Kinetics of calcium release by immunoaffinity-purified inositol 1,4,5-trisphosphate receptor in reconstituted lipid vesicles

Abstract

The kinetics of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release of the immunoaffinity-purified IP3 receptor (IP3R), reconstituted into lipid vesicles, was investigated using the fluorescent Ca2+ indicator fluo-3. IP3R was purified from mouse cerebellar microsomal fraction by using an immunoaffinity column conjugated with an anti-IP3R type 1 (IP3R1) antibody. The immunoblotting analysis using monoclonal antibodies against each IP3R type showed that the purified IP3R is almost homogeneous, composed of IP3R1. Ca2+ efflux from the proteoliposomes was monitored as fluorescence changes of 10 μm fluo-3, whose concentration was high enough to buffer released Ca2+ and to keep deviations of extravesicular free Ca2+ concentration within 30 nM, excluding the possibility of Ca2+-mediated regulation of IP3-induced Ca2+ release. We also examined IP3-induced Ca2+ release using 1 μm fluo-3, where the deviations of free Ca2+ concentration were within 300 nM. At both fluo-3 concentrations, IP3-induced Ca2+ release showed similar kinetic properties, i.e. little Ca2+ regulation of Ca2+ release was observed in this system. IP3-induced Ca2+ release of the purified IP3R exhibited positive cooperativity; the Hill coefficient was 1.8 ± 0.1. The halfmaximal initial rate for Ca2+ release occurred at 100 nM IP3. At the submaximal concentrations of IP3, the purified IP3R showed quantal Ca2+ release, indicating that a single type of IP3R is capable of producing the phenomenon of quantal Ca2+ release. The profiles of the IP3-induced Ca2+ release of the purified IP3R were found to be biexponential with the fast and slow rate constants (kfast = 0.3 ∼ 0.7 s-1, kslow = 0.03 ∼ 0.07 s-1)» indicating that IP3R has two states to release Ca2+. The amount of released Ca2+ by the slow phase was constant over the range of 10-5000 niw IP3 concentrations, whereas that by the fast phase increased in proportion to added IP3. This provides evidence to support the view that the fast phase of Ca2+ release is mediated by the low affinity state and the slow phase by the high affinity state of the IP3R. This also suggests that the fast component of Ca2+ release is responsible for the process of quantal Ca2+ release.