Differential modulation of inositol 1,4,5-trisphosphate receptor type 1 and type 3 by ATP

Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP3R) results in a more pronounced Ca2+ release in the presence of inositol 1,4,5-trispho sphate (IP3). Two recently published studies demonstrated a different ATP s ensitivity of IP3-induced Ca2+ release in cell types expressing different I P3R isoforms. Cell types expressing mainly IP(3)R3 were less sensitive to A TP than cell types expressing mainly IP(3)R1 (Missiaen L, Parys JB, Sienaer t I et al. Functional properties of the type 3 InsP(3) receptor in 16HBE14o - bronchial mucosal cells. J Biol Chem 1998; 273. 8983-8986; Miyakawa T, Ma eda A, Yamazawa T et al. Encoding of Ca2+ signals by differential expressio n of IP3 receptor subtypes. EMBO J 1999; 18: 1303-1308). In order to invest igate the difference in ATP sensitivity between IP3R isoforms at the molecu lar level, microsomes of Sf9 insect cells expressing full-size IP(3)R1 or I P(3)R3 were covalently labeled with ATP by using the photoaffinity label 8- azido[alpha-P-32]ATP. ATP labeling of the IP3R was measured after immunopre cipitation of IP(3)Rs with isoform-specific antibodies, SDS-PAGE and Phosph orimaging. Unlabeled ATP inhibited covalent linking of 8-azido[alpha-P-32]A TP to the recombinant IP(3)R1 and IP(3)R3 with an IC50 of 1.6 mu M and 177 mu M, respectively. MgATP was as effective as ATP in displacing 8-azido[alp ha-P-32]ATP from the ATP-binding sites on IP(3)R1 and IP(3)R3, and in stimu lating IP3-induced Ca2+ release from permeabilized A7r5 and 16HBE14o- cells . The interaction of ATP with the ATP-binding sites on IP(3)R1 and IP(3)R3 was different from its interaction with the IP3-binding domains, since ATP inhibited IP3 binding to the N-terminal 581 amino acids of IP(3)R1 and IP(3 )R3 with an IC50 of 353 mu M and 4.0 mM, respectively. The ATP-binding site s of IP(3)R1 bound much better ATP than ADP, AMP and particularly GTP, whil e IP(3)R3 displayed a much broader nucleotide specificity. These results th erefore provide molecular evidence for a differential regulation of IP(3)R1 and IP(3)R3 by ATP. (C) 2000 Harcourt Publishers Ltd.