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-IP ₃R1 membrane correspond to the cytosolic domain of IP₃R1. Our study provides the first 3D representation of individual IP₃R1 particles in an aqueous solution.