High‐Affinity Cannabinoid Binding Site: Regulation by Ions, Ascorbic Acid, and Nucleotides

Abstract: The high‐affinity cannabinoid site in rat brain is an integral component of brain membranes that recognizes cannabinoids with inhibitory constants (K_i) in the nanomolar range. To clarify its physiological role, we studied the regulation of [³H]5′‐trimethylammonium Δ⁸‐tetrahydrocannabinol ([³H]TMA) binding. The site is inhibited by heavy metal ions, such as La³⁺, at low micromolar concentrations; divalent cations, such as Ca²⁺ and Mg²⁺, inhibit [³H]TMA binding, though at somewhat higher concentrations. In contrast, [³H]TMA binding is stimulated by Fe²⁺, Cu²⁺, and Hg²⁺ ions. Ascorbic acid and its analogs are also stimulators of cannabinoid binding at low micromolar concentrations. Stimulation of [³H]TMA binding by ascorbate or ions is dependent upon molecular oxygen, but is not inhibited by metabolic poisons. Metabolically stable nucleoside triphosphate analogs enhance [³H]TMA binding by different mechanisms, with hydrolysis of a high‐energy phosphate bond apparently requisite for these influences. These results suggest that the cannabinoid binding site is associated with a nucleotide‐utilizing protein possessing multiple regulatory subsites.