Visualization of inositol 1,4,5-trisphosphate receptor by atomic force microscopy

Inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) acts as a ligand-gated channel that mediates neuronal signals by releasing Ca2+ from the endoplasmic reticulum. The three-dimensional (3D) structure of tetrameric IP3R has been demonstrated by using electron microscopy (EM) with static specimens; however, the dynamic aspects of the IP3R structure have never been visualized in a native environment. Here we attempt to measure the surface topography of IP3R in solution using atomic force microscopy (AFM). AFM revealed large protrusions extending ∼4.3 nm above a flat membrane prepared from Spodoptera frugiperda (Sf9) cells overexpressing mouse type 1 IP3R (Sf9-IP3R1). The average diameter of the large protrusions was ∼32 nm. A specific antibody against a cytosolic epitope close to the IP3-binding site enabled us to gold-label the Sf9-IP3R1 membrane as confirmed by EM. AFM images of the gold-labeled membrane revealed 7.7-nm high protrusions with a diameter of ∼30 nm, which should be IP3R1-antibody complexes. Authentic IP3R1 immuno-purified from mouse cerebella had approximately the same dimensions as those of the IP3R-like protrusions on the membrane. Altogether, these results suggest that the large protrusions on the Sf9-IP3R1 membrane correspond to the cytosolic domain of IP3R1. Our study provides the first 3D representation of individual IP3R1 particles in an aqueous solution.