Immunogold localization of inositol 1,4,5-trisphosphate (InsP3) receptor in mouse cerebellar Purkinje cells using three monoclonal antibodies.

Abstract

ABSTRACT. Ultrastructural localization of I11SP3 receptor in mouse cerebellar Purkinje cells was investigated by immunogold technique using three monoclonal antibodies (mab 10A6, 4C11 and 18A10). The epitopes of the three antibodies were numerously detected on the smooth endoplasmic reticulum (ER) (especially, on the stacks of flattened smooth ER, subsurface cisterns and spine apparatus), scantily on the rough ER and on the outer nuclear membrane, but were not detectable on either the plasmalemma, synaptic densities, mitochondria or Golgi apparatus. Not only mab 4C11 and 10A6 which bind to the N-terminal region of the receptor but also 18A10 which binds to the C-terminal region were localized on the cytoplasmic surface of the ER membranes. This indicates that the C terminus of InsP3 receptor is localized on the cytoplasmic surface of the ER. Wenoticed that gold particles are usually localized on the fuzzy structure of the cytoplasmic surface of smooth ER, which is suggested to correspond to the feet structure of the ryanodine receptor. In the Nissl body, gold particles were found not only on the ER membranes but also in the cytoplasmic matrix between the rough ER cisterns. Wesuggest that the peculiar structure of Nissl body, which is composed of parallel cisterns of rough ER, sandwiching a number of free polyribosomes between the cisternal elements, is due to the fact that the major proteins like I11SP3 receptor are synthesized mostly on the free polyribosomes and become membrane bound only at the later stage of the biosynthesis.Inositol

Inositol 1,4,5-trisphosphate receptors in endocrine cells: localization and association in hetero- and homotetramers.

“…Subcellular localization studies using antibodies to purified cerebellar receptor, or to peptides from the cloned type I receptor, indicate that IP3Rs are present in the ER and possibly in caveolae (Ross et al, 1989;Otsu et al, 1990;Fujimoto et al, 1992). A recent study using antibodies to peptides derived from the type III IP3R suggests that it may be localized to secretory granules in pancreatic endocrine cells (Blondel et al, 1994).…”

Molecular Cloning of Mouse Type 2 and Type 3 Inositol 1,4,5-Trisphosphate Receptors and Identification of a Novel Type 2 Receptor Splice Variant

“…Because localized groups of IP 3 R within the range of diffusion of the Ca 2ϩ released are likely to be linked together as functional units to generate Ca 2ϩ puffs, the spatial arrangement of IP 3 Rs may be a critical component in controlling the spatiotemporal patterns of intracellular Ca 2ϩ . IP 3 R is mainly localized on the endoplasmic reticulum (ER) (31). The ER is normally contiguous throughout the cell and forms a membranous network that is the site for Ca 2ϩ storage, lipid biosynthesis, and the processing and quality control of newly synthesized proteins.…”

Ca2+ stores in Purkinje neurons: endoplasmic reticulum subcompartments demonstrated by the heterogeneous distribution of the InsP3 receptor, Ca(2+)-ATPase, and calsequestrin

“…The molecular characterization of two intracellular second messenger-gated Ca*+ channels, the ryanodine receptor (Fleischer and Inui, 1989;Takeshima et al, 1989;Zorzato et al, 1990) and the InsP, receptor (Supattapone et al, 1988;Furuichi et al, 1989;Mignery et al, 1989;Mignery and Siidhof, 1990) and the availability of antibodies specific for these proteins made possible the further investigation of the nature of second messenger-responsive intracellular Ca*+ stores in nonmuscle cells (Mignery et al, 1989;Ross et al, 1989;Ellisman et al, 1990;Otsu et al, 1990;Satoh et al, 1990;Walton et al, 1991). These studies have focused on Purkinje cells (PCs) of the cerebellum because PCs contain particularly high concentrations of both Ca2+ channels.…”