Abstract

Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) are intracellular Ca²⁺ channels. Their opening is initiated by binding of IP₃ to the IP₃-binding core (IBC; residues 224–604 of IP₃R1) and transmitted to the pore via the suppressor domain (SD; residues 1–223). The major conformational changes leading to IP₃R activation occur within the N terminus (NT; residues 1–604). We therefore developed a high-throughput fluorescence polarization (FP) assay using a newly synthesized analog of IP₃, fluorescein isothiocyanate (FITC)-IP₃, to examine the thermodynamics of IP₃ and adenophostin A binding to the NT and IBC. Using both single-channel recording and the FP assay, we demonstrate that FITC-IP₃ is a high-affinity partial agonist of the IP₃R. Conventional [³H]IP₃ and FP assays provide similar estimates of the K_D for both IP₃ and adenophostin A in cytosol-like medium at 4°C. They further establish that the isolated IBC retains the ability of full-length IP₃R to bind adenophostin A with ∼10-fold greater affinity than IP₃. By examining the reversible effects of temperature on ligand binding, we established that favorable entropy changes (TΔS) account for the greater affinities of both ligands for the IBC relative to the NT and for the greater affinity of adenophostin A relative to IP₃. The two agonists differ more substantially in the relative contribution of ΔH and TΔS to binding to the IBC relative to the NT. This suggests that different initial binding events drive the IP₃R on convergent pathways toward a similar open state.