Determinants of adenophostin A binding to inositol trisphosphate receptors

Inositol 1,4,5-trisphosphate (IP₃) receptors from cerebellum and recombinant type 1 IP₃ receptors expressed in Sf9 cells had indistinguishable affinities for IP₃ (K_d = 6.40±0.48nM) and adenophostin A (K_d = 0.89±0.05nM). In cytosol-like medium, each of the three mammalian IP₃ receptor subtypes when expressed in Sf9 cells bound adenophostin A with greater affinity than IP₃. It has been suggested that adenophostin A binds with high affinity only in the presence of ATP, but we found that adenophostin A similarly displaced [³H]IP₃ from type 1 IP₃ receptors whatever the ATP concentration. N-terminal fragments of the type 1 receptor were expressed with and without the S1 splice site; its removal had no effect on [³H]IP₃ binding to the 1—604 protein, but abolished binding to the 224—604 protein. The 1—604 fragment and full-length receptor bound adenophostin A with the same affinity, but the fragment had 3-fold greater affinity for IP₃, suggesting that C-terminal residues selectively inhibit IP₃ binding. The 224—604S1⁺ fragment bound IP₃ and adenophostin A with increased affinity, but as with the 1—604 fragment it bound adenophostin A with only 2-fold greater affinity than IP₃. High-affinity binding of adenophostin A may be partially determined by its 2′-phosphate interacting more effectively than the 1-phosphate of IP₃ with residues within the IP₃-binding core. This may account for the 2-fold greater affinity of adenophostin A relative to IP₃ for the minimal IP₃-binding domain. In addition we suggest that C-terminal residues, which impede access of IP₃, may selectively interact with adenophostin A to allow it unhindered access to the IP₃-binding domain.