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 InsP(3) comprise a family of closely related ion channels that release Ca(2+) from intracellular stores, most prominently the endoplasmic reticulum and its extension into the nuclear envelope. The precise sub-cellular localization of InsP(3)Rs and the spatial relationships among them are important for the initiation, spatial and temporal properties and propagation of local and global Ca(2+) signals, but the spatial organization of InsP(3)Rs in Ca(2+) 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 InsP(3)R located on the cytoplasmic side of the nuclear envelope. Identification of approximately 15 nm structures as the cytoplasmic domain of InsP(3)R was indirectly supported by a marked increase in their frequency after transient transfections with cDNAs for rat types 1 and 3 InsP(3)R, and directly confirmed by gold labeling either with heparin or a specific anti-InsP(3)R antibody. Over-expression of InsP(3)R 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 InsP(3)Rs in native nuclear envelopes and can be used to determine their spatial distribution and density.