ATP modulation of Ca2+ release by type-2 and type-3 inositol (1,4,5)-triphosphate receptors -: Differing ATP sensitivities and molecular determinants of action

ATP enhances Ca2+ release from inositol (1,4,5)-trisphosphate receptors (InsP(3)R). However, the three isoforms of InsP(3)R are reported to respond to ATP with differing sensitivities. Ca2+ release through InsP(3)R(1) is positively regulated at lower ATP concentrations than InsP(3)R(3), and InsP(3)R(2) has been reported to be insensitive to ATP modulation. We have reexamined these differences by studying the effects of ATP on InsP(3)R(2) and InsP(3)R(3) expressed in isolation on a null background in DT40 InsP(3)R knock-out cells. We report that the Ca2+-releasing activity as well as the single channel open probability of InsP(3)R(2) was enhanced by ATP, but only at submaximal InsP(3) levels. Further, InsP(3)R(2) was more sensitive to ATP modulation than InsP(3)R(3) under similar experimental conditions. Mutations in the ATPB sites of InsP(3)R(2) and InsP(3)R(3) were generated, and the functional consequences of these mutations were tested. Surprisingly, mutation of the ATPB site in InsP(3)R(3) had no effect on ATP modulation, suggesting an additional locus for the effects of ATP on this isoform. In contrast, ablation of the ATPB site of InsP(3)R(2) eliminated the enhancing effects of ATP. Furthermore, this mutation had profound effects on the patterns of intracellular calcium signals, providing evidence for the physiological significance of ATP binding to InsP(3)R(2).