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 (IP(3)) binding to its receptors (IP(3)R) in the endoplasmic reticulum (ER) activates Ca(2+) release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca(2+) concentration signals including temporal oscillations and propagating waves. IP(3)-mediated Ca(2+) release is also controlled by cytoplasmic Ca(2+) concentration with both positive and negative feedback. Single-channel properties of the IP(3)R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP(3)R on cytoplasmic Ca(2+) and IP(3) concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP(3), even as [Ca(2+)](i) was raised to high concentrations, displaying two distinct types of functional Ca(2+) binding sites: activating sites with half-maximal activating [Ca(2+)](i) (K(act)) of 210 nM and Hill coefficient (H(act)) ≈2; and inhibitory sites with half-maximal inhibitory [Ca(2+)](i) (K(inh)) of 54 μM and Hill coefficient (H(inh)) ≈4. Lowering IP(3) concentration was without effect on Ca(2+) activation parameters or H(inh), but decreased K(inh) with a functional half-maximal activating IP(3) concentration (K(IP(3))) of 50 nM and Hill coefficient (H(IP(3))) of 4 for IP(3). These results demonstrate that Ca(2+) is a true receptor agonist, whereas the sole function of IP(3) is to relieve Ca(2+) inhibition of IP(3)R. Allosteric tuning of Ca(2+) inhibition by IP(3) enables the individual IP(3)R Ca(2+) channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca(2+) concentration signaling and quantal Ca(2+) release.