A CEREBELLAR PURKINJE-CELL MARKER P400 PROTEIN IS AN INOSITOL 1,4,5-TRISPHOSPHATE (INSP3) RECEPTOR PROTEIN - PURIFICATION AND CHARACTERIZATION OF INSP3 RECEPTOR COMPLEX

P400 protein is a 250 kd glycoprotein, characteristic of the cerebellum, which is accumulated at the endoplasmic reticulum, at the plasma membrane and at the postsynaptic density of Purkinje cells. In this study, we purified inositol 1,4,5-trisphosphate (InsP3) receptor from mouse cerebellum and examined the possibility that P400 protein is identical with cerebellar InsP3 receptor protein. InsP3 receptor was solubilized with Triton X-100 from a post-nuclear fraction of ddY mouse cerebellum and was purified With high yield by sequential column chromatography on DE52, hepArin-agarose, lentil lectin-Sepharose and hydroxylapatite. In these chromatographies, P400 protein co-migrated completely with the InsP3 binding activity. The purified receptor is a 250 kd protein with a B(max) of 2.1 pmol/μg and a K(D) of 83 nM. IT reacted with three different monoclonal antibodies against P400 protein, indicating that P400 protein is the same substance as the InsP3 receptor (P400/InsP3 receptor protein). Electron microscopy of the purified receptor showed a square shape with sides ~25 nm long. Binding assays of the cerebella of Purkinje cell-degeneration (pcd) mice with [3H]INsP3 demonstrated that the InsP3 binding sites in the cerebellum are distributed exclusively on the Purkinje cells. Immunohistochemical analysis indicated that P400/InsP3 receptor is present at the dendrites, cell bodies, axons and synaptic boutons of the Purkinje cells.