Location, Structure, and Dynamics of the Synthetic Cannabinoid Ligand CP-55,940 in Lipid Bilayers

Abstract

The widely used hydrophobic cannabinoid ligand CP-55,940 partitions with high efficiency into biomembranes. We studied the location, orientation, and dynamics of CP-55,940 in POPC bilayers by solid-state NMR. Chemical-shift perturbation of POPC protons from the aromatic ring-current effect, as well as 1H NMR cross-relaxation rates, locate the hydroxyphenyl ring of the ligand near the lipid glycerol, carbonyls, and upper acyl-chain methylenes. Order parameters of the hydroxyphenyl ring determined by the 1H-13C DIPSHIFT experiment indicate that the bond between the hydroxyphenyl and hydroxycyclohexyl rings is oriented perpendicular to the bilayer normal. 2H NMR order parameters of the nonyl tail are very low, indicating that the hydrophobic chain maintains a high level of conformational flexibility in the membrane. Lateral diffusion rates of CP-55,940 and POPC were measured by 1H magic-angle spinning NMR with pulsed magnetic field gradients. The rate of CP-55,940 diffusion is comparable to the rate of lipid diffusion. The magnitude of cross-relaxation and diffusion rates suggests that associations between CP-55,940 and lipids are with lifetimes of a fraction of a microsecond. With its flexible hydrophobic tail, CP-55,940 may efficiently approach the binding site of the cannabinoid receptor from the lipid-water interface by lateral diffusion.

A Lipid Pathway for Ligand Binding Is Necessary for a Cannabinoid G Protein-coupled Receptor

“…These studies have shown that the C-3 side chain of classical cannabinoids is aligned parallel with the membrane acyl chains (60,62) and that the fatty acid chain of endogenous cannabinoids orients parallel to membrane acyl chains with terminal methyl near the center of the bilayer (61). Cannabinoid ligand entry at the TMH6/7 interface is supported by isothiocyanate labeling studies of CB2 using the classical cannabinoid, AM841 functionalized at the C-3 dimethylheptyl side chain terminal carbon (1).…”

“…The orientation of both classical and endogenous cannabinoid ligands in the lipid bilayer has been established by small angle x-ray diffraction/differential calorimetry experiments (60), as well as by NMR (61,62). These studies have shown that the C-3 side chain of classical cannabinoids is aligned parallel with the membrane acyl chains (60,62) and that the fatty acid chain of endogenous cannabinoids orients parallel to membrane acyl chains with terminal methyl near the center of the bilayer (61).…”

Recombinant Cannabinoid Type 2 Receptor in Liposome Model Activates G Protein in Response to Anionic Lipid Constituents

“…CB 2 receptor is activated upon specific binding of cannabinoid agonists to a putative binding site formed by transmembrane helices III, V, VI, and VII near the extracellular membrane surface (21,45). Current models propose that ligand binding induces a conformational change in CB 2 receptor, resulting in formation of a catalytic surface on the cytoplasmic side of the receptor where binding and activation of cognate G proteins takes place.…”

A Key Agonist-induced Conformational Change in the Cannabinoid Receptor CB1 Is Blocked by the Allosteric Ligand Org 27569

“…Thus, the more hydrophobic CP55940 would have a slower apparent rate of repartitioning from an empty micelle to a micelle containing a receptor, and this could thus contribute to the slower observed fluorescence change/conformational change in the labeled CB1. Similarly, the faster rate of change observed for the antagonist SR141716A might be partially due to its greater aqueous solubility (lower octanol/water partition coefficient when compared with agonist -1 ϫ 10 5 versus 1.6 ϫ 10 6 , respectively) (45,46). Notably, multistep binding models have previously been proposed for cannabinoid ligands to account for their interaction with membranes (47,48).…”