Pharmacological characterization of the specific binding of [H]ryanodine to rat brain microsomal membranes

High-affinity binding of ³H]ryanodine has been characterized in rat brain microsomal fractions. Membrane fractions from 4 brain regions (cerebral cortex, cerebellum, hippocampus and brainstem) have been isolated using sucrose density gradient purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed the presence of a high-molecular weight protein (M_r 320kDa), similar to that of ryanodine receptor from muscle sarcoplasmic reticulum (SR). In the presence of high salt (1 M KCl), ³H]ryanodine binds to low density (0.8 M sucrose) cortical microsomal fraction with high affinity (K_d 1.5nM), and with the highest capacity (B_max 330fmol/mg protein). Kinetic analysis of the binding suggests multiple available binding sites for ryanodine. Binding of ryanodine is Ca²⁺ dependent (ED₅₀ 1 μM) and inhibited by Mg²⁺ and Ruthenium red. Adenine nucleotides have a biphasic effect on the binding of ³H]ryanodine. At low Ca²⁺ concentration caffeine and daunorubicin enhance the binding of ³H]ryanodine. The inositol 1,4,5-trisphosphate (IP₃) binding inhibitor, heparin, has no effect on ryanodine binding, and ryanodine and caffeine do not influence the binding of ³H]IP₃, which is enriched in the cerebellar fractions. These data demonstrate significant quantitative differences in the pharmacology of brain and muscle receptors and raise the question as to the physiological role of ryanodine binding proteins in the central nervous system and whether it is coupled to an endoplasmatic reticulum (ER) Ca²⁺ release channel.