CYCLIC-AMP-DEPENDENT PHOSPHORYLATION OF AN IMMUNOAFFINITY-PURIFIED HOMOTETRAMERIC INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR (TYPE-I) INCREASES CA2+ FLUX IN RECONSTITUTED LIPID VESICLES

We established a novel method to isolate a single type of inositol 1,4 ,5-trisphosphate receptor (IP(3)R) among the heterogeneous population of receptors to study the regulatory mechanism of Ca2+ release. We rai sed in the rabbit a polyclonal antibody against synthetic peptide corr esponding to amino acids 2736-2747 (pep 6) of type I IP(3)R (IP(3)R-I) that is most abundant in cerebellum. We purified IP(3)R-I from a 1% c holamidopropyl)dimethylammonio]-1-propanesulfonic acid solubilized mou se cerebellar microsomal fraction by immunoaffinity chromatography on an anti-pep 6 antibody-Sepharose 4B column with specific elution by th e pep 6 peptide (GHPPHMNVNPQQ) of the IP(3)R-I C terminus. Immunoaffin ity-purified IP(3)R reconstituted into lipid vesicles formed a homotet ramer structure. Monoclonal antibody 18A10, which partially blocks the Ca2+ release from cerebellar microsome, almost completely inhibited I P3-induced Ca-45(2+) influx into proteoliposomes, whereas monoclonal a ntibody that recognizes other regions did not inhibit Ca2+ influx. Bot h the rate and extent of Ca-45(2+) influx into proteoliposomes increas ed 20% after incubation with the catalytic subunit of cyclic AMP-depen dent protein kinase, accompanied by stoichiometric phosphorylation of IP(3)R protein.