Visualization of inositol 1,4,5-trisphosphate receptors on the nuclear envelope outer membrane by freeze-drying and rotary shadowing for electron microscopy

The receptors for the second messenger InsP3 comprise a family of closely related ion channels that release Ca2+ from intracellular stores, most prominently the endoplasmic reticulum and its extension into the nuclear envelope. The precise sub-cellular localization of InsP3Rs and the spatial relationships among them are important for the initiation, spatial and temporal properties and propagation of local and global Ca2+ signals, but the spatial organization of InsP3Rs in Ca2+ stores is poorly characterized. Using nuclei isolated from insect Sf9 cells and freeze-dry rotary shadowing, we have addressed this by directly visualizing the cytoplasmic domain of InsP3R located on the cytoplasmic side of the nuclear envelope. Identification of ∼15 nm structures as the cytoplasmic domain of InsP3R was indirectly supported by a marked increase in their frequency after transient transfections with cDNAs for rat types 1 and 3 InsP3R, and directly confirmed by gold labeling either with heparin or a specific anti-InsP3R antibody. Over-expression of InsP3R did not result in the formation of arrays or clusters with channels touching each other. Gold-labeling suggests that the channel amino terminus resides near the center of the cytoplasmic tetrameric quaternary structure. The combination of nuclear isolation with freeze-drying and rotary shadow techniques allows direct visualization of InsP3Rs in native nuclear envelopes and can be used to determine their spatial distribution and density.