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.

Figure 1

Chemical structure of (a) synthetic cannabinoid ligand CP-55,940 and its deuteration at the nonyl tail (b) CP-55,940-d₁₉ and (c) CP-55,940-d₆.

Figure 2

Order parameters S(n) of the POPC-d₃₁ palmitoyl chain at 0, 15, and 30 mol % of CP-55,940 at 25°C (top panel). Ligand-induced changes of order parameters, ΔS(n), are shown in the bottom panel.

Figure 3

Solid-state ¹H MAS NMR spectra of (a) POPC-d₃₁ MLVs and (b) POPC-d₃₁ MLVs with 30 mol % CP-55,940. (c) High-resolution solution-state ¹H NMR spectrum of 1 mM CP-55,940 in methanol-d₄.

Figure 4

¹H chemical-shift changes (Δδ) of POPC-d₃₁ induced upon incorporation of 15 mol % (slashed bars) and 30 mol % (solid bars) of CP-55,940.

Figure 5

Normalized ¹H NMR cross-relaxation rates (per proton) between the hydroxyphenyl ring of CP-55,940 and POPC. Open bars are for the ring signal (h) at 6.67 ppm, and solid bars are for the ring signal (l) at 6.86 ppm. Values for C3, -(CH₂)_n-, and ω-CH₃ were not obtained, due to superposition with ligand protons. The use of CP-55,940-d₁₉ allowed the overlap for ω-CH₃ to be circumvented, and cross relaxation at this site was confirmed to be negligible.

Figure 6

Natural-abundance ¹³C MAS NMR spectra of the hydroxyphenyl ring of CP-55,940 measured in the DIPSHIFT experiment at the MAS frequency of 3,994 Hz: (a) without MREV-8 cycles (t₁ = 0, cross-polarization only), and (b) with four MREV-8 cycles (t₁ = 148.6 μs). The chemical structure above the spectra shows the orientations of the hydroxyphenyl ring with respect to the bilayer normal (x, y, or z axis) that could possibly yield identical ¹H-¹³C dipolar interactions for sites 1–3 (see text).

Figure 7

¹H-¹³C dipolar dephasing curves (solid lines) for the carbon atoms at sites (a) 1, (b) 2, and (c) 3 of the hydroxyphenyl ring of CP-55,940 obtained with the DIPSHIFT experiment; see Fig. 6 for the carbon assignments. Intensities of the ¹³C NMR signals were plotted as a function of the evolution period t₁ of the ¹³C magnetization under ¹H-¹³C dipolar interaction; the length of t₁ corresponds to the number of MREV-8 cycles (from 0 to 7) applied within one rotor period τ_r = 250 μs. Signal intensities were normalized to the intensity at t₁ = 0. The broken lines above and below the solid line show the effect of a ±1 kHz change of the dipolar interaction from the best fit (see text). The error bars represent the spectral noise level.

Figure 8

Schematic illustration of the structural features of CP-55,940 in PC bilayers determined by solid-state NMR. The location of the hydroxyphenyl ring with respect to the bilayer normal axis is highlighted with a gray band. The fast lateral diffusion of the ligand takes place along this band. The orientation of the hydroxylphenyl ring is shown by an arrow with the degree of director fluctuation. The nonyl tail has high conformational flexibility.