Use-Dependent Inhibition of P2X sub(3) Receptors by Nanomolar Agonist

• Published in: The Journal of neuroscience 2005-08, Vol.25 (32), p.7359-7365
• Main Authors: Pratt, Emily B, Brink, Thaddeus S, Bergson, Pamela, Voigt, Mark M, Cook, Sean P
• Format: Article
• Language: English
• ISSN: 0270-6474; 1529-2401

P2X sub(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 (< 1 nM) of desensitized P2X sub(3) receptors for agonist. We found that agonist binding to the desensitized state provided a mechanism for potent inhibition of P2X sub(3) current. Sustained applications of 0.5 nM ATP inhibited >50% of current to repetitive applications of P2X sub(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 sub(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 sub(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 [ super(32)P]ATP from desensitized P2X sub(3) receptors mirrored the rate of recovery from desensitization. As expected, disruption of agonist binding by site-directed mutagenesis increased the IC sub(50) for inhibition and increased the rate of recovery.