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 (IP3R) among the heterogeneous population of receptors to study the regulatory mechanism of Ca2+ release. We raised in the rabbit a polyclonal antibody against synthetic peptide corresponding to amino acids 2736-2747 (pep 6) of type I IP3R (IP3-R-I) that is most abundant in cerebellum. We purified IP3R-I from a 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid solubilized mouse cerebellar microsomal fraction by immunoaffinity chromatography on an anti-pep 6 antibody-Sepharose 4B column with specific elution by the pep 6 peptide (GHPPHMNVNPQQ) of the IP3R-I C terminus. Immunoaffinity-purified IP3R reconstituted into lipid vesicles formed a homotetramer structure. Monoclonal antibody 18A10, which partially blocks the Ca2+ release from cerebellar microsome, almost completely inhibited IP3-induced 45Ca2+ influx into proteoliposomes, whereas monoclonal antibody that recognizes other regions did not inhibit Ca2+ influx. Both the rate and extent of 45Ca2+ influx into proteoliposomes increased 20% after incubation with the catalytic subunit of cyclic AMP-dependent protein kinase, accompanied by stoichiometric phosphorylation of IP3R protein.