Temperature-dependence and conformational basis of inositol 1,4,5-trisphosphate receptor regulated by Ca2+

The inositol 1,4,5-trisphosphate (InsP(3)) receptor was purified from bovin e cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (1:1) successfully. No effect of Ca 2+ concentration on [H-3]-InsP(3) binding to unreconstituted InsP(3) recept or could be observed either at 4 degrees C or at 25 degrees C, whereas the effect of [Ca2+] on reconstituted InsP(3) receptor depended on the temperat ure. The Ca2+ concentration outside the proteolipsome ([Ca2+](o)) had no de tectable effect on InsP(3) binding to InsP(3) receptor at 4 degrees C. In c ontrast, with increase of [Ca2+](o) from 0 to 100 nmol/L at 25 degrees C, t he InsP(3) binding activity increased gradually. Then the InsP3 binding act ivity was decreased drastically at higher [Ca2+](o) and inhibited entirely at 50 mu mol/L [Ca2+](o). Conformational studies on intrinsic fluorescence of the reconstituted InsP(3) receptor and its quenching by KI and HB indica ted that the global conformation of reconstituted InsP(3) receptor could no t be affected by [Ca2+](o) at 4 degrees C. While at 25 degrees C, the effec ts of 10 mu mol/L [Ca2+](o) on global, membrane and cytoplasmic conformatio n of the reconstituted InsP(3) receptor were different significantly from t hat of 100 nmol/L [Ca2+](o).