Stimulation of Inositol 1,4,5-Trisphosphate (IP(3)) Receptor Subtypes by Adenophostin A and Its Analogues

Inositol 1,4,5-trisphosphate receptors (IP(3)R) are intracellular Ca(2+) channels. Most animal cells express mixtures of the three IP(3)R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP(3)R and it shares with IP(3) the essential features of all IP(3)R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP(3). The two essential phosphate groups contribute to closure of the clam-like IP(3)-binding core (IBC), and thereby IP(3)R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP(3)R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP(3)R. We measured Ca(2+) release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP(3)R and stably expressing single subtypes of mammalian IP(3)R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP(3)R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP(3)R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP(3). These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP(3)R subtypes. They demonstrate that differences in the Ca(2+) signals evoked by AdA analogues are unlikely to be due to selective regulation of IP(3)R subtypes.