KINETICS OF CALCIUM-RELEASE BY IMMUNOAFFINITY-PURIFIED INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR IN RECONSTITUTED LIPID VESICLES

The kinetics of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ releas e of the immunoaffinity-purified IP3 receptor (IP(3)R), reconstituted into lipid vesicles, was investigated using the fluorescent Ca2+ indic ator fluo-3. IP(3)R was purified from mouse cerebellar microsomal frac tion by using an immunoaffinity column conjugated with an anti-IP(3)R type 1 (IP(3)R1) antibody. The immunoblotting analysis using monoclona l antibodies against each IP(3)R type showed that the purified IP(3)R is almost homogeneous, composed of IP(3)R1. Ca2+ efflux from the prote oliposomes was monitored as fluorescence changes of 10 mu M fluo-3, wh ose concentration was high enough to buffer released Ca2+ and to keep deviations of extravesicular free Ca2+ concentration within 30 nM, exc luding the possibility of Ca2+-mediated regulation of IP3-induced Ca2 release. We also examined IP3-induced Ca2+ release using 1 mu M fluo- 3, where the deviations of free Ca2+ concentration were within 300 nM. At both fluo-3 concentrations, IP3-induced Ca2+ release showed simila r kinetic properties, i.e. little Ca2+ regulation of Ca2+ release was observed in this system. IP3-induced Ca2+ release of the purified IP(3 )R exhibited positive cooperativity; the Hill coefficient was 1.8 +/- 0.1. The half-maximal initial rate for Ca2+ release occurred at 100 nM IP3. At the submaximal concentrations of IP3, the purified IP(3)R sho wed quantal Ca2+ release, indicating that a single type of IP(3)R is c apable of producing the phenomenon of quantal Ca2+ release. The profil es of the IP3-induced Ca2+ release of the purified IP(3)R were found t o be biexponential with the fast and slow rate constants (k(fast) = 0. 3 similar to 0.7 s(-1), k(slow) = 0.03 similar to 0.07 s(-1)), indicat ing that IP(3)R has two states to release Ca2+. The amount of released Ca2+ by the slow phase was constant over the range of 10-5000 nM IP3 concentrations, whereas that by the fast phase increased in proportion to added IP3. This provides evidence to support the view that the fas t phase of Ca2+ release is mediated by the low affinity state and the slow phase by the high affinity state of the IP(3)R. This also suggest s that the fast component of Ca2+ release is responsible for the proce ss of quantal Ca2+ release.