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

Wakako Suhara, Mime Kobayashi, Hiroshi Sagara, Kozo Hamada, Touichiro Goto, Ichiro Fujimoto, Keiichi Torimitsu, Katsuhiko Mikoshiba

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)102-107
Number of pages6
JournalNeuroscience Letters
Volume391
Issue number3
DOIs
Publication statusPublished - 2006 Jan 2
Externally publishedYes

Keywords

  • Atomic force microscopy
  • Calcium signaling
  • Inositol 1,4,5-trisphosphate receptor
  • Molecular imaging
  • Protein structure

ASJC Scopus subject areas

  • Neuroscience(all)

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