Structural and functional characterization of inositol 1,4,5-trisphosphate receptor channel from mouse cerebellum.
The cerebellar inositol 1,4,5-trisphosphate (InsP3) receptor is a high molecular weight glycoprotein abundantly expressed in Purkinje cells. The subunit structure of the InsP3 receptor protein was examined by cross-linking experiments. Agarose-polyacrylamide gel electrophoresis of the cross-linked materials demonstrated that the cerebellar InsP3 receptor protein is composed of four noncovalently bound identical subunits each with a Mr of 320,000 in both purified and microsome-bound states. Chromatography of the purified receptor on a calmodulin-Sepharose column demonstrated a Ca2(+)-dependent interaction of the InsP3 receptor with calmodulin. Photoaffinity labeling of the cerebellar microsomal fraction with [alpha-32P]8-azidoadenosine 5'-triphosphate revealed the presence of ATP-binding site in the InsP3 receptor. Scatchard analysis of the purified InsP3 receptor revealed the Bmax and Kd values for ATP binding of 2.3 pmol/micrograms and 17 microM, respectively. Reconstitution of the purified InsP3 receptor into the planar lipid bilayer indicated channel activity in the purified receptor. It exhibited a calcium conductance (26 pS in 53 mM Ca2+) and sodium conductance (21 pS in 100-500 mM asymmetric Na+ solutions) with permeability ratios of PCa/PTris = 6.3 and PNa/PCl = 5.4. The purified channel was activated with submillimolar ATP in the presence of InsP3 and modified to reach a large conductance state.
Maeda
N
N
Division of Regulation of Macromolecular Function, Osaka University, Japan.
Kawasaki
T
T
Nakade
S
S
Yokota
N
N
Taguchi
T
T
Kasai
M
M
Mikoshiba
K
K
D000345:Affinity Labels; D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry; D015220:Calcium Channels; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004553:Electric Conductivity; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear
D000345:Affinity Labels; D015220:Calcium Channels; D003432:Cross-Linking Reagents; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D008051:Lipid Bilayers; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear