Abstract
Inositol 1,4,5-trisphosphate receptor (IP3R) is a highly controlled calcium (Ca2+) channel gated by inositol 1,4,5-trisphosphate (IP3). Multiple regulators modulate IP3-triggered pore opening by binding to discrete allosteric sites within IP3R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca2+, the most pivotal regulator, induced marked structural changes in the tetrameric IP3R purified from mouse cerebella. Electron microscopy of the IP3R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca2+ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP3-binding domains, assigned by binding to heparin-gold. Ca2+-dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca2+ appeared to regulate IP3 gating activity through the rearrangement of functional domains.
Highlights
We improved rapid purification of the IP3R1 channel so that we could use electron microscopic study to visualize the domain arrangement and to investigate its structural change by Ca2ϩ
From the ‡Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan, the ¶Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita, Osaka, 565-0874, Japan, the ʈDivision of Molecular Neurobiology, Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan, and the ‡‡Calcium Oscillation Project, International Cooperative Research Project (ICORP), Japan Science and Technology Corporation (JST), 3-144, Shirokanedai, Minato-ku, Tokyo 108-0071, Japan
We purified IP3R1 to apparent homogeneity as judged by gel electrophoresis (Fig. 1A) from mouse cerebella, which was functionally active as estimated by the specific activity of maximum binding to IP3 (3 nmol/mg of protein)
Summary
We improved rapid purification of the IP3R1 channel so that we could use electron microscopic study to visualize the domain arrangement and to investigate its structural change by Ca2ϩ. Comparison with the dimensions of ryanodine receptor, another intracellular Ca2ϩ channel [16], provides support that the projected size of the square structure in this study is reasonable because of the ratio in the molecular mass of the protomer. We tried to capture a Ca2ϩ-dependent transition between the dual structures by imaging IP3R1 particles injected into 1 mM CaCl2, 1 mM EDTA, or 1 mM EGTA.
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