Inositol 1,4,5‐trisphosphate‐stimulated calcium release from permeabilized cerebellar granule cells

Abstract

1 Muscarinic cholinoceptor stimulation of phosphoinositide hydrolysis in rat cultured cerebellar granule cells results in a rapid, transient accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃), which has been implicated in the release of non-mitochondrial intracellular Ca²⁺stores. In the present study, the release of Ca²⁺from intracellular stores and the Ins(1,4,5)P₃ receptor responsible for this process have been investigated. 2 Monolayers of saponin-permeabilized granule cells accumulate ⁴⁵Ca²⁺in an ATP-dependent manner and the sequestered ⁴⁵Ca²⁺can be concentration-dependently released by Ins(1,4,5)P₃ by a stereospecific and heparin-sensitive mechanism. The EC₅₀ for Ins(1,4,5)P₃-stimulated ⁴⁵Ca²⁺release was 80 ± 3 nm. 3 Radioligand binding studies performed on a crude granule cell membrane fraction indicated the presence of an apparently homogeneous population of stereo-specific Ins(1,4,5)P₃ receptors (K_D 54.7 ± 2.0 nm; B_max 1.37 ± 0.29 pmol mg⁻¹ protein). 4 This study provides evidence for Ins(1,4,5)P₃-sensitive intracellular Ca²⁺stores in primary cultures of cerebellar granule cells and suggest that these cells provide an excellent model neuronal system in which to study the relative functional roles of Ca²⁺release from intracellular stores and Ca²⁺-entry in neuronal Ca²⁺homeostasis.