics Committee approval, a retrospective chart review was performed for patients who underwent surgery at seven university hospitals in Japan in 2013. A total of 511 patients who underwent lung cancer surgery and 298 patients who underwent TKA were included. The prevalence of chronic postsurgical pain (CPSP) at 3 and 6 months was 18 and 12% after lung surgery and 49 and 33% after TKA, respectively. The prevalence of analgesic use at 3 and 6 months was 16 and 9% after lung surgery and 34 and 22% after TKA, respectively. In both groups, preoperative analgesic use was associated with CPSP. Anesthetic methods or techniques during both types of surgery did not significantly affect the prevalence of CPSP. This is the first study in which the prevalence of CPSP after lung surgery and TKA in Japanese population was extensively evaluated in a multicenter trial. Further prospective studies are needed to confirm the prevalence of CPSP in the Japanese population and to identify risk factors and prevention methods.

Effects of dexmedetomidine on hemodynamics and respiration in intubated, spontaneously breathing patients after endoscopic submucosal dissection for cervical esophageal or pharyngeal cancer.

PURPOSE: We evaluated the hemodynamic and respiratory effects of dexmedetomidine in intubated, spontaneously breathing patients after endoscopic submucosal dissection (ESD) for cervical esophageal or pharyngeal cancer. METHODS: This retrospective study included 129 patients aged 66.5 ± 8.3 years, who underwent ESD under general anesthesia, and who were kept intubated overnight to prevent airway obstruction, receiving sedation with dexmedetomidine. Constant dexmedetomidine infusion at 0.51 ± 0.16 µg/kg/h was started intraoperatively (n = 109) or postoperatively (n = 20), following (n = 29) or not following (n = 100) loading doses, and continued until extubation. Hemodynamic and respiratory variables, and Richmond Agitation-Sedation Scale (RASS) score, were recorded. RESULTS: Postoperatively, 129 patients remained intubated while breathing spontaneously for 16.4 ± 3.3 h, and 124 patients could be sedated solely with dexmedetomidine, whereas 5 required rescue sedatives. During infusion, blood pressure decreased progressively until 12 h, whereas heart rate decreased only at 3 h. Hemodynamic alterations during dexmedetomidine infusion greatly depended not only on its hemodynamic effects but also on baseline hemodynamics before anesthesia. No serious adverse effect was noted. CONCLUSION: Dexmedetomidine in intubated, spontaneously breathing patients after ESD was safe and effective. Patient baseline hemodynamics could significantly affect hemodynamics during drug infusion. Without loading doses, plasma drug concentrations were expected to increase progressively. A progressive decrease in blood pressure and unchanged heart rate after an initial decrease suggested that hemodynamic effects of dexmedetomidine in our patients might differ from those reported in young volunteers, although further studies are required to elucidate these points.

[With the Characteristics of Chronic Pain Patients, Can the Therapeutic Effect of Antidepressant Duloxetine be Predicted?].

BACKGROUND: Duloxetine, an antidepressant, is used for treatment of pain, but the factors related to its effectiveness are not well known, and therefore we have performed a retrospective study. METHODS: Over a 22-month period from June 2012 patients with pain lasting for 3 months or more, with an NRS of 4 or higher, and given duloxetine within 3 months from their first diagnosis, were extracted from the medical records. These patients were compared and studied regarding their scores of the HADS (hos- pital anxiety and depression scale) at the time of first visit, duration of the disease, type of patient, and treat- ment effect after 1 month. RESULTS: The subjects were 61 patients, and they were categorized based on the presence of anxiety, the presence of dysphoria whether from organic or inor- ganic condition, and the duration of the disease, and no significant difference in the effectiveness of duloxetine was found. CONCLUSIONS: Duloxetine had an overall effectiveness of 50.8%, regardless of the presence of anxiety or depression, the duration of the disease and the type of diseases.

Effects of the volatile anesthetic sevoflurane on tonic GABA currents in the mouse striatum during postnatal development.

The volatile anesthetic sevoflurane, which is widely used in pediatric surgery, has proposed effects on GABAA receptor-mediated extrasynaptic tonic inhibition. In the developing striatum, medium-sized spiny projection neurons have tonic GABA currents, which function in the excitatory/inhibitory balance and maturation of striatal neural circuits. In this study, we examined the effects of sevoflurane on the tonic GABA currents of medium spiny neurons in developing striatal slices. Sevoflurane strongly increased GABAA receptor-mediated tonic conductance at postnatal days 3-35. The antagonist of the GABA transporter-1, 1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride further increased tonic GABA conductance during the application of sevoflurane, thereby increasing the total magnitude of tonic currents. Both GABA (5 µM) and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride, the δ-subunit-containing GABAA receptor agonist, induced tonic GABA currents in medium spiny neurons but not in cholinergic neurons. However, sevoflurane additively potentiated the tonic GABA currents in both cells. Interestingly, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride-sensitive neurons made a large current response to sevoflurane, indicating the contribution of the δ-subunit on sevoflurane-enhanced tonic GABA currents. Our findings suggest that sevoflurane can affect the tone of tonic GABA inhibition in a developing striatal neural network.

Successful one-lung ventilation in a patient after laryngectomy by inserting a long spiral single-lumen tube into the left main bronchus: a case report.

Background: Patients who have had laryngectomy require a thorough preoperative assessment for potential stomal stenosis, and an action plan for possible inadvertent displacement of the voice prosthesis (VP) must be considered. We report the anesthetic management of a post-laryngectomy patient undergoing lung resection surgery. The patient had both a laryngectomy and a VP in situ. Case Description: A 66-year-old man with Parkinson's disease, who had previously undergone total laryngectomy for supraglottic laryngeal cancer, had a cuffed tracheostomy tube and a VP inserted into the tracheoesophageal fistula below it. He was scheduled for segmentectomy combined with lymph node dissection under combined epidural-general anesthesia due to lung cancer in the apical segment of the right lung. Following induction of general anesthesia, instead of using a double-lumen endotracheal tube, we inserted a long spiral single-lumen tube (SLT) (6 mm inner diameter, 8.7 mm outer diameter) through the tracheostoma under the guidance of a 4 mm bronchoscope because of concerns about airway injury due to the narrowed diameter of the stoma and potential dislodgement of the VP. The tube was carefully advanced and smoothly placed into the left main bronchus, and the surgery was completed using one-lung ventilation (OLV). Conclusions: For post-total laryngectomy patients, it is important to assess the size and condition of the tracheostoma and the usage of a VP, and choose an appropriate endotracheal tube. A long spiral SLT might be an option for OLV in patients after laryngectomy with a tracheoesophageal VP.

Usefulness of stroke volume index obtained with the FloTrac/ Vigileo system for the prediction of acute kidney injury after radical esophagectomy.

PURPOSE: To assess the impact of stroke volume index (SVI) at the end of esophagectomy upon postoperative renal function. METHODS: We reviewed medical records of 128 patients undergoing esophagectomy. Intraoperative hemodynamics were monitored with the FloTrac sensor/Vigileo monitor system in addition to standard monitors. Patients were divided into two groups according to SVI at the end of surgery: the normal SVI group (n = 76), with SVI ≥ 35 ml/m2, and the low SVI group (n = 52), with SVI<35 ml/m2. We compared postoperative renal function, indicated by serum creatinine and estimated glomerular filtration rate, on post-operative days 0 through 3. We also compared numbers of patients who developed postoperative acute kidney injury (AKI). RESULTS: Although there were no intergroup differences in preoperative renal function or other intraoperative hemodynamic variables, including arterial pressure, central venous pressure, stroke volume variation, a volume of infusion, urine output, and the total intraoperative in-out balance, estimated glomerular filtration rate was significantly lower and serum creatinine was significantly higher in the low SVI group than in the normal SVI group on postoperative days 1 and 2 (P<0.05). In addition, more patients developed postoperative AKI in the low SVI group than in the normal SVI group (12 of 52 vs. 5 of 76, P = 0.015). CONCLUSIONS: Low SVI at the end of esophagectomy may represent a risk factor for AKI in the early postoperative period. Further studies are required to examine whether maintaining SVI above 35 ml/m2 reduces the incidence of AKI after esophagectomy.

Antiemetic effect of naloxone in combination with dexamethasone and droperidol in patients undergoing laparoscopic gynecological surgery.

PURPOSE: We examined the effects of dexamethasone, droperidol, naloxone, and a combination of these three agents on postoperative nausea and vomiting (PONV) in female patients. METHODS: In this randomized, controlled study, 120 female patients with ASA PS I or II undergoing laparoscopic gynecological surgery were randomly allocated into four groups. Patients received dexamethasone 8 mg (Dx group) or droperidol 1 mg (Dr group) before induction of general anesthesia. Anesthesia was induced and maintained with propofol and remifentanil. Postoperative analgesia was provided by intravenous patient-controlled analgesia using a disposable infusion pump filled with fentanyl 20 µg/kg alone (Dx group), fentanyl 20 µg/kg with droperidol 2 mg (Dr group), fentanyl 20 µg/kg with naloxone 0.1 mg (Nx group), or fentanyl 20 µg/kg with droperidol 2 mg and naloxone 0.1 mg (Cm group) in a total volume of 80 ml, with a constant infusion rate of 4 ml/h and a bolus dose 2 ml with a 30-min lockout time. RESULTS: The number of patients who developed PONV and required a rescue antiemetic was significantly less in the Cm group than in the Nx group (p < 0.001 for all). The incidence of PONV was 43, 43, 70, and 17 % in the Dx, Dr, Nx, and Cm groups, respectively. CONCLUSION: A combination of naloxone, droperidol, and dexamethasone was effective for preventing PONV in patients receiving fentanyl for postoperative analgesia after laparoscopic gynecological surgery, although further investigations are required to examine the effect of adding naloxone to other antiemetics.

Effects of sevoflurane and propofol on pulmonary inflammatory responses during lung resection.

PURPOSE: Pulmonary inflammatory reactions are affected by one-lung ventilation (OLV) and anesthetic agents. However, the effects of anesthetic agents on pulmonary inflammatory reactions may vary. Our previous investigations suggested that inflammatory reactions were more pronounced in the dependent lung during lung resection under general anesthesia with propofol and remifentanil. Therefore, in the present study we attempted to determine the difference in pulmonary inflammatory reaction using either sevoflurane or propofol in both dependent and nondependent lungs during OLV. METHODS: Forty adult patients undergoing elective lung resection were randomized to receive either propofol (n = 20) or sevoflurane (n = 20) as the main anesthetic agent. Intraoperative analgesia was provided by remifentanil in both groups. Epithelial lining fluid (ELF) was obtained from each lung using a bronchoscopic microsampling method. ELF and plasma levels of inflammatory cytokines were measured using multiplexed bead-based immunoassays before and after OLV. RESULTS: Epithelial lining fluid levels of interleukin (IL)-1ß, IL-6, and IL-8 were significantly increased in the dependent lung and the nondependent lung after OLV compared with baseline levels (P < 0.05). Moreover, IL-6 ELF level in the dependent lung was significantly higher in the propofol group than in the sevoflurane group after OLV (P < 0.001). CONCLUSION: One-lung ventilation induced inflammatory responses of the bronchial epithelia in the dependent lung and the nondependent lung during lung resection. Moreover, this inflammatory response was significantly suppressed by sevoflurane compared with propofol. Furthermore, the antiinflammatory effect of sevoflurane was more pronounced in the dependent lung than in the nondependent lung during OLV.

Lysosomal GPCR-like protein LYCHOS signals cholesterol sufficiency to mTORC1.

Lysosomes coordinate cellular metabolism and growth upon sensing of essential nutrients, including cholesterol. Through bioinformatic analysis of lysosomal proteomes, we identified lysosomal cholesterol signaling (LYCHOS, previously annotated as G protein-coupled receptor 155), a multidomain transmembrane protein that enables cholesterol-dependent activation of the master growth regulator, the protein kinase mechanistic target of rapamycin complex 1 (mTORC1). Cholesterol bound to the amino-terminal permease-like region of LYCHOS, and mutating this site impaired mTORC1 activation. At high cholesterol concentrations, LYCHOS bound to the GATOR1 complex, a guanosine triphosphatase (GTPase)-activating protein for the Rag GTPases, through a conserved cytoplasm-facing loop. By sequestering GATOR1, LYCHOS promotes cholesterol- and Rag-dependent recruitment of mTORC1 to lysosomes. Thus, LYCHOS functions in a lysosomal pathway for cholesterol sensing and couples cholesterol concentrations to mTORC1-dependent anabolic signaling.

The effect of one-lung ventilation upon pulmonary inflammatory responses during lung resection.

PURPOSE: One-lung ventilation (OLV) is commonly used during thoracic surgery. Clinical studies using bronchoalveolar lavage fluid analysis have demonstrated that OLV induces pulmonary inflammatory reactions in the ventilated dependent lung. However, few clinical studies have investigated such inflammatory reactions in the dependent lung compared with the collapsed nondependent lung. Here we used a bronchoscopic microsampling method to obtain epithelial lining fluid (ELF) from each lung, and then compared the inflammatory reactions in the dependent lung and the nondependent lung during thoracic surgery. METHODS: Twenty adult patients were studied. All patients underwent thoracic surgery using OLV. Propofol and remifentanil were used for total intravenous anesthesia. A double-lumen endotracheal tube was used to perform OLV. ELF was obtained from each lung using the bronchoscopic microsampling method. ELF levels of inflammatory mediators, tumor necrosis factor α, interleukin (IL)-1ß, IL-6, IL-8, IL-10, and IL-12p70 were measured using ELISA before and after OLV. RESULTS: ELF levels of IL-1ß, IL-6, and IL-8 were significantly increased in the dependent lung and the nondependent lung at the end of surgery compared with their baseline levels (p < 0.05). ELF level of IL-6 was significantly higher in the dependent lung than in the nondependent lung at the end of surgery (p = 0.019). CONCLUSIONS: One-lung ventilation induced inflammatory responses of the bronchial epithelia in the dependent lung and the nondependent lung during thoracic surgery. In addition, these inflammatory responses were more augmented in the dependent lung than in the nondependent lung.

[Successful anesthetic management of a postpartum patient with amniotic fluid embolism].

We report a case of amniotic fluid embolism (AFE) after cesarean section (C/S). A 35-year-old primigravida with placenta previa and myoma underwent C/S because of nonreassuring fetal status caused by medical induction of labor. C/S was performed smoothly under general anesthesia and the baby had no problems. Immediately after the end of C/S, she went into sudden cardiovascular collapse and massive postpartum hemorrhage (PPH) became apparent. The mechanical ventilation with 100% oxygen was continued. Cardiovascular stabilization was attained with immediate administration of noradrenaline and blood transfusion. As her clinical course indicated coagulopathy due to disseminated intravascular coagulation (DIC), we gave transfusion of fresh frozen plasma and red cell concentrate before the diagnosis of DIC was established by laboratory tests. Since we thought that manual pressure and uterotonics were not adequate to stop PPH, we performed uterine artery embolization additionally. The PPH with DIC was stopped by these measures seven hours after C/S. The patient and her baby left the hospital with no complications. AFE is a rare and often fatal obstetric condition, characterized by sudden cardiovascular collapse, and massive bleeding with DIC. The prompt awareness and initiation of appropriate measures are mandatory for patient's survival.

[Anesthetic management of cardiac pheochromocytoma resection and coronary artery bypass grafting under cardiopulmonary bypass].

We experienced pheochromocytoma resection and coronary artery bypass grafting under cardiopulmonary bypass (CPB). The patient was a 69-year-old man who was first diagnosed with atherosclerotic angina. During operation, his blood pressure increased at induction and manipulation of the tumor under CPB, associated with an increased serum noradrenaline concentration. Starting operation, we monitored using transesophageal echocardiography (TEE), and used that view for diagnosis and anesthetic or hemodynamic management. It was especially useful after tumor resection. Surgical and hemodynamic management was facilitated by TEE. TEE was useful to make a diagnosis of cardiac pheochromocytoma, to determine the area of resection, to determine the surgical repair, and to make a decision of hemodynamic management in this complicated patient. We suggest that perfoming these cases under CPB and TEE is recommended for stabilization of hemodynamic states.

Validation of Trifluoromethylphenyl Diazirine Cholesterol Analogues As Cholesterol Mimetics and Photolabeling Reagents.

Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization.

This study examines how site-specific binding to three identified neurosteroid-binding sites in the α1ß3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3ß-epimer epi-allopregnanolone, binds to the canonical ß3(+)-α1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the ß3 subunit, promoting receptor desensitization and the α1 subunit promoting effects that vary between neurosteroids. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABAAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid-binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABAARs.

Common binding sites for cholesterol and neurosteroids on a pentameric ligand-gated ion channel.

Cholesterol is an essential component of cell membranes, and is required for mammalian pentameric ligand-gated ion channel (pLGIC) function. Computational studies suggest direct interactions between cholesterol and pLGICs but experimental evidence identifying specific binding sites is limited. In this study, we mapped cholesterol binding to Gloeobacter ligand-gated ion channel (GLIC), a model pLGIC chosen for its high level of expression, existing crystal structure, and previous use as a prototypic pLGIC. Using two cholesterol analogue photolabeling reagents with the photoreactive moiety on opposite ends of the sterol, we identified two cholesterol binding sites: an intersubunit site between TM3 and TM1 of adjacent subunits and an intrasubunit site between TM1 and TM4. In both the inter- and intrasubunit sites, cholesterol is oriented such that the 3­OH group points toward the center of the transmembrane domains rather than toward either the cytosolic or extracellular surfaces. We then compared this binding to that of the cholesterol metabolite, allopregnanolone, a neurosteroid that allosterically modulates pLGICs. The same binding pockets were identified for allopregnanolone and cholesterol, but the binding orientation of the two ligands was markedly different, with the 3­OH group of allopregnanolone pointing to the intra- and extracellular termini of the transmembrane domains rather than to their centers. We also found that cholesterol increases, whereas allopregnanolone decreases the thermal stability of GLIC. These data indicate that cholesterol and neurosteroids bind to common hydrophobic pockets in the model pLGIC, GLIC, but that their effects depend on the orientation and specific molecular interactions unique to each sterol.