Abstract
Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel involved in various cellular signaling. Type 3 IP3R (IP3R3) retains ligand-gated Ca2+ channel properties differing from other subtypes in terms of IP3-binding affinity and regulation of its channel activity by effector molecules. In this study, we found the natural Pro335 --> Leu polymorphism of mouse IP3R3 between BALB/c and C57BL/6J. We investigated the functional differences between Pro335IP3R3 and Leu335IP3R3 with purified receptors reconstituted into proteoliposomes as well as with soluble ligand binding domains. Pro335IP3R3 exhibited significantly higher IP3-binding affinity and IP3-induced Ca2+ release than those of Leu335IP3R3 in both forms of the receptor. Moreover, the polymorphic change caused differences in the effect of external Ca2+ on IP3-induced Ca2+ release. The Pro335 --> Leu substitution alters the conformation of soluble ligand binding domain as revealed by intrinsic fluorescence and circular dichroism spectra with or without Ca2+. The results indicate that the polymorphism of IP3R3 causes changes in receptor function, presumably affecting intracellular Ca2+ signaling.
Highlights
Inositol 1,4,5-trisphosphate (IP3)1 is a well known second messenger that is produced by hydrolysis of phosphoinositol 4,5-bisphosphate involving phospholipase (1)
Polymorphism of IP3R3 Found in BC and B6 Mice—The coding sequences of the IP3R3 of BC and B6 mice were compared with the search for polymorphism that may potentially be associated with functional variation
IP3R3, we investigated their functional differences with the whole receptors and their ligand-binding domain (LBD)
Summary
Inositol 1,4,5-trisphosphate (IP3) is a well known second messenger that is produced by hydrolysis of phosphoinositol 4,5-bisphosphate involving phospholipase (1). DT 40 cells expressing only IP3R3 exhibit a single Ca2ϩ peak upon stimulation of the B-cell receptor, whereas type 1 or type 2 generates Ca2ϩ oscillation with the same signal (13). In IP3R1-knockdown, COS-7 cells, expressing primarily the type 3 receptor, exhibited a single Ca2ϩ peak, whose amplitude was reduced relative to that of the untreated cell (14). A study using a microsome purified from COS-7 cells expressing IP3R3 (17) and a patch clamping with Xenopus oocyte demonstrated the biphasic dependence of the channel activity by Ca2ϩ (18). We observed that BALB/c (BC) mice exhibited a higher defecation than C57BL/6J (B6) mice under stress conditions (19) It has been known in Caenorhabditis elegans that IP3R regulates the defecation rhythm (20). The results indicate that functional difference exists between receptor variants when tested with their ligand binding domains as well as the whole receptor
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