Stimulation of inositol 1,4,5-trisphosphate (IP3) receptor subtypes by analogues of IP3. PLoS ONE

Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca 2+ channels. Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the a- and b-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca2+ release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-p interaction between the adenine of AdA and a conserved arginine within the IBC a-domain contributing to closure of the IBC. The two complementary contacts between AdA and the a-domain (cation-p interaction and 30-phosphate) allow activation of IP3R by an analogue of AdA (30-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of al