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Structural and functional characterization of inositol 1,4,5-trisphosphate receptor channel from mouse cerebellum.

1991; Elsevier BV; Volume: 266; Issue: 2 Linguagem: Inglês

10.1016/s0021-9258(17)35289-4

1083-351X

Nobuaki Maeda, Takashi Kawasaki, Shinji Nakade, Naoto Yokota, Takahisa Taguchi, Michio Kasai, Katsuhiko Mikoshiba,

Trace Elements in Health

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 InsPs receptor protein is composed of four noncovalently bound identical subunits each with a M, 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 [ ~t -~~P ] 8azidoadenosine 5'-triphosphate revealed the presence of ATP-binding site in the InsP3 receptor.Scatchard analysis of the purified InsP3 receptor revealed the B,,, and K , values for ATP binding of 2.3 pmol/pg and 17 p ~, respectively.Reconstitution of the purified InsPs receptor into the planar lipid bilayer indicated channel activity in the purified receptor.It exhibited a calcium conductance (26 pS in 53 m M Ca2+) and sodium conductance (21 pS in 100-500 m M asymmetric Na+ solutions) with permeability ratios of P ~J P T ~~~ = 6.3 and P N J P ~, = 5.4.The purified channel was activated with submillimolar ATP in the presence of InsP3 and modified to reach a large conductance state.Inositol 1,4,5-trisphosphate (InsP3)' is a well known second messenger, which causes the liberation of calcium ions from intracellular storage sites (1).The calcium release has been thought to be mediated by a calcium channel directly coupled to the InsP3-specific receptor.Recently, an InsP3 receptor protein was purified from mouse and rat cerebella, where it