Use-Dependent Inhibition of P2X₃Receptors by Nanomolar Agonist

Abstract

P2X 3 receptors desensitize within 100 ms of channel activation, yet recovery from desensitization requires several minutes. The molecular basis for this slow rate of recovery is unknown. We designed experiments to test the hypothesis that this slow recovery is attributable to the high affinity (Ͻ1 nM) of desensitized P2X 3 receptors for agonist. We found that agonist binding to the desensitized state provided a mechanism for potent inhibition of P2X 3 current. Sustained applications of 0.5 nM ATP inhibited Ͼ50% of current to repetitive applications of P2X 3 agonist. Inhibition occurred at 1000-fold lower agonist concentrations than required for channel activation and showed strong use dependence. No inhibition occurred without previous activation and desensitization. Our data are consistent with a model whereby inhibition of P2X 3 by nanomolar [agonist] occurs by the rebinding of agonist to desensitized channels before recovery from desensitization. For several ATP analogs, the concentration required to inhibit P2X 3 current inversely correlated with the rate of recovery from desensitization. This indicates that the affinity of the desensitized state and recovery rate primarily depend on the rate of agonist unbinding. Consistent with this hypothesis, unbinding of [ 32 P]ATP from desensitized P2X 3 receptors mirrored the rate of recovery from desensitization. As expected, disruption of agonist binding by site-directed mutagenesis increased the IC 50 for inhibition and increased the rate of recovery.

Activation and Regulation of Purinergic P2X Receptor Channels

“…ATP is a full agonist for rP2X3R with estimated EC 50 values of 1.2 M (Chen et al, 1995), 2.6 M (Pratt et al, 2005), 4.1 M (Asatryan et al, 2008), and 7.3 M (Grote et al, 2005), but the precision of these estimates is limited by profound desensitization (Khmyz et al, 2008). Similar to the rP2X1R, ␣␤-meATP is a full agonist at rP2X3R, acting with a potency similar to (Pratt et al, 2005;Asatryan et al, 2008) or slightly lower than (Chen et al, 1995) that of ATP. 2-meSATP is also a full agonist for this receptor, whereas ATP␥S acts as a partial agonist Liu et al, 2001a).…”

Cutaneous Sensory Neurons Expressing the Mrgprd Receptor Sense Extracellular ATP and Are Putative Nociceptors

“…Because application of P2X3 agonists produce rapidly desensitizing current that can take several minutes or longer to fully recover (Pratt et al 2005;Sokolova et al 2004), a protocol was used in which ␣␤Me-ATP was applied every 60 s to study the P2X3-like current at the same time point on its recovery from desensitization (Fig. 6C).…”

Molecular mechanism for opioid dichotomy: bidirectional effect of μ-opioid receptors on P2X3 receptor currents in rat sensory neurones

“…This concentration of meATP is close to saturating for the activation of P2X 3 receptors, whereas 2-to 3-min intervals between agonist applications ensure >90 % recovery of P2X 3 receptors from desensitization [30]. In all experiments, application of Enk was preceded by 2-3 applications of meATP performed to obtain a stable control current.…”

“…Its rate depends on the agonist (with recovery being the slowest when ATP is used for activation [30]) and the temperature [31]; the recovery from desensitization is inhibited by purotoxin-1 from spider venom [32]. To reveal possible effects of Enk on P2X 3 receptors recovery from desensitization, the meATP application protocol was modified.…”