Inositol 1,4,5-trisphosphate (IP3) receptors from cerebellum and recombinant type 1 IP3 receptors expressed in Sf9 cells had indistinguishable affinities for IP3 (Kd = 6.40±0.48nM) and adenophostin A (Kd = 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.
Abbreviations used: CLM, cytosol-like medium; IP3, inositol 1,4,5-trisphosphate; Kd, equilibrium dissociation constant; TEM, Tris/EDTA medium.