Small angle X-ray diffraction studies on the topography of cannabinoids in synaptic plasma membranes

In a previous publication, we have described in detail how we used small angle x-ray diffraction to determine the topography of (-)-Δ8-tetrahydrocannabinol (Δ8-THC) in dimyristoylphophatidylcholine (DMPC) bilayers, and to deduce the conformation of the THC side chain by using the iodo-analog (5′-I-Δ8-THC) in the model membrane. We have now extended our studies to synaptic plasma membrane systems where the cannabinoids are believed to exert part of their pharmacological effects. Synaptic plasma membranes (SPM) were isolated from fresh bovine brains and Δ8-THC was incorporated into the membranes. By comparing the electron density profiles of drug free and drug-containing SPM preparations, we observed an electron density increase due to the presence of Δ8-THC in a region centered at 9.2 Å from the terminal methyl groups of the membrane bilayer. In an attempt to dissect the effects of different membrane components on the topography of Δ8-THC, we carried out parallel experiments using membrane preparations from the synaptosomal membrane total lipid extract (TLX) as well as from bovine brain phosphatidyl choline extract (PCX) containing 30 mole percent cholesterol (Chol). Our results regarding the topography of Δ8-THC and 5′-I-Δ8-THC in these lipid membranes show that the TLX bilayer simulates the natural membrane environment very closely whereas in the PCX/Chol bilayer Δ8-THC resides at a location approximately 4 Å closer to the membrane interface, similar to that found in our previous study using DMPC model membrane. These x-ray diffraction results provide insights regarding the location of the binding sites on the cannabinoid receptor and indicate that preparations of the total lipid extract from synaptosomal membranes duplicate very well the properties of the intact membrane preparation