Assay and purification of neuronal receptors for inositol 1,4,5-trisphosphate

Receptors for inositol 1,4,5-trisphosphate (IP₃) serve as ligand-activated, Ca²⁺-release channels found in intracellular membrane compartments. This chapter describes procedures for rapid purification of the IP₃ receptor complex and an assay to measure quantitatively, the binding properties of [³H]IP₃ to both native and purified preparations. The mammalian brain, particularly the cerebellum, is an extraordinarily rich source of IP₃ receptor proteins, facilitating development of binding assays, methods of purification and reconstitution, and molecular cloning. The highly charged nature of IP₃ suggests the basis for an assay involving ion exchange to adsorb unbound ligand and size exclusion to exclude ligand bound to receptor. The purification protocols exploit some unusual biochemical properties of cerebellar IP₃ receptors including a complex interaction of the receptor with wheat germ agglutinin and the tendency of the receptor to associate in homotetramers, a property reminiscent of its skeletal muscle homolog, the ryanodine receptor/Ca²⁺ release channel.