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

Abstract

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.