Regulation and drug modulation of a voltage-gated sodium channel: Pivotal role of the S4-S5 linker in activation and slow inactivation.

Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4-S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4-S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4-S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed "door-wedge" action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.

Functional Human α7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coli.

Human Cys-loop receptors are important therapeutic targets. High-resolution structures are essential for rational drug design, but only a few are available due to difficulties in obtaining sufficient quantities of protein suitable for structural studies. Although expression of proteins in E. coli offers advantages of high yield, low cost, and fast turnover, this approach has not been thoroughly explored for full-length human Cys-loop receptors because of the conventional wisdom that E. coli lacks the specific chaperones and post-translational modifications potentially required for expression of human Cys-loop receptors. Here we report the successful production of full-length wild type human α7nAChR from E. coli Chemically induced chaperones promote high expression levels of well-folded proteins. The choice of detergents, lipids, and ligands during purification determines the final protein quality. The purified α7nAChR not only forms pentamers as imaged by negative-stain electron microscopy, but also retains pharmacological characteristics of native α7nAChR, including binding to bungarotoxin and positive allosteric modulators specific to α7nAChR. Moreover, the purified α7nAChR injected into Xenopus oocytes can be activated by acetylcholine, choline, and nicotine, inhibited by the channel blockers QX-222 and phencyclidine, and potentiated by the α7nAChR specific modulators PNU-120596 and TQS. The successful generation of functional human α7nAChR from E. coli opens a new avenue for producing mammalian Cys-loop receptors to facilitate structure-based rational drug design.

ELIC-α7 Nicotinic acetylcholine receptor (α7nAChR) chimeras reveal a prominent role of the extracellular-transmembrane domain interface in allosteric modulation.

The native α7 nicotinic acetylcholine receptor (α7nAChR) is a homopentameric ligand-gated ion channel mediating fast synaptic transmission and is of pharmaceutical interest for treatment of numerous disorders. The transmembrane domain (TMD) of α7nAChR has been identified as a target for positive allosteric modulators (PAMs), but it is unclear whether modulation occurs through changes entirely within the TMD or changes involving both the TMD and the extracellular domain (ECD)-TMD interface. In this study, we constructed multiple chimeras using the TMD of human α7nAChR and the ECD of a prokaryotic homolog, ELIC, which is not sensitive to these modulators, and for which a high resolution structure has been solved. Functional ELIC-α7nAChR (EA) chimeras were obtained when their ECD-TMD interfaces were modified to resemble either the ELIC interface (EAELIC) or α7nAChR interface (EAα7). Both EAα7 and EAELIC show similar activation response and desensitization characteristics, but only EAα7 retained the unique pharmacology of α7nAChR evoked by PAMs, including potentiation by ivermectin, PNU-120596, and TQS, as well as activation by 4BP-TQS. This study suggests that PAM modulation through the TMD has a more stringent requirement at the ECD-TMD interface than agonist activation.

NMR structures of the human α7 nAChR transmembrane domain and associated anesthetic binding sites.

The α7 nicotinic acetylcholine receptor (nAChR), assembled as homomeric pentameric ligand-gated ion channels, is one of the most abundant nAChR subtypes in the brain. Despite its importance in memory, learning and cognition, no structure has been determined for the α7 nAChR TM domain, a target for allosteric modulators. Using solution state NMR, we determined the structure of the human α7 nAChR TM domain (PDB ID: 2MAW) and demonstrated that the α7 TM domain formed functional channels in Xenopus oocytes. We identified the associated binding sites for the anesthetics halothane and ketamine; the former cannot sensitively inhibit α7 function, but the latter can. The α7 TM domain folds into the expected four-helical bundle motif, but the intra-subunit cavity at the extracellular end of the α7 TM domain is smaller than the equivalent cavity in the α4ß2 nAChRs (PDB IDs: 2LLY; 2LM2). Neither drug binds to the extracellular end of the α7 TM domain, but two halothane molecules or one ketamine molecule binds to the intracellular end of the α7 TM domain. Halothane and ketamine binding sites are partially overlapped. Ketamine, but not halothane, perturbed the α7 channel-gate residue L9'. Furthermore, halothane did not induce profound dynamics changes in the α7 channel as observed in α4ß2. The study offers a novel high-resolution structure for the human α7 nAChR TM domain that is invaluable for developing α7-specific therapeutics. It also provides evidence to support the hypothesis: only when anesthetic binding perturbs the channel pore or alters the channel motion, can binding generate functional consequences.

Photoaffinity labeling the propofol binding site in GLIC.

Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homologue from Gloeobacter violaceus . In the structure of GLIC cocrystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices (Nury, H, et al. (2011) Nature 469, 428-431). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol or AziPm) that acts as an anesthetic in vivo and potentiates GABAAR in vitro. For GLIC expressed in Xenopus oocytes, propofol and AziPm inhibited current responses at pH 5.5 (EC20) with IC50 values of 20 and 50 µM, respectively. When [(3)H]AziPm (7 µM) was used to photolabel detergent-solubilized, affinity-purified GLIC at pH 4.4, protein microsequencing identified propofol-inhibitable photolabeling of three residues in the GLIC transmembrane domain: Met-205, Tyr-254, and Asn-307 in the M1, M3, and M4 transmembrane helices, respectively. Thus, for GLIC in solution, propofol and AziPm bind competitively to a site in proximity to these residues, which, in the GLIC crystal structure, are in contact with the propofol bound in the intrasubunit pocket.

Reversal of ion-charge selectivity renders the pentameric ligand-gated ion channel GLIC insensitive to anaesthetics.

pLGICs (pentameric ligand-gated ion channels) are a family of structurally homologous cation and anion channels involved in neurotransmission. Cation-selective members of the pLGIC family are typically inhibited by general anaesthetics, whereas anion-selective members are potentiated. GLIC is a prokaryotic cation pLGIC and can be inhibited by clinical concentrations of general anaesthetics. The introduction of three mutations, Y221A (Y-3'A), E222P (E-2'P) and N224R (N0'R), at the selectivity filter and one, A237T (A13'T), at the hydrophobic gate, converted GLIC into an anion channel. The mutated GLIC (GLIC4) became insensitive to the anaesthetics propofol and etomidate, as well as the channel blocker picrotoxin. MD (molecular dynamics) simulations revealed changes in the structure and dynamics of GLIC4 in comparison with GLIC, particularly in the tilting angles of the pore-lining helix [TM2 (transmembrane helix 2)] that consequently resulted in different pore radius and hydration profiles. Propofol binding to an intra-subunit site of GLIC shifted the tilting angles of TM2 towards closure at the hydrophobic gate region, consistent with propofol inhibition of GLIC. In contrast, the pore of GLIC4 was much more resilient to perturbation from propofol binding. The present study underscores the importance of pore dynamics and conformation to anaesthetic effects on channel functions.

Unconventional PDZ Recognition Revealed in α7 nAChR-PICK1 Complexes.

PDZ domains are modular domains that conventionally bind to C terminal or internal motifs of target proteins to control cellular functions through the regulation of protein complex assemblies. Almost all reported structures of PDZ-target protein complexes rely on fragments or peptides as target proteins. No intact target protein complexed with PDZ was structurally characterized. In this study, we used NMR spectroscopy and other biochemistry and biophysics tools to uncover insights into structural coupling between the PDZ domain of protein interacting with C-kinase 1 (PICK1) and α7 nicotinic acetylcholine receptors (α7 nAChR). Notably, the intracellular domains of both α7 nAChR and PICK1 PDZ exhibit a high degree of plasticity in their coupling. Specifically, the MA helix of α7 nAChR interacts with residues lining the canonical binding site of the PICK1 PDZ, while flexible loops also engage in protein-protein interactions. Both hydrophobic and electrostatic interactions mediate the coupling. Overall, the resulting structure of the α7 nAChR-PICK1 complex reveals an unconventional PDZ binding mode, significantly expanding the repertoire of functionally important PDZ interactions.

NMR structures of the transmembrane domains of the α4ß2 nAChR.

The α4ß2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4ß2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and ß2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and ß2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na(+) flux assay revealed that α4ß2 formed Na(+) permeable channels in lipid vesicles. Efflux of Na(+) through the α4ß2 channels reduced intra-vesicle Sodium Green™ fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4ß2. The study provides structural insight into the TM domains of the α4ß2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4ß2 nAChR and for designing new therapeutic modulators.

NMR structure and dynamics of a designed water-soluble transmembrane domain of nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828-1833, 2004; Ma et al., PNAS, 105: 16537-42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ~83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T(2) dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.

SARS-CoV-2 Spike Protein Downregulates Cell Surface α7nAChR through a Helical Motif in the Spike Neck.

A deficiency of the functional α7 nicotinic acetylcholine receptor (α7nAChR) impairs neuronal and immune systems. The SARS-CoV-2 spike protein (S12) facilitates virus cell entry during COVID-19 infection and can also independently disrupt cellular functions. Here, we found that S12 expression significantly downregulated surface expression of α7nAChR in mammalian cells. A helical segment of S12 (L1145-L1152) in the spike neck was identified to be responsible for the downregulation of α7nAChR, as the mutant S12AAA (L1145A-F1148A-L1152A) had minimal effects on surface α7nAChR expression. This S12 segment is homologous to the α7nAChR intracellular helical motif known for binding chaperone proteins RIC3 and Bcl-2 to promote α7nAChR surface expression. Competition from S12 for binding these proteins likely underlies suppression of surface α7nAChR. Considering the critical roles of α7nAChR in cellular functions, these findings provide a new perspective for improving mRNA vaccines and developing treatment options for certain symptoms related to long COVID.

Structural Elucidation of Ivermectin Binding to α7nAChR and the Induced Channel Desensitization.

The α7 nicotinic acetylcholine receptor (α7nAChR) mediates signaling in the central nervous system and cholinergic anti-inflammatory pathways. Ivermectin is a positive allosteric modulator of a full-length α7nAChR and an agonist of the α7nAChR construct containing transmembrane (TMD) and intracellular (ICD) domains, but structural insights of the binding have not previously been determined. Here, combining nuclear magnetic resonance as a primary experimental tool with Rosetta comparative modeling and molecular dynamics simulations, we have revealed details of ivermectin binding to the α7nAChR TMD + ICD and corresponding structural changes in an ivermectin-induced desensitized state. Ivermectin binding was stabilized predominantly by hydrophobic interactions from interfacial residues between adjacent subunits near the extracellular end of the TMD, where the inter-subunit gap was substantially expanded in comparison to the apo structure. The ion-permeation pathway showed a profile distinctly different from the resting-state profile but similar to profiles of desensitized α7nAChR. The ICD also exhibited structural changes, including reorientation of the MX and h3 helices relative to the channel axis. The resulting structures of the α7nAChR TMD + ICD in complex with ivermectin provide opportunities for discovering new modulators of therapeutic potential and exploring the structural basis of cytoplasmic signaling under different α7nAChR functional states.

Structures of highly flexible intracellular domain of human α7 nicotinic acetylcholine receptor.

The intracellular domain (ICD) of Cys-loop receptors mediates diverse functions. To date, no structure of a full-length ICD is available due to challenges stemming from its dynamic nature. Here, combining nuclear magnetic resonance (NMR) and electron spin resonance experiments with Rosetta computations, we determine full-length ICD structures of the human α7 nicotinic acetylcholine receptor in a resting state. We show that ~57% of the ICD residues are in highly flexible regions, primarily in a large loop (loop L) with the most mobile segment spanning ~50 Å from the central channel axis. Loop L is anchored onto the MA helix and virtually forms two smaller loops, thereby increasing its stability. Previously known motifs for cytoplasmic binding, regulation, and signaling are found in both the helices and disordered flexible regions, supporting the essential role of the ICD conformational plasticity in orchestrating a broad range of biological processes.

NMR structure of the transmembrane domain of the n-acetylcholine receptor beta2 subunit.

Nicotinic acetylcholine receptors (nAChRs) are involved in fast synaptic transmission in the central and peripheral nervous system. Among the many different types of subunits in nAChRs, the beta2 subunit often combines with the alpha4 subunit to form alpha4beta2 pentameric channels, the most abundant subtype of nAChRs in the brain. Besides computational predictions, there is limited experimental data available on the structure of the beta2 subunit. Using high-resolution NMR spectroscopy, we solved the structure of the entire transmembrane domain (TM1234) of the beta2 subunit. We found that TM1234 formed a four-helix bundle in the absence of the extracellular and intracellular domains. The structure exhibited many similarities to those previously determined for the Torpedo nAChR and the bacterial ion channel GLIC. We also assessed the influence of the fourth transmembrane helix (TM4) on the rest of the domain. Although secondary structures and tertiary arrangements were similar, the addition of TM4 caused dramatic changes in TM3 dynamics and subtle changes in TM1 and TM2. Taken together, this study suggests that the structures of the transmembrane domains of these proteins are largely shaped by determinants inherent in their sequence, but their dynamics may be sensitive to modulation by tertiary and quaternary contacts.

NMR studies of a channel protein without membranes: structure and dynamics of water-solubilized KcsA.

Structural studies of polytopic membrane proteins are often hampered by the vagaries of these proteins in membrane mimetic environments and by the difficulties in handling them with conventional techniques. Designing and creating water-soluble analogues with preserved native structures offer an attractive alternative. We report here solution NMR studies of WSK3, a water-soluble analogue of the potassium channel KcsA. The WSK3 NMR structure (PDB ID code 2K1E) resembles the KcsA crystal structures, validating the approach. By more stringent comparison criteria, however, the introduction of several charged residues aimed at improving water solubility seems to have led to the possible formations of a few salt bridges and hydrogen bonds not present in the native structure, resulting in slight differences in the structure of WSK3 relative to KcsA. NMR dynamics measurements show that WSK3 is highly flexible in the absence of a lipid environment. Reduced spectral density mapping and model-free analyses reveal dynamic characteristics consistent with an isotropically tumbling tetramer experiencing slow (nanosecond) motions with unusually low local ordering. An altered hydrogen-bond network near the selectivity filter and the pore helix, and the intrinsically dynamic nature of the selectivity filter, support the notion that this region is crucial for slow inactivation. Our results have implications not only for the design of water-soluble analogues of membrane proteins but also for our understanding of the basic determinants of intrinsic protein structure and dynamics.

Functional Tolerance to Cysteine Mutations in Human α7 Nicotinic Acetylcholine Receptors.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is involved in various intracellular signaling pathways that mediate addiction, chronic pain, and other diseases, but its intracellular domain structures remain undetermined. The presence of 17 native cysteines in α7 nAChR provides opportunities for extracting structural information through site-directed labeling of chemical probes in strategic locations, but it also creates uncertainties in channel function when those native cysteines must be mutated. Using site-directed mutagenesis and two-electrode voltage clamp electrophysiology measurements, we found that α7 nAChR's function was well tolerated for mutations of all 13 cysteines as long as two pairs of disulfide-bond cysteines remained in the extracellular domain. Furthermore, surface plasmon resonance measurements showed that the cysteine mutations did not affect α7 nAChR binding to the intracellular protein PICK1. The study suggests that a high native cysteine content does not necessarily preclude the use of single cysteine labeling for acquiring structural information on functional proteins.

A Thermal Place Preference Test for Discovery of Neuropathic Pain Drugs.

Developing potent non-opioid pain medications is an integral part of the battle to conquer both chronic pain and the current opioid crisis. Although most screening approaches use in vitro surrogate targets, in vivo screening of analgesic candidates is a necessary preclinical step in drug discovery. Here, we report the design of a new automated behavioral testing apparatus based on the principle of a thermal place preference test (TPPT). This new design can detect, quantify, and differentiate behavioral responses to cold stimuli between sham and chronic constriction injury (CCI) rodents with up to 12 animals tested simultaneously. At an optimized temperature pair of 12.5 °C vs 30.0 °C (±0.5 °C), the TPPT design has captured the antinociceptive effects of morphine and pregabalin on CCI rats in individual 10 min tests. Moreover, it can differentiate analgesic effects by morphine or pregabalin from anxiolytic effects by diazepam. The results, along with the relatively low cost to construct the apparatus and moderately high throughput, make our TPPT design applicable for behavioral studies of chronic pain in rodents and for high-throughput in vivo screening of the next generation of pain medications.

Structural basis of neurosteroid anesthetic action on GABAA receptors.

Type A γ-aminobutyric acid receptors (GABAARs) are inhibitory pentameric ligand-gated ion channels in the brain. Many anesthetics and neurosteroids act through binding to the GABAAR transmembrane domain (TMD), but the structural basis of their actions is not well understood and no resting-state GABAAR structure has been determined. Here, we report crystal structures of apo and the neurosteroid anesthetic alphaxalone-bound desensitized chimeric α1GABAAR (ELIC-α1GABAAR). The chimera retains the functional and pharmacological properties of GABAARs, including potentiation, activation and desensitization by alphaxalone. The apo-state structure reveals an unconventional activation gate at the intracellular end of the pore. The desensitized structure illustrates molecular determinants for alphaxalone binding to an inter-subunit TMD site. These structures suggest a plausible signaling pathway from alphaxalone binding at the bottom of the TMD to the channel gate in the pore-lining TM2 through the TM1-TM2 linker. The study provides a framework to discover new GABAAR modulators with therapeutic potential.

Structural Basis of Alcohol Inhibition of the Pentameric Ligand-Gated Ion Channel ELIC.

The structural basis for alcohol modulation of neuronal pentameric ligand-gated ion channels (pLGICs) remains elusive. We determined an inhibitory mechanism of alcohol on the pLGIC Erwinia chrysanthemi (ELIC) through direct binding to the pore. X-ray structures of ELIC co-crystallized with 2-bromoethanol, in both the absence and presence of agonist, reveal 2-bromoethanol binding in the pore near T237(6') and the extracellular domain (ECD) of each subunit at three different locations. Binding to the ECD does not appear to contribute to the inhibitory action of 2-bromoethanol and ethanol as indicated by the same functional responses of wild-type ELIC and mutants. In contrast, the ELIC-α1ß3GABAAR chimera, replacing the ELIC transmembrane domain (TMD) with the TMD of α1ß3GABAAR, is potentiated by 2-bromoethanol and ethanol. The results suggest a dominant role of the TMD in modulating alcohol effects. The X-ray structures and functional measurements support a pore-blocking mechanism for inhibitory action of short-chain alcohols.

Sequence and helicity requirements for the proapoptotic activity of Bax BH3 peptides.

Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-XL is commonly observed in human malignancies and contributes to chemotherapy and radiation resistance. Bcl-2 and Bcl-XL inhibit apoptosis by binding to proapoptotic proteins such as Bax, thereby preventing chemotherapy-induced or radiation-induced release of cytochrome c from mitochondria and subsequent activation of the caspase protease cascade. Efforts to inhibit Bcl-2 or Bcl-XL function in tumor cells have focused on developing agents to inhibit the interactions of these proteins with proapoptotic proteins. Peptides derived from the BH3 domains of proapoptotic proteins have been shown to disrupt the interactions of Bcl-2 and Bcl-XL with key binding partners in cell-free reactions and to promote cellular apoptosis. However, less is known about the targets of BH3 peptides in intact cells as well as the sequence, length, and conformational requirements for peptide biological activity. In this report, we show that cell-permeable Bax BH3 peptides physically disrupt Bax/Bcl-2 heterodimerization in intact cells and that this disruption correlates with peptide-induced cell death. A point-mutant, control peptide that failed to disrupt intracellular Bax/Bcl-2 interactions also failed to promote apoptosis. To determine important sequence, length, and structural requirements for peptide activity, we generated and systematically analyzed the biological activities of 17 Bax BH3 peptide variants. Peptides were quantitatively examined for their ability to inhibit Bax/Bcl-2 and Bax/Bcl-XL heterodimerization in vitro and to promote cytochrome c release from mitochondria isolated from Jurkat, HL-60, U937, and PC-3 cells. Our results define 15 amino acids as the minimal length required for Bax BH3 peptide biological activity and show that amino acids COOH terminal to the BH3 core sequence are less critical than those located NH2 terminal to the core. In addition, circular dichroism spectroscopy revealed that high alpha-helical content generally correlated with, but was not sufficient for, peptide activity. Taken together, these studies provide a basis for future optimization of Bax BH3 peptide as a therapeutic anticancer agent.

Ensemble-based virtual screening for cannabinoid-like potentiators of the human glycine receptor α1 for the treatment of pain.

The human glycine receptors (hGlyRs) are chloride-selective ion channels that mediate inhibitory neurotransmission in the brain stem and spinal cord. They are also targets for compounds of potential use in analgesic therapies. Here, we develop a strategy to discover analgesic drugs via structure-based virtual screening based on the recently published NMR structure of the hGlyR-α1 transmembrane domain (PDB ID: 2M6I ) and the critical role of residue S296 in hGlyR-α1 potentiation by Δ(9)-tetrahydrocannabinol (THC). We screened 1549 FDA-approved drugs in the DrugBank database on an ensemble of 180 hGlyR-α1 structures generated from molecular dynamics simulations of the NMR structure of the hGlyR-α1 transmembrane domain in different lipid environments. Thirteen hit compounds from the screening were selected for functional validation in Xenopus laevis oocytes expressing hGlyR-α1. Only one compound showed no potentiation effects; seven potentiated hGlyR-α1 at a level greater than THC at 1 µM. Our virtual screening protocol is generally applicable to drug targets with lipid-facing binding sites.