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The currents of α7 nicotinic acetylcholine receptors activated by acetylcholine (ACh) are brief. The channel has high permeability to calcium relative to monovalent cations and shows inward rectification. It has been previously noted that in the presence of positive allosteric modulators (PAMs), currents through the channels of α7 receptors differ from normal α7 currents both in sensitivity to specific channel blockers and their current-voltage (I-V) relationships, no longer showing inward rectification. Linear I-V functions are often associated with channels lacking calcium permeability, so we measured the I-V functions of α7 receptors activated by ACh when PAMs were bound to the allosteric binding site in the transmembrane domain. Currents were recorded in chloride-free Ringer’s solution with low or high concentrations of extracellular calcium to determine the magnitude of the reversal potential shift in the two conditions as well as the I-V relationships. ACh-evoked currents potentiated by the allosteric agonist–PAMs (ago-PAMs) (3aR,4S,9bS)-4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (GAT107) and 3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propenamide (B-973B) showed reduced inward rectification and calcium-dependent reversal potential shifts decreased by 80%, and 50%, respectively, compared with currents activated by ACh alone, indicative of reduced calcium permeability. Currents potentiated by 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide were also linear and showed no calcium-dependent reversal potential shifts. The ago-PAMs GAT-107 and B-973B stimulated increases in intracellular calcium in stably transfected HEK293 cells. However, these calcium signals were delayed relative to channel activation produced by these agents and were insensitive to the channel blocker mecamylamine. Our results indicate that, although allosterically activated α7 nicotinic ACh receptor may affect intracellular calcium levels, such effects are not likely due to large channel-dependent calcium influx. SIGNIFICANCE STATEMENT: Positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptor can increase channel activation by two or more orders of magnitude, raising the concern that, due to the relatively high calcium permeability of α7 receptors activated by acetylcholine alone, such efficacious PAMs may have cytotoxic side effects. We show that PAMs alter the ion conduction pathway and, in general, reduce the calcium permeability of the channels. This supports the hypothesis that α7 effects on intracellular calcium may be independent of channel-mediated calcium influx.

Many synthetic compounds to which we attribute specific activities are produced as racemic mixtures of stereoisomers, and it may be that all the desired activity comes from a single enantiomer. We have previously shown this to be the case with the α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM) 3a,4,5,9b-Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) and the α7 ago-PAM 4BP-TQS. Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (2,3,5,6TMP-TQS), previously published as a "silent allosteric modulator" and an antagonist of α7 allosteric activation, shares the same scaffold with three chiral centers as the aforementioned compounds. We isolated the enantiomers of 2,3,5,6TMP-TQS and determined that the (-) isomer was a significantly better antagonist than the (+) isomer of the allosteric activation of both wild-type α7 and the nonorthosterically activatible C190A α7 mutant by the ago-PAM GAT107 (the active isomer of 4BP-TQS). In contrast, (+)2,3,5,6TMP-TQS proved to be an α7 PAM. (-)2,3,5,6TMP-TQS was shown to antagonize the allosteric activation of α7 by the structurally unrelated ago-PAM B-973B as well as the allosteric activation of the TQS-sensitive α4β2L15'M mutant. In silico docking of 2,3,5,6TMP-TQS in the putative allosteric activation binding site suggested a specific interaction of the (-) enantiomer with α7T106, and allosteric activation of α7T106 mutants was not inhibited by (-)2,3,5,6TMP-TQS, confirming the importance of this interaction and supporting the model of the allosteric binding site. Comparisons and contrasts between 2,3,5,6TMP-TQS isomers and active and inactive enantiomers of other TQS-related compounds identify the orientation of the cyclopentenyl ring to the plane of the core quinoline to be a crucial determinate of PAM activity. SIGNIFICANCE STATEMENT: Many synthetic ligands are in use as racemic preparations. We show that one enantiomer of the TQS analog Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, originally reported to lack activity when used as a racemic preparation, is an α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM). The other enantiomer is not a PAM, but it is an effective allosteric antagonist. In silico studies and structural comparisons identify essential elements of both the allosteric ligands and receptor binding sites important for these allosteric activities.

Homomeric α7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by α7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to α7 at the 15′ position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L15′M) in heteromeric nAChR receptors containing α4 and β2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the α7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea. The presence of the mutation in the β2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the α4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant β subunit, regardless of the position of the mutant β subunit within the pentameric complex. Similar results were obtained when β2L15′M was coexpressed with α2 or α3 and when the L15′M mutation was placed in β4 and coexpressed with α2, α3, or α4. Functional receptors were not observed when β1L15′M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the α4β2L15′M receptor compared with α7 and the availability of high-resolution α4β2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of α7 subunits but are insensitive to PAMs, which greatly increase the open probability of α7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in α7. Specifically, the mutation of the TM2 15′ leucine to methionine in α subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal β subunits allows heteromeric receptors to respond to select α7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.

Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels. Typically, channel activation follows the binding of agonists to the orthosteric binding sites of the receptor. α7 nAChRs have a very low probability of channel activation, which can be reversed by the binding of α7 selective positive allosteric modulators (PAMs) to putative sites within the transmembrane domains. Although typical PAMs, like PNU-120596, require coapplication of an orthosteric agonist to produce large channel activations, some, like GAT107 and B-973B [(S)-3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propanamide], are characterized as allosteric activating PAMs, which also bind to an allosteric activation (AA) site in the extracellular domain and activate the α7 ion channel by themselves. We had previously characterized N,N-diethyl-N′-phenylpiperazine analogs with various functions. In this work, we docked members of this family to a homology model of the α7 receptor extracellular domain. The compound 1,1-diethyl-4(naphthalene-2-yl)piperazin-1-ium (2NDEP) a weak partial agonist, showed particularly favorable docking and binding energies at the putative AA site of the receptor. We hypothesized that 2NDEP could couple with PAMs through the AA site. This hypothesis was tested with the α7 mutant C190A, which is not activated by orthosteric agonists but is effectively activated by GAT107. The results showed that 2NDEP acts as an allosteric agonist of α7C190A when coapplied with the PAM PNU-120596. Also, the allosteric activity was nearly abolished upon coapplication with the AA site–selective antagonist 2,3,5,6MP-TQS (cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), consistent with AA site involvement. Overall, our findings show a novel mode of agonism through an allosteric site in the extracellular domain of α7 nAChR.

B-973 is an efficacious type II positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptors that, like 4BP-TQS and its active isomer GAT107, can produce direct allosteric activation in addition to potentiation of orthosteric agonist activity, which identifies it as an allosteric activating (ago)-PAM. We compared the properties of B-973B, the active enantiomer of B-973, with those of GAT107 regarding the separation of allosteric potentiation and activation. Both ago-PAMs can strongly activate mutants of α7 that are insensitive to standard orthosteric agonists like acetylcholine. Likewise, the activity of both ago-PAMs is largely eliminated by the M254L mutation in the putative transmembrane PAM-binding site. Allosteric activation by B-973B appeared more protracted than that produced by GAT107, and B-973B responses were relatively insensitive to the noncompetitive antagonist mecamylamine compared with GAT107 responses. Similar differences are also seen in the single-channel currents. The two agents generate unique profiles of full-conductance and subconductance states, with B-973B producing protracted bursts, even in the presence of mecamylamine. Modeling and docking studies suggest that the molecular basis for these effects depends on specific interactions in both the extracellular and transmembrane domains of the receptor.

Daily treatment with cannabinoids results in tolerance to many, but not all, of their behavioral and physiologic effects. The present studies investigated the effects of 7-day exposure to 10 mg/kg daily of Δ(9)-tetrahydrocannabinol (THC) on the diuretic and antinociceptive effects of THC and the synthetic cannabinoid AM4054. Comparison studies determined diuretic responses to the κ-opioid agonist U50,488 and furosemide. After determination of control dose-response functions, mice received 10 mg/kg daily of THC for 7 days, and dose-response functions were re-determined 24 hours, 7 days, or 14 days later. THC and AM4054 had biphasic diuretic effects under control conditions with maximum effects of 30 and 35 ml/kg of urine, respectively. In contrast, antinociceptive effects of both drugs increased monotonically with dose to >90% of maximal possible effect. Treatment with THC produced 9- and 7-fold rightward shifts of the diuresis and antinociception dose-response curves for THC and, respectively, 7- and 3-fold rightward shifts in the AM4054 dose-response functions. U50,488 and furosemide increased urine output to >35 ml/kg under control conditions. The effects of U50,488 were attenuated after 7-day treatment with THC, whereas the effects of furosemide were unaltered. Diuretic effects of THC and AM4054 recovered to near-baseline levels within 14 days after stopping daily THC injections, whereas tolerance to the antinociceptive effects persisted longer than 14 days. The tolerance induced by 7-day treatment with THC was accompanied by a 55% decrease in the Bmax value for cannabinoid receptors (CB1). These data indicate that repeated exposure to THC produces similar rightward shifts in the ascending and descending limbs of cannabinoid diuresis dose-effect curves and to antinociceptive effects while resulting in a flattening of the U50,488 diuresis dose-effect function.

Cannabinoid receptor 1 (CB(1)) inverse agonists (e.g., rimonabant) have been reported to produce adverse effects including nausea, emesis, and anhedonia that limit their clinical applications. Recent laboratory studies suggest that the effects of CB(1) neutral antagonists differ from those of such inverse agonists, raising the possibility of improved clinical utility. However, little is known regarding the antagonist properties of neutral antagonists. In the present studies, the CB(1) inverse agonist SR141716A (rimonabant) and the CB(1) neutral antagonist AM4113 were compared for their ability to modify CB(1) receptor-mediated discriminative stimulus effects in nonhuman primates trained to discriminate the novel CB(1) full agonist AM4054. Results indicate that AM4054 serves as an effective CB(1) discriminative stimulus, with an onset and time course of action comparable with that of the CB(1) agonist Δ(9)-tetrahydrocannabinol, and that the inverse agonist rimonabant and the neutral antagonist AM4113 produce dose-related rightward shifts in the AM4054 dose-effect curve, indicating that both drugs surmountably antagonize the discriminative stimulus effects of AM4054. Schild analyses further show that rimonabant and AM4113 produce highly similar antagonist effects, as evident in comparable pA(2) values (6.9). Taken together with previous studies, the present data suggest that the improved safety profile suggested for CB(1) neutral antagonists over inverse agonists is not accompanied by a loss of antagonist action at CB(1) receptors.

Behavioral studies of chronic CB(1) receptor activation may provide a pharmacological approach to understanding efficacy-related differences among CB(1) ligands as well as mechanistic commonalities between cannabinoid and noncannabinoid drugs. In the present studies, the effects of CB(1) agonists [(6aR,10aR)-3-(1-adamantyl)-6,6,9-trimethyl-6a,7,10,10a-tetrahydrobenzo[c]chromen-1-ol (AM411), 9β-(hydroxymethyl)-3-(1-adamantyl)-hexahydrocannabinol (AM4054), R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate (WIN55,212.2), Δ(9)-tetrahydrocannabinol (Δ(9)-THC), (R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide (methanandamide)], CB(1) antagonists [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (SR141716A), 5-(4-alkylphenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (AM4113)], and dopamine (DA)-related [methamphetamine, (±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF82958), (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390), (6aR)-5,6,6a,7-tetrahydro-6-propyl-4H-dibenzo[de,g]quinoline-10,11-diol (R-(-)-NPA), haloperidol] and opioid (morphine, naltrexone) drugs on scheduled-controlled responding under a 30-response fixed ratio schedule of stimulus-shock termination in squirrel monkeys were compared before and during chronic treatment with the long-acting CB(1) agonist AM411 (1.0 mg/kg per day, i.m.). Prechronic treatment with all drugs except naltrexone (1-10 mg/kg) produced dose-related decreases in responses rates. Dose-response re-determinations during chronic treatment revealed the following: 1) >250-fold (AM411, methanandamide) and >45-fold (AM4054, WIN55,212.2, Δ(9)-THC) rightward shifts in the ED(50) values for CB(1) agonists; 2) >100-fold and >20-fold leftward shifts in the ED(50) values for SR141716A and AM4113, respectively; and 3) approximately 4.8-fold and 10-fold rightward shifts in the ED(50) values for methamphetamine and the DA D(2) agonist R-(-)-NPA, respectively. Dose-response relationships for other DA-related and opioid drugs were unchanged by chronic CB(1) agonist treatment. Differences in the magnitude of tolerance among CB(1) agonists during chronic treatment may be indicative of differences in their pharmacological efficacy, whereas the enhanced sensitivity to behaviorally disruptive effects of CB(1) antagonists may provide evidence for CB(1)-related behavioral and/or physical dependence. Finally, the development of cross-tolerance to methamphetamine and R-(-)-NPA bolsters previous evidence of interplay between CB(1) and DA D(2) signaling mechanisms.

Activation of cannabinoid CB2 receptors suppresses neuropathic pain induced by traumatic nerve injury. The present studies were conducted to evaluate the efficacy of cannabinoid CB2 receptor activation in suppressing painful peripheral neuropathy evoked by chemotherapeutic treatment with the antitumor agent paclitaxel. Rats received paclitaxel (2 mg/kg i.p./day) on 4 alternate days to induce mechanical hypersensitivity (mechanical allodynia). Mechanical allodynia was defined as a lowering of the threshold for paw withdrawal to stimulation of the plantar hind paw surface with an electronic von Frey stimulator. Mechanical allodynia developed in paclitaxel-treated animals relative to groups receiving the Cremophor EL/ethanol/saline vehicle at the same times. Two structurally distinct cannabinoid CB2 agonists, the aminoalkylindole ( R , S )-AM1241 [( R , S )-(2-iodo-5-nitrophenyl)-[1-((1-methyl-piperidin-2-yl)methyl)-1 H -indol-3-yl]-methanone] and the cannabilactone AM1714 (1,9-dihydroxy-3-(1′,1′-dimethylheptyl)-6 H -benzo[ c ]chromene-6-one), produced a dose-related suppression of established paclitaxel-evoked mechanical allodynia after systemic administration. Pretreatment with the CB2 antagonist SR144528 [5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)- N -(1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl)-1 H -pyrazole-3-carboxamide], but not the CB1 antagonist SR141716 [5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl- N -(piperidin-1-yl)-1 H -pyrazole-3-carboxamide], blocked the antiallodynic effects of both ( R , S )-AM1241 and AM1714. Moreover, ( R )-AM1241, but not ( S )-AM1241, suppressed paclitaxel-evoked mechanical allodynia relative to either vehicle treatment or preinjection thresholds, consistent with mediation by CB2. Administration of either the CB1 or CB2 antagonist alone failed to alter paclitaxel-evoked mechanical allodynia. Moreover, ( R , S )-AM1241 did not alter paw withdrawal thresholds in rats that received the Cremophor EL vehicle in lieu of paclitaxel, whereas AM1714 induced a modest antinociceptive effect. Our data suggest that cannabinoid CB2 receptors may be important therapeutic targets for the treatment of chemotherapy-evoked neuropathy.

Amino acid residues in the transmembrane domains of the CB(1) receptor are important for ligand recognition and signal transduction. We used site-directed mutagenesis to identify the role of two novel and adjacent residues in the transmembrane helix II domain, Ile2.62 and Asp2.63. We investigated the role of the conserved, negatively charged aspartate at position 2.63 in cannabinoid receptor (CB(1)) function by substituting it with asparagine (D2.63N) and glutamate (D2.63E). In addition, the effect of the mutant I2.62T alone and in combination with D2.63N (double mutant) on the affinity and potency of structurally diverse ligands was investigated. Recombinant human CB(1) receptors, stably expressed in human embryonic kidney 293 cells, were assayed for ligand affinity and agonist-stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding. The charge-conserved mutant D2.63E behaved similar to wild type. The charge-neutralization mutation D2.63N attenuated the potency of (-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP,55940), (R)-(-)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55212-2), (-)-11beta-hydroxy-3-(1',1'-dimethylheptyl) hexahydrocannabinol (AM4056), and (-)-11-hydroxyldimethylheptyl-Delta(8)-tetrahydrocannabinol (HU210) for the stimulation of GTPgammaS binding, without affecting their binding affinities. Likewise, the I2.62T mutant selectively altered agonist potency without altering agonist affinity. It was surprising to note that the double mutant (I2.62T-D2.63N) displayed a drastic and synergistic increase (by approximately 50-fold) in the EC(50) for agonist-mediated activation. The profound loss of function in the I2.62T-D2.63N double mutant suggests that, although these residues are not obligatory for agonist recognition, they play a synergistic and crucial role in modulating signal transduction.

Ligands of structurally diverse natures are able to bind at the CB 1 cannabinoid receptor, suggesting the existence of multiple binding sites on the receptor. Modeling studies have implicated Ser2.60(173) and Ser7.39(383) as possible interaction site(s) for CB 1 agonists. To test the importance of these residues for receptor recognition, recombinant human CB 1 receptors, stably expressed in human embryonic kidney 293 cells, were used to investigate the consequences of mutating Ser2.60 (to S2.60A) or Ser7.39 (to S7.39A) in radioligand binding and guanosine 5′-3- O -(thio)triphosphate functional assays. The S7.39A mutant resulted in a total ablation of [ 3 H](–)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP55,940) high-affinity binding. However, [ 3 H]( R )-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]-pyrrolo[1,2,3- de ]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55,212-2) binding properties at S7.39A were comparable with those of the wild-type (WT) receptor. The binding affinity of (–)-11β-hydroxy-3-(1′,1′-dimethylheptyl)hexahydrocannabinol (AM4056) and (–)-11-hydroxydimethylheptyl-Δ 8 -tetrahydrocannabinol (HU210) were drastically reduced (50- to 100-fold) at the S7.39A mutant. Likewise, the EC 50 for HU210 and AM4056-mediated activation of the S7.39A receptor was increased by >200-fold. In contrast, the binding affinity and potency of WIN55,212-2, CP55,940, HU210, and AM4056 were unaltered at the S2.60A mutant compared with WT human CB 1 receptors. These results clearly suggest that Ser7.39, but not Ser2.60, plays a crucial role in mediating ligand specific interactions for CP55,940, HU210, and AM4056 at the human CB 1 receptor. Our modeling studies predict that Ser7.39 in a g –χ1 conformation may induce a helix bend in TMH7 that provides docking space for CP55,940 binding; the S7.39A mutation may alter this binding space, precluding CP55,940 binding.

The CB1 cannabinoid receptor has been shown to play important physiological roles in the central nervous system, as well as peripherally, and is a target for development of therapeutic medications. To gain insight on the ligand binding site(s) and structural features of activation, we designed and synthesized (-)-7′-isothiocyanato-11-hydroxy-1′,1′-dimethylheptylhexahydrocannabinol (AM841), a classical cannabinoid affinity label that incorporates an isothiocyanate substituent as an electrophilic reactive group capable of interacting irreversibly with a suitably located and properly oriented nucleophilic amino acid residue at or near the binding site. To obtain evidence for the site of covalent attachment of AM841, C6.47, identified in part by interactive ligand docking, was mutated to serine, alanine, and leucine to reduce or eliminate the nucleophilic character. Wild-type (WT) and mutant CB1 receptors were evaluated for their abilities to recognize a series of cannabinergic ligands. Each bound comparably to WT, excluding C6.47L, which displayed a reduced affinity for 3 H-labeled (1 R ,3 R ,4 R )-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55940), AM841, 11-hydroxy-1′,1′-dimethylheptylhexahydrocannabinol (AM4056), and (-)-7′-bromo-11-hydroxy-1′,1′-dimethylheptylhexahydrocannabinol (AM4043) and an improvement in affinity for (-)- trans -Δ 9 -tetrahydrocannabinol (Δ 9 -THC). The affinity of 3 H-labeled [2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo-[1,2,3- de ]-1,4-benzoxazin-6-yl](naphthyl)methanone (WIN55212-2) was unchanged across all mutants. It is noteworthy that AM841 was shown to bind irreversibly to WT CB1 but exhibited no covalent attachment with the mutants and behaved as an agonist suggesting irreversible attachment to C6.47 maintains CB1 in its active state. The evidence presented identifies C6.47 as the site of covalent bond formation with AM841 and combined with the binding data fully supports the molecular modeling. These studies present the first report of tandem applications of affinity labeling, site-directed mutagenesis, and interactive ligand docking for CB1.