The effect of the endogenous cannabinoid ligand anandamide on the function of the cloned α₇ subunit of the nicotinic acetylcholine (ACh) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Anandamide reversibly inhibited nicotine (10 μM) induced-currents in a concentration-dependent manner (10 nM to 30 μM), with an IC₅₀ value of 229.7 ± 20.4 nM. The effect of anandamide was neither dependent on the membrane potential nor meditated by endogenous Ca²⁺ dependent Cl^- channels since it was unaffected by intracellularly injected BAPTA and perfusion with Ca²⁺-free bathing solution containing 2 mM Ba²⁺. Anandamide decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on nicotinic acetylcholine (nACh) α₇ receptors. This effect was not mediated by CB₁ or CB₂ receptors, as neither the selective CB₁ receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) nor CB₂ receptor antagonist N-((1S)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528) reduced the inhibition by anandamide. In addition, inhibition of nicotinic responses by anandamide was not sensitive to either pertussis toxin treatment or to the membrane permeable cAMP analog 8-Br-cAMP (0.2 mM). Inhibitors of enzymes involved in anandamide metabolism including phenylmethylsulfonyl fluoride, superoxide dismutase, and indomethacin, or the anandamide transport inhibitor AM404 did not prevent anandamide inhibition of nicotinic responses, suggesting that anandamide itself acted on nicotinic receptors. In conclusion, these results demonstrate that the endogenous cannabinoid anandamide inhibits the function of nACh α₇ receptors expressed in Xenopus oocytes in a cannabinoid receptor-independent and noncompetitive manner.

Endogenous cannabinoids (endocannabinoids), produced from membrane-bound precursors via calcium and/or G-protein dependent processes, mimic the effects of cannabinoids by activating cannabinoid CB1 and/or CB2 receptors. Several reports however, also indicate that endocannabinoids can produce effects that are independent of cannabinoid receptors. Thus, in pharmacologically relevant concentrations, endocannabinoids have been demonstrated to modulate the functional properties of voltage-gated ion channels including Ca2+ channels, Na+ channels and various types of K+ channels, and ligand-gated ion channels such as 5-HT3, and nicotinic ACh receptors. In addition, the functional modulations by endocannabinoids of other ion-transporting membrane proteins such as transient potential receptor-class channels, gap junctions, and neurotransmitter transporters have also been reported. These findings indicate that additional molecular targets for endocannabinoids exist and that these targets may represent important sites for cannabinoids to alter either the excitability of the neurons or the response of the neuronal systems. This review focuses on the results of recent studies indicating that beyond their receptor-mediated effects, endocannabinoids alter the function of ion channels directly.

The effects of endogenous and synthetic cannabinoid receptor agonists, including 2-arachidonoylglycerol (2-AG), R-methanandamide, WIN55,212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one], and CP 55,940 [1α,2β-(R)-5α]-(-)-5-(1,1-dimethyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl-phenol], and the psychoactive constituent of marijuana, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), on the function of homomeric α₇-nicotinic acetylcholine (nACh) receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp technique. The endogenous cannabinoid receptor ligands 2-AG and the metabolically stable analog of anandamide (arachidonylethanolamide), R-methanandamide, reversibly inhibited currents evoked with ACh (100 μM) in a concentration-dependent manner (IC₅₀ values of 168 and 183 nM, respectively). In contrast, the synthetic cannabinoid receptor agonists CP 55,940, WIN55,212-2, and the phytochemical Δ⁹-THC did not alter α₇-nACh receptor function. The inhibition of α₇-mediated currents by 2-AG was found to be non-competitive and voltage-independent. Additional experiments using endocannabinoid metabolites suggested that arachidonic acid, but not ethanolamine or glycerol, could also inhibit the α₇-nACh receptor function. Whereas the effects of arachidonic acid were also noncompetitive and voltage-independent, its potency was much lower than 2-AG and anandamide. Results of studies with chimeric α₇-nACh-5-hydroxytryptamine (5-HT)₃ receptors comprised of the amino-terminal domain of the α₇-nACh receptor and the transmembrane and carboxyl-terminal domains of 5-HT₃ receptors indicated that the site of interaction of the endocannabinoids with the α₇-nAChR was not located on the N-terminal region of the receptor. These data indicate that cannabinoid receptor ligands that are produced in situ potently inhibit α₇-nACh receptor function, whereas the synthetic cannabinoid ligands, and Δ⁹-THC, are without effect, or are relatively ineffective at inhibiting these receptors.