Visualization of inositol 1,4,5-trisphosphate receptor by atomic force microscopy.
Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) acts as a ligand-gated channel that mediates neuronal signals by releasing Ca(2+) from the endoplasmic reticulum. The three-dimensional (3D) structure of tetrameric IP(3)R has been demonstrated by using electron microscopy (EM) with static specimens; however, the dynamic aspects of the IP(3)R structure have never been visualized in a native environment. Here we attempt to measure the surface topography of IP(3)R in solution using atomic force microscopy (AFM). AFM revealed large protrusions extending approximately 4.3 nm above a flat membrane prepared from Spodoptera frugiperda (Sf9) cells overexpressing mouse type 1 IP(3)R (Sf9-IP(3)R1). The average diameter of the large protrusions was approximately 32 nm. A specific antibody against a cytosolic epitope close to the IP(3)-binding site enabled us to gold-label the Sf9-IP(3)R1 membrane as confirmed by EM. AFM images of the gold-labeled membrane revealed 7.7-nm high protrusions with a diameter of approximately 30 nm, which should be IP(3)R1-antibody complexes. Authentic IP(3)R1 immuno-purified from mouse cerebella had approximately the same dimensions as those of the IP(3)R-like protrusions on the membrane. Altogether, these results suggest that the large protrusions on the Sf9-IP(3)R1 membrane correspond to the cytosolic domain of IP(3)R1. Our study provides the first 3D representation of individual IP(3)R1 particles in an aqueous solution.
Suhara
Wakako
W
The Division of Neural Signal Information NTT-IMSUT, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
Kobayashi
Mime
M
Sagara
Hiroshi
H
Hamada
Kozo
K
Goto
Touichiro
T
Fujimoto
Ichiro
I
Torimitsu
Keiichi
K
Mikoshiba
Katsuhiko
K
D015220:Calcium Channels / Q000737:chemistry* / Q000648:ultrastructure*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018625:Microscopy, Atomic Force / Q000379:methods*; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000648:ultrastructure*; D012996:Solutions; D014867:Water / Q000737:chemistry*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D012996:Solutions; D014867:Water