Inositol 1,4,5-tris-phosphate activation of inositol tris-phosphate receptor Ca 2+ channel by ligand tuning of Ca 2+ inhibition

Inositol 1,4,5-tris-phosphate (IP3 ) binding to its receptors (IP3 R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals including temporal oscillations and propagating waves. IP3 -mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3 R in its native ER membrane were investigated by patch clamp electrophysiology of isolatedXenopus oocyte nuclei to determine the dependencies of IP3 R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+ ]i ) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP3 , even as [Ca2+ ]i was raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites with half-maximal activating [Ca2+ ]i (K act ) of 210 nM and Hill coefficient (H act ) ≈2; and inhibitory sites with half-maximal inhibitory [Ca2+ ]i (K inh ) of 54 μM and Hill coefficient (H inh ) ≈4. Lowering IP3 concentration was without effect on Ca2+ activation parameters orH inh , but decreasedK inh with a functional half-maximal activating IP3 concentration (K IP3 ) of 50 nM and Hill coefficient (H IP3 ) of 4 for IP3 . These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3 R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3 R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release.