The present work is devoted to the investigation of structural and functional characteristics of P2X3 receptors related to the process of desensitization. ATP-activated P2X3 receptors expressed in nociceptive sensory neurons play an important role in pain signaling. Basic properties of this receptor subtype, including very strong desensitization, depend on the rate of dissociation of the agonist from the binding site. Even though the rough structure of the ATP binding site has been proposed on the basis of the X-ray structure of the zebrafish P2X4 receptor and mutagenesis studies, the fine subunit-specific structural properties predisposing the receptor to tight capture of the agonist inside the binding pocket have not been elucidated. In this work, by exploring in silico the functional role for the left flipper located in the ectodomain region, we identified within this loop a candidate residue S275, which could contribute to the closure of the agonist-binding pocket. Testing of the S275 mutants using the patch?clamp technique revealed a crucial role for S275 in agonist binding and receptor desensitization. The S275A mutant showed a reduced rate of onset of desensitization and accelerated resensitization and was weakly inhibited by nanomolar agonist. Extracellular calcium application produced inhibition instead of facilitation of membrane currents. Moreover, some full agonists became only partial agonists when applied to the S275A receptor. These effects were stronger with the more hydrophobic mutants S275C and S275V. Taken together, our data suggest that S275 contributes to the closure of the agonist-binding pocket and that effective capture of the agonist provided by the left flipper in calcium-dependent manner determines the high rate of desensitization, slow recovery, and sensitivity to nanomolar agonist of the P2X3 receptor. In addition to investigation of fundamental processes observed in P2X3 receptors, we also studied some pharmacological sensitivity of P2X3 receptor. P2X3 receptors are highly susceptible to the fast and long-term desensitization, and this property can be used as an alternative and very promising method to reduce the ATP-mediated participation of P2X3 receptors in the process of chronic pain. Drugs available nowadays lack specificity, versatility and potency. Synthetic, non-hydrolysable analogue of diadenosine tetraphosphate (Ap4A), tested in the current work, could become potent analgesic drugs. It was shown that AppNHppA selectively inhibited P2X3 receptor transmembrane current in the patch clamp experiments. AppNHppA induces current depression at concentrations that don't generate macroscopic current, affecting desensitized receptor. Inhibitory effect of AppNHppA on fast current of P2X3 receptors correlates with expression of such in trigeminal, dorsal root and nodose (with no inhibition of slow current) ganglions. The same depressant action was observed on recombinant receptors in HEK293 cells: inhibition of current through the P2X3 receptors and no significant effect on P2X2 receptors. Our results indicate that in in vitro experiments diadenosine tetraphosphates analogues show prospective features to be used as a prototype for the creation of anti-nociceptive drugs in future. Another example of a drug affecting desensitized receptor is naproxen. Naproxen is one of the most commonly used medicines for the treatment of migraine attack. Using patch clamp recordings from HEK293 cells expressing P2X3 receptors, for the first time we tested the direct action of naproxen on P2X3 receptor-mediated membrane current. In clinically relevant concentrations of 0.5 mM, naproxen produced a use-dependent blocking effect on ATP receptors. Naproxen inhibited P2X3 receptors via facilitation of desensitization, which determines current decay in the continuous presence of the agonist. In summary, we present a novel fast mechanism for the antimigraine action of naproxen, which can act in synergy with the cyclooxygenase inhibition to attenuate headaches.
