Abstract
Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R) are intracellular Ca(2+) channels. Their opening is initiated by binding of IP(3) to the IP(3)-binding core (IBC; residues 224-604 of IP(3)R1) and transmitted to the pore via the suppressor domain (SD; residues 1-223). The major conformational changes leading to IP(3)R 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 IP(3), fluorescein isothiocyanate (FITC)-IP(3), to examine the thermodynamics of IP(3) and adenophostin A binding to the NT and IBC. Using both single-channel recording and the FP assay, we demonstrate that FITC-IP(3) is a high-affinity partial agonist of the IP(3)R. Conventional [(3)H]IP(3) and FP assays provide similar estimates of the K(D) for both IP(3) and adenophostin A in cytosol-like medium at 4 degrees C. They further establish that the isolated IBC retains the ability of full-length IP(3)R to bind adenophostin A with approximately 10-fold greater affinity than IP(3). By examining the reversible effects of temperature on ligand binding, we established that favorable entropy changes (T Delta S) 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 IP(3). The two agonists differ more substantially in the relative contribution of Delta H and T Delta S to binding to the IBC relative to the NT. This suggests that different initial binding events drive the IP(3)R on convergent pathways toward a similar open state.