title: Endogenous inhibitors of InsP3-induced Ca2+ release in neuroblastoma cells.
abstract: Cerebellar Purkinje neurons and neuroblastoma N1E-115 cells require 10-50 times more InsP3 to induce Ca2+ release than do a variety of non-neuronal cells (including astrocytes, hepatocytes, endothelial cells, or smooth muscle cells). Given the importance of InsP3-induced Ca2+ release for the development of synaptic plasticity in Purkinje neurons, a low InsP3 sensitivity may facilitate the integration of numerous synaptic inputs before initiating a change in synaptic strength. In the present study, attention is directed at the mechanism underlying this low InsP3 sensitivity of Ca2+ release. We show that permeabilization of neuroblastoma cells with saponin increased InsP3 sensitivity of Ca2+ release, indicating the presence of a diffusible, cytosolic inhibitor(s) of Ca2+ release. Consistent with this hypothesis, gel filtration of the neuroblastoma cytosol yielded three peaks that inhibited InsP3-induced Ca2+ release from permeabilized cells. The prominent inhibitory peak decreased the InsP3 sensitivity of Ca2+ release from permeabilized cells, did not bind 3H-InsP3, and was present in sufficient levels to account for the low InsP3 sensitivity of Ca2+ release in intact neuroblastoma cells. Purification of this prominent inhibitory fraction yielded a protein band that was identified by mass spectrometry as stress-induced phosphoprotein 1 (mSTI1). Furthermore, immunoprecipitation of mSTI1 decreased the inhibitory activity of N1E-115 cytosol, indicating that mSTI1 contributes to the inhibition of InsP3-induced Ca2+ release. Thus, the low InsP3 sensitivity of Ca2+ release in neuroblastoma cells can be explained by the presence of cytosolic inhibitors of Ca2+ release and include stress-induced phosphoprotein 1.
authors.affiliation: Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06032, USA. watras@nso2.uchc.edu
authors.forename: James
authors.initials: J
authors.lastname: Watras
authors.forename: Charles C
authors.initials: CC
authors.lastname: Fink
authors.forename: Leslie M
authors.initials: LM
authors.lastname: Loew
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004622:Embryo, Mammalian; D006360:Heat-Shock Proteins / Q000378:metabolism; D047468:Immunoprecipitation / Q000379:methods; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000187:drug effects; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008970:Molecular Weight; D032342:Muscle Cells / Q000187:drug effects / Q000378:metabolism; D009447:Neuroblastoma; D011689:Purkinje Cells / Q000187:drug effects / Q000378:metabolism; D012503:Saponins / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
chemical_list: D006360:Heat-Shock Proteins; D012503:Saponins; C489791:Stip1 protein, mouse; D015544:Inositol 1,4,5-Trisphosphate; D014357:Trypsin; D002118:Calcium