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 microM 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 Ca(2+)-mediated regulation of IP3-induced Ca2+ release. We also examined IP3-induced Ca2+ release using 1 microM 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 half-maximal 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+ of 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 (k(fast) = 0.3 approximately 0.7 s-1, k(slow) = 0.03 approximately 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 nM 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.
Highlights
Education, Scienceand Culture, the Japan Societyof the Promotion of Science, the Intractable Diseases Research Foundation, and the Human Frontier ScienceProgram
Inositol 1,4,5-trisphosphate (IPs)! is the second messenger derived from the hydrolysis of phosphatidylinositol bisphosphate via activation of phospholipase C, phospholipase C activity is enhanced by the activation of G protein-linked and tyrosine kinase-linked cell surface membrane receptors by various extracellular stimuli, such as hormones, growth factors, neurotransmitters, odorants, lights, etc
This IPa-mediated Ca2 + signaling plays a critical role in a variety of cell functions, including fertilization, cell proliferation, metabolism, secretion, contraction of smooth muscle, and neural signals [1]. For these multiple cell signaling, the mechanisms of transducing the IP3 signal into a Ca2 + signal, i.e. IPs-induced Ca2 + release, may be diverse in each cell type
Summary
Vol 270, No 32, Issue of August 11, pp. 19046-19051, 1995 Printed in U.S.A. Kinetics of Calcium Release by Immunoaffinity-purified Inositol 1,4,S-Trisphosphate Receptor in Reconstituted Lipid Vesicles*. The IPs signal is converted into a Ca 2 + signal by binding to its specific receptor, i.e. the IPs receptor (IPsR), which is an IPa-induced Ca 2 + _ releasing channel located on intracellular Ca 2 + stores such as the endoplasmic reticulum This IPa-mediated Ca2 + signaling plays a critical role in a variety of cell functions, including fertilization, cell proliferation, metabolism, secretion, contraction of smooth muscle, and neural signals [1]. To further characterize the channel opening mechanism, the kinetic study of lICR should be examined using a purified single type of the IP sR
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