Identification of Significant Amino Acids in Multiple Transmembrane Domains of Human Transient Receptor Potential Ankyrin 1 (TRPA1) for Activation by Eudesmol,an Oxygenized Sesquiterpene in Hop Essential Oil

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is activated by various noxious or irritant substances in nature, including spicy compounds. Many TRPA1 chemical activators have been reported; however, only limited information is available regarding the amino acid residues that contribute to the activation by non-electrophilic activators, whereas activation mechanisms by electrophilic ligands have been well characterized. We used intracellular Ca²⁺ measurements and whole-cell patch clamp recordings to show that eudesmol, an oxygenated sesquiterpene present at high concentrations in the essential oil of hop cultivar Hallertau Hersbrucker, could activate human TRPA1. Gradual activation of inward currents with outward rectification by eudesmol was observed in human embryonic kidney-derived 293 cells expressing human TRPA1. This activation was completely blocked by a TRPA1-specific inhibitor, HC03–0031. We identified three critical amino acid residues in human TRPA1 in putative transmembrane domains 3, 4, and 5, namely threonine at 813, tyrosine at 840, and serine at 873, for activation by β-eudesmol in a systematic mutational study. Our results revealed a new TRPA1 activator in hop essential oil and provide a novel insight into mechanisms of human TRPA1 activation by non-electrophilic chemicals.     

TRPA1 channels: novel targets of 1,4-dihydropyridines

Transient receptor potential type A1 (TRPA1) channels are cation permeable channels activated by irritant chemicals and pungent natural compounds. Their location in peptidergic sensory terminals innervating the skin and blood vessels makes them important effectors of vasodilator responses of neural origin. 1,4-dihydropyridines are a class of L-type calcium channel antagonists commonly used in the treatment of hypertension and ischemic heart disease. Here we show that four different 1,4-dihydropyridines (nifedipine, nimodipine, nicardipine and nitrendipine), and the structurally related L-type calcium channel agonist BayK8644, exert powerful excitatory effects on TRPA1 channels. The activation does not depend on elevated Ca2+ levels and cross-desensitizes with that produced by other TRPA1 agonists. The activation produced by nifedipine was reduced by camphor and the selective TRPA1 antagonist HC03001. In a subclass of mouse nociceptors expressing TRPA1 channels, assessed by responses to the TRPA1 agonist mustard oil, nifedipine also produced large elevations in [Ca2+](i). These responses were fully abrogated in TRPA1(-/-) mice. These findings identify TRPA1 channels as a new molecular target for the 1,4-dihydropyridine class of calcium channel modulators.     

Molecular architecture and subunit organization of TRPA1 ion channel revealed by electron microscopy

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective ion channel, which is expressed in nociceptor sensory neurons and transduces chemical, inflammatory, and neuropathic pain signals. Numerous non-reactive compounds and electrophilic compounds, such as endogenous inflammatory mediators and exogenous pungent chemicals, can activate TRPA1. Here we report a 16-? resolution structure of purified, functional, amphipol-stabilized TRPA1 analyzed by single-particle EM. Molecular models of the N and C termini of the channel were generated using the I-TASSER protein structure prediction server and docked into the EM density to provide insight into the TRPA1 subunit organization. This structural analysis suggests a location for critical N-terminal cysteine residues involved in electrophilic activation at the interface between neighboring subunits. Our results indicate that covalent modifications within this pocket may alter interactions between subunits and promote conformational changes that lead to channel activation.     

Molecular Basis for Benzodiazepine Agonist Action at the Type 1 Cholecystokinin Receptor

Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu^7.39 that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a “trigger” for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu^7.39, whereas the role of this residue was less clear for chemically distinct agonists.     

Voltage-dependent modulation of TRPA1 currents by diphenhydramine

Diphenhydramine (DPH) has been broadly used to treat allergy. When used as a topical medicine, DPH temporarily relieves itching and pain. Although transient receptor potential type A1 (TRPA1) channel is known to play roles in both acute and chronic itch and pain, whether DPH affects the activities of TRPA1 remains unclear. Using whole-cell patch clamp recordings, we demonstrated that DPH modulates the voltage-dependence of TRPA1. When co-applied with a TRPA1 agonist, DPH significantly enhanced the inward currents while suppressing the outward currents of TRPA1, converting the channel from outwardly rectifying to inwardly rectifying. This effect of DPH occurred no matter TRPA1 was activated by an electrophilic or non-electrophilic agonist and for both mouse and human TRPA1. The modulation of TRPA1 by DPH was maintained in the L906C mutant, which by itself also causes inward rectification of TRPA1, indicating that additional acting sites are present for the modulation of TRPA1 currents by DPH. Our recordings also revealed that DPH partially blocked capsaicin evoked TRPV1 currents. These data suggest that DPH may exert its therapeutic effects on itch and pain, through modulation of TRPA1 in a voltage-dependent fashion.     

Structure and activity of membrane receptors: Modeling and computational simulation of ligand recognition in a three-dimensional model of the 5-hydroxytryptamine1A receptor

A three-dimensional molecular model of the transmembrane domain of the 5-HT_1A receptor (5-HT_1AR) is presented in the context of a general strategy for modeling the macromolecular structure of a guanine nucleotide binding, regulatory protein coupled receptor (GPCR). The model of the 5-HT_1AR rests on the definition of the putative residues of the ligand-binding site guided by criteria based on specific models proposed from structure-activity studies and on published results of modifications of GPCRs using methods of molecular biology. The resulting requirements for matching recognition sites in the agonist-binding pocket define the molecular details of the interaction between the agonist 5-HT and the human 5-HT_1AR that includes: (1) the interaction between the protonated amine moiety and the conserved negative Asp-116, located in TMH 3; (2) the hydrogen bond between the hydroxyl group and Thr-199, located in TMH 5; and (3) the interaction complex between the aromatic ring portion of the ligand and the neutral form of His-192, located in TMH 5. Results from quantum mechanical calculations of the interaction between an agonist and the proposed recognition pocket of the 5-HT_1AR model suggest a trigger of the receptor activation mechanism resulting from ligand binding. The antagonist-binding pocket of the human 5-HT_1AR is inferred from the interaction sites of pindolol with the receptor model: (1) the ionic interaction between the protonated amine of the ligand and the side chain of the conserved Asp-116, located in TMH 3; and (2) the hydrogen bonds between the ether oxygen and the hydroxyl group of the ligand and Asn-385, located in TMH 7. Use of the model is proposed to facilitate the identification of the structural elements of agonists and antagonists that are key for their specific functions, in order to achieve the design of new compounds with predetermined pharmacological properties.     

Diallyl sulfides in garlic activate both TRPA1 and TRPV1

We searched for novel agonists of TRP receptors especially for TRPA1 and TRPV1 in foods. We focused attention on garlic compounds, diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). In TRPA1 or TRPV1 heterogeneously expressed CHO cells, all of those compounds increased [Ca²⁺]_i in concentration-dependent manner. The EC₅₀ values of DADS and DATS were similar to that of allyl isothiocyanate (AITC) and that of DAS was 170-fold larger than that of AITC. Maximum responses of these sulfides were equal to that of AITC. The EC₅₀ values of these compounds for TRPV1 were around 100 μM against that of capsaicin (CAP), 25.6 nM and maximum responses of garlic compounds were half to that of CAP. The Ca²⁺ responses were significantly suppressed by co-application of antagonist. We conclude that DAS, DADS, and DATS are agonist of both TRPA1 and TRPV1 but with high affinity for TRPA1.     

Salak Plum Peel Extract as a Safe and Efficient Antioxidant Appraisal for Cosmetics

We searched in this study for novel agonists of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and transient receptor potential cation channel, subfamily A, member 1 (TRPA1) in pepper, focusing attention on 19 compounds contained in black pepper. Almost all the compounds in HEK cells heterogeneously expressed TRPV1 or TRPA1, increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a concentration-dependent manner. Among these, piperine, isopiperine, isochavicine, piperanine, pipernonaline, dehydropipernonaline, retrofractamide C, piperolein A, and piperolein B relatively strongly activated TRPV1. The EC₅₀ values of these compounds for TRPV1 were 0.6–128 μM. Piperine, isopiperine, isochavicine, piperanine, piperolein A, piperolein B, and N-isobutyl-(2E,4E)-tetradeca-2,4-diamide also relatively strongly activated TRPA1, the EC₅₀ values of these compounds for TRPA1 were 7.8–148 μM. The Ca²⁺ responses of these compounds for TRPV1 and TRPA1 were significantly suppressed by co-applying each antagonist. We identified in this study new transient receptor potential (TRP) agonists present in black pepper and found that piperine, isopiperine, isochavicine, piperanine, piperolein A, and piperolein B activated both TRPV1 and TRPA1.     

Novel menthol-derived cooling compounds activate primary and second-order trigeminal sensory neurons and modulate lingual thermosensitivity

We presently investigated 2 novel menthol derivatives GIV1 and GIV2, which exhibit strong cooling effects. In previous human psychophysical studies, GIV1 delivered in a toothpaste medium elicited a cooling sensation that was longer lasting compared with GIV2 and menthol carboxamide (WS-3). In the current study, we investigated the molecular and cellular effects of these cooling agents. In calcium flux studies of TRPM8 expressed in HEK cells, both GIV1 and GIV2 were approximately 40- to 200-fold more potent than menthol and WS-3. GIV1 and GIV2 also activated TRPA1 but at levels that were 400 times greater than those required for TRPM8 activation. In calcium imaging studies, subpopulations of cultured rat trigeminal ganglion and dorsal root ganglion cells responded to GIV1 and/or GIV2; the majority of these were also activated by menthol and some were additionally activated by the TRPA1 agonist cinnamaldehyde and/or the TRPV1 agonist capsaicin. We also made in vivo single-unit recordings from cold-sensitive neurons in rat trigeminal subnucleus caudalis (Vc). GIV 1 and GIV2 directly excited some Vc neurons, GIV1 significantly enhanced their responses to cooling, and both GIV1 and GIV2 reduced responses to noxious heat. These novel cooling compounds provide additional molecular tools to investigate the neural processes of cold sensation.     

Expression of functional TRPA1 receptor on human lung fibroblast and epithelial cells

The transient receptor potential subfamily A member 1 (TRPA1) is a non-selective cation channel implicated in the pathogenesis of several airway diseases like asthma and chronic obstructive pulmonary disease (COPD). Most of the research on TRPA1 focuses on its expression and function in neuronal context; studies investigating non-neuronal expression of TRPA1 are lacking. In the present study, we show functional expression of TRPA1 in human lung fibroblast cells (CCD19-Lu) and human pulmonary alveolar epithelial cell line (A549). We demonstrate TRPA1 expression at both mRNA and protein levels in these cell types. TRPA1 selective agonists like allyl isothiocyanate (AITC), 4-hydroxynonenal (4-HNE), crotonaldehyde and zinc, induced a concentration-dependent increase in Ca⁺² influx in CCD19-Lu and A549 cells. AITC-induced Ca⁺² influx was inhibited by Ruthenium red (RR), a TRP channel pore blocker, and by GRC 17536, a TRPA1 specific antagonist. Furthermore, we also provide evidence that activation of the TRPA1 receptor by TRPA1 selective agonists promotes release of the chemokine IL-8 in CCD19-Lu and A549 cells. The IL-8 release in response to TRPA1 agonists was attenuated by TRPA1 selective antagonists. In conclusion, we demonstrate here for the first time that TRPA1 is functionally expressed in cultured human lung fibroblast cells (CCD19-Lu) and human alveolar epithelial cell line (A549) and may have a potential role in modulating release of this important chemokine in inflamed airways.     

Expression of functional TRPA1 receptor on human lung fibroblast and epithelial cells

The transient receptor potential subfamily A member 1 (TRPA1) is a non-selective cation channel implicated in the pathogenesis of several airway diseases like asthma and chronic obstructive pulmonary disease (COPD). Most of the research on TRPA1 focuses on its expression and function in neuronal context; studies investigating non-neuronal expression of TRPA1 are lacking. In the present study, we show functional expression of TRPA1 in human lung fibroblast cells (CCD19-Lu) and human pulmonary alveolar epithelial cell line (A549). We demonstrate TRPA1 expression at both mRNA and protein levels in these cell types. TRPA1 selective agonists like allyl isothiocyanate (AITC), 4-hydroxynonenal (4-HNE), crotonaldehyde and zinc, induced a concentration-dependent increase in Ca+2 influx in CCD19-Lu and A549 cells. AITC-induced Ca+2 influx was inhibited by Ruthenium red (RR), a TRP channel pore blocker, and by GRC 17536, a TRPA1 specific antagonist. Furthermore, we also provide evidence that activation of the TRPA1 receptor by TRPA1 selective agonists promotes release of the chemokine IL-8 in CCD19-Lu and A549 cells. The IL-8 release in response to TRPA1 agonists was attenuated by TRPA1 selective antagonists. In conclusion, we demonstrate here for the first time that TRPA1 is functionally expressed in cultured human lung fibroblast cells (CCD19-Lu) and human alveolar epithelial cell line (A549) and may have a potential role in modulating release of this important chemokine in inflamed airways.     

Structural insights into ligand-binding pocket formation in Nurr1 by molecular dynamics simulations

Abstract

The nuclear receptor Nurr1 (NR4A2) has been identified as a potential target for the treatment of Parkinson’s disease. In contrast to most other nuclear receptors, the X-ray crystal structure of the Nurr1 ligand-binding domain (LBD) lacks any ligand-binding pocket (LBP). However, NMR spectroscopy measurements have revealed that the known Nurr1 agonist docosahexaenoic acid (DHA) binds to a region within the LBD that corresponds to the classical NR ligand-binding pocket (LBP). In order to investigate the structural dynamics of the Nurr1 LBD and to study potential LBP formation, the conformational space of the receptor was sampled using a molecular dynamics (MD) simulation. Docking of DHA into 50,000 LBD structures extracted from the simulation revealed the existence of a transient LBP that is capable to fully harbor the compound. The location of the identified pocket overlaps with the ligand-binding site suggested by NMR experiments. Structural analysis of the protein-ligand complex showed that only modest structural rearrangements within the Nurr1 LBD are required for LBP formation. These findings may support structure-based drug discovery campaigns for the development of receptor-specific agonists.     

Artepillin C,a Major Ingredient of Brazilian Propolis,Induces a Pungent Taste by Activating TRPA1 Channels

Brazilian green propolis is a popular health supplement because of its various biological properties. The ethanol extract of Brazilian green propolis (EEBP) is characteristic for its herb-like smell and unique pungent taste. However, the ingredients responsible for its pungency have not yet been identified. This study provides the first evidence that artepillin C is the main pungent ingredient in EEBP and that it potently activates human transient receptor potential ankyrin 1 (TRPA1) channels. EEBP was fractionated using column chromatography with a step gradient elution of an ethanol-water solution, and the fractions having the pungent taste were determined by sensory tests. HPLC analysis revealed that the pungent fraction was composed primarily of artepillin C, a prenylated derivative of cinnamic acid. Artepillin C was also identified as the pungent compound of EEBP by organoleptic examiners. Furthermore, the effects of artepillin C and other cinnamic acids found in EEBP on TRPA1 channels were examined by calcium imaging and plate reader-based assays in human TRPA1-expressing cells to investigate the molecular mechanisms underlying their pungent tastes. Artepillin C and baccharin activated the TRPA1 channel strongly, whereas drupanin caused a slight activation and p-coumaric acid showed no activation. Because the EC₅₀ values of artepillin C, baccharin, and allyl isothiocyanate were 1.8 µM, 15.5 µM, and 6.2 µM, respectively, artepillin C was more potent than the typical TRPA1 agonist allyl isothiocyanate. These findings strongly indicate that artepillin C is the main pungent ingredient in EEBP and stimulates a pungent taste by activating TRPA1 channels.     

Mechanism of partial agonism at the GluR2 AMPA receptor: Measurements of lobe orientation in solution

The mechanism by which the binding of a neurotransmitter to a receptor leads to channel opening is a central issue in molecular neurobiology. The structure of the agonist binding domain of ionotropic glutamate receptors has led to an improved understanding of the changes in structure that accompany agonist binding and have provided important clues about the link between these structural changes and channel activation and desensitization. However, because the binding domain has exhibited different structures under different crystallization conditions, understanding the structure in the absence of crystal packing is of considerable importance. The orientation of the two lobes of the binding domain in the presence of a full agonist, an antagonist, and several partial agonists was measured using NMR spectroscopy by employing residual dipolar couplings. For some partial agonists, the solution conformation differs from that observed in the crystal. A model of channel activation based on the results is discussed.     

Identification of in vivo disulfide conformation of TRPA1 ion channel

TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys-622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 μM versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.     

Plasticity of the ligand binding pocket in the bitter taste receptor T2R7

Bitter taste receptors (T2Rs) are a group of 25 G protein-coupled receptors (GPCRs) in humans. The cognate agonists and the mechanism of ligand binding to the majority of the T2Rs remain unknown. Here we report the first structure-function analysis of T2R7 and study the ability of this receptor to bind to different agonists by site-directed mutagenesis. Screening of ligands for T2R7 in calcium based assays lead to the identification of novel compounds that activate this receptor. Quinine, diphenidol, dextromethorphan and diphenhydramine showed substantial activation of T2R7. Interestingly, these bitter compounds showed different pharmacological characteristics. To investigate the structural features in T2R7 that might contribute to the observed differences in agonist specificities, molecular model guided ligand docking and site-directed mutagenesis was pursued. Amino acids D65, D86, W89, N167, T169, W170, S181, T255 and E271 in the ligand-binding pocket were replaced and the mutants characterized pharmacologically. Our results suggest D86, S181 and W170 present on the extracellular side of transmembrane 3 (TM3), TM5 and in extracellular loop 2 (ECL2) are essential for agonist binding in T2R7. Mutations of these amino acids lead to loss-of-function. We also identified gain-of-function residues that are agonist specific. These results suggest that agonists bind at an extracellular site rather than deep within the TM core involving residues present in both ECL2 and TM helices in T2R7. Similar to majority of the Class A GPCRs, ECL2 in T2R7 plays a significant role in agonist binding and activation.     

Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation

Background

The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1.

Methodology/Principal Findings

By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells and fibroblasts, acrolein and CS extract evoked IL-8 release, a response selectively reduced by TRPA1 antagonists. Capsaicin, agonist of the transient receptor potential vanilloid 1 (TRPV1), a channel co-expressed with TRPA1 by airway sensory nerves, and acrolein or CS (TRPA1 agonists), or the neuropeptide substance P (SP), which is released from sensory nerve terminals by capsaicin, acrolein or CS), produced neurogenic inflammation in mouse airways. However, only acrolein and CS, but not capsaicin or SP, released the keratinocyte chemoattractant (CXCL-1/KC, IL-8 analogue) in bronchoalveolar lavage (BAL) fluid of wild-type mice. This effect of TRPA1 agonists was attenuated by TRPA1 antagonism or in TRPA1-deficient mice, but not by pharmacological ablation of sensory nerves.

Conclusions

Our results demonstrate that, although either TRPV1 or TRPA1 activation causes airway neurogenic inflammation, solely TRPA1 activation orchestrates an additional inflammatory response which is not neurogenic. This finding suggests that non-neuronal TRPA1 in the airways is functional and potentially capable of contributing to inflammatory airway diseases.     

The Met268Pro mutation of mouse TRPA1 changes the effect of caffeine from activation to suppression

The transient receptor potential A1 channel (TRPA1) is activated by various compounds, including isothiocyanates, menthol, and cinnamaldehyde. The sensitivities of the rodent and human isoforms of TRPA1 to menthol and the cysteine-attacking compound CMP1 differ, and the molecular determinants for these differences have been identified in the 5th transmembrane region (TM5) for menthol and TM6 for CMP1. We recently reported that caffeine activates mouse TRPA1 (mTRPA1) but suppresses human TRPA1 (hTRPA1). Here we aimed to identify the molecular determinant that is responsible for species-specific differences in the response to caffeine by analyzing the functional properties of various chimeras expressed in Xenopus oocytes. We initially found that the region between amino acids 231 and 287, in the distal N-terminal cytoplasmic region of mTRPA1, is critical. In a mutagenesis study of this region, we subsequently observed that introduction of a Met268Pro point mutation into mTRPA1 changed the effect of caffeine from activation to suppression. Because the region including Met-268 is different from other reported ligand-binding sites and from the EF-hand motif, these results suggest that the caffeine response is mediated by a unique mechanism, and confirm the importance of the distal N-terminal region for regulation of TRPA1 channel activity.     

Homology model of the CB1 cannabinoid receptor: sites critical for nonclassical cannabinoid agonist interaction

Association of cannabimimetic compounds such as cannabinoids, aminoalkylindoles (AAIs), and arachidonylethanolamide (anandamide) with the brain cannabinoid (CB(1)) receptor activates G-proteins and relays signals to regulate neuronal functions. A CB(1) receptor homology model was constructed using the published x-ray crystal structure of bovine rhodopsin (Palczewski et al., Science, 2000, Vol. 289, pp. 739-745) in the conformation most likely to represent the "high-affinity" state for agonist binding to G-protein coupled receptors (GPCRs). A molecular docking approach that combined Monte Carlo and molecular dynamics simulations was used to identify the putative binding conformations of nonclassical cannabinoid agonists, including AC-bicyclic CP47497 and CP55940, and ACD-tricyclic CP55244. Placement of these ligands was based upon the assumption of a critical hydrogen bond between the A-ring OH and the side chain N of Lys192 in transmembrane helix 3. We evaluated two alternative binding conformations, C3-in and C3-out, denoting the directionality of the ligand C3 side chain within the receptor with respect to the inside or the outside of the cell. Assuming both the C3-in or C3-out conformation, the calculated ligand-receptor binding energy (DeltaE(bind)) was correlated with the experimentally observed binding affinity (K(i)) for a series of nonclassical cannabinoid agonists. The C3-in conformation was marginally better than the alternative C3-out conformation in predicting the rank order of the tested nonclassical cannabinoid analogs. Adopting the C3-in conformation due to the greater number of receptor interactions with known pharmacophoric elements of the ligand, key residues were identified comprising the presumed hydrophobic pocket that interacts with the C3 side chain of cannabinoid agonists. Key hydrogen bonds would form between both K3.28(192) and E(258) and the A-ring OH, and between Q(261) and the C-ring C-12 hydroxypropyl. In summary, the present study represents one of the first attempts to construct a homology model of the CB(1) cannabinoid receptor based upon the published bovine rhodopsin x-ray crystal structure and to elucidate the putative ligand binding site for nonclassical cannabinoid agonists. We postulated sites of the CB(1) receptor critical for the ligand interaction, including the hydrophobic pocket interacting with the key pharmacophoric moiety, the C3 side chain. More work is needed to delineate between two alternative (and possibly other) binding conformations of the nonclassical cannabinoid ligands within the CB(1) receptor. The present study provides a consistent framework for further investigation of the CB(1) receptor-ligand interaction and for the study of CB(1) receptor activation.