Atp-Dependent Adenophostin Activation of Inositol 1,4,5-Trisphosphate Receptor Channel Gating: Kinetic Implications for the Durations of Calcium Puffs in Cells

The inositol 1,4,5-trisphosphate (InsP₃) receptor (InsP₃R) is a ligand-gated intracellular Ca²⁺ release channel that plays a central role in modulating cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i). The fungal metabolite adenophostin A (AdA) is a potent agonist of the InsP₃R that is structurally different from InsP₃ and elicits distinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities of the InsP₃R in the outer membrane of isolated Xenopus laevis oocyte nuclei. InsP₃R activated by either AdA or InsP₃ have identical channel conductance properties. Furthermore, AdA, like InsP₃, activates the channel by tuning Ca²⁺ inhibition of gating. However, gating of the AdA-liganded InsP₃R has a critical dependence on cytoplasmic ATP free acid concentration not observed for InsP₃-liganded channels. Channel gating activated by AdA is indistinguishable from that elicited by InsP₃ in the presence of 0.5 mM ATP, although the functional affinity of the channel is 60-fold higher for AdA. However, in the absence of ATP, gating kinetics of AdA-liganded InsP₃R were very different. Channel open time was reduced by 50%, resulting in substantially lower maximum open probability than channels activated by AdA in the presence of ATP, or by InsP₃ in the presence or absence of ATP. Also, the higher functional affinity of InsP₃R for AdA than for InsP₃ is nearly abolished in the absence of ATP. Low affinity AdA analogues furanophostin and ribophostin activated InsP₃R channels with gating properties similar to those of AdA. These results provide novel insights for interpretations of observed effects of AdA on calcium signaling, including the mechanisms that determine the durations of elementary Ca²⁺ release events in cells. Comparisons of single-channel gating kinetics of the InsP₃R activated by InsP₃, AdA, and its analogues also identify molecular elements in InsP₃R ligands that contribute to binding and activation of channel gating.