Agonist Antagonist Interactions at the Rapidly Desensitizing P2X3 Receptor

Abstract

P2X3 receptors (P2XRs), as members of the purine receptor family, are deeply involved in chronic pain sensation and therefore, specific, competitive antagonists are of great interest for perspective pain management. Heretofore, Schild plot analysis has been commonly used for studying the interaction of competitive antagonists and the corresponding receptor. Unfortunately, the steady-state between antagonist and agonist, as a precondition for this kind of analysis, cannot be reached at fast desensitizing receptors like P2X3R making Schild plot analysis inappropriate. The aim of this study was to establish a new method to analyze the interaction of antagonists with their binding sites at the rapidly desensitizing human P2X3R. The patch-clamp technique was used to investigate the structurally divergent, preferential antagonists A317491, TNP-ATP and PPADS. The P2X1,3-selective α,β-methylene ATP (α,β-meATP) was used as an agonist to induce current responses at the wild-type (wt) P2X3R and several agonist binding site mutants. Afterwards a Markov model combining sequential transitions of the receptor from the closed to the open and desensitized mode in the presence or absence of associated antagonist molecules was developed according to the measured data. The P2X3R-induced currents could be fitted correctly with the help of this Markov model allowing identification of amino acids within the binding site which are important for antagonist binding. In conclusion, Markov models are suitable to simulate agonist antagonist interactions at fast desensitizing receptors such as the P2X3R. Among the antagonists investigated, TNP-ATP and A317491 acted in a competitive manner, while PPADS was identified as a (pseudo)irreversible blocker.

P2Y2 receptor antagonists as anti‐allodynic agents in acute and sub‐chronic trigeminal sensitization: Role of satellite glial cells

“…Next, to directly evaluate the role of P2YRs in the development of facial allodynia in CFA‐injected rats, we exposed animals to purinergic antagonists. First, the P2Y 1 R‐selective antagonist MRS2179 (5 or 15 mg kg −1 ) was utilized in parallel with the non‐selective antagonist PPADS (25 mg kg −1 ), which is known to inhibit several P2X nucleotide receptor channels (including the pro‐algogenic neuronal P2X3R subtype; Helms et al, ; Martucci et al, ) and various P2YRs (Abbracchio et al, ). Drug administration and assessment of allodynia were performed as described above.…”

“…Recently published data showed an anti‐allodynic effect of P2Y 2 R inhibition in neuropathic TG pain (Li et al, ). Accordingly, the non‐selective P2XR and P2YR antagonist PPADS (Abbracchio et al, ; Helms et al, ) has proven analgesic in a rodent model of neuropathic pain (Martucci et al, ). Nevertheless, P2Y 1 R and P2Y 2 R activation was reported to inhibit pro‐algogenic P2X3 receptors in sensory neurons (Gerevich et al, ; Mo et al, ), suggesting a complex modulation of pain pathways by P2YRs, possibly depending upon algogenic stimuli and pain models.…”

“…Recently published data showed an anti-allodynic effect of P2Y 2 R inhibition in neuropathic TG pain (Li et al, 2014). Accordingly, the non-selective P2XR and P2YR antagonist PPADS (Abbracchio et al, 2006;Helms et al, 2013) has proven analgesic in a rodent model of neuropathic pain (Martucci et al, 2008). Nevertheless, P2Y 1 R and P2Y 2 R activation was reported to inhibit pro-algogenic P2X3 receptors FIGURE 8: The anti-allodynic effect of AR-C118925 is maintained in sub-chronic conditions, along with the inhibition of SGC activation.…”

Characterization of purinergic receptor expression in ARPKD cystic epithelia

“…α,β-Methylene adenosine 5′-triphosphate (αβmeATP), a phosphonic analog of ATP and a common agonist of the P2X receptors, was further used to detect specific P2 receptors involved in elevation of [Ca 2+ ] i in cystic cells. αβmeATP has the EC 50 ~1 μM for P2X 1 and P2X 3 and ~1000-fold less potent for P2X 2 and P2X 4,5,6,7 receptors [38][39][40]. Similarly to ATP, high concentrations of αβmeATP (~400 μM) were required for the elevation of [Ca 2+ ] i in the cystic monolayer (Fig.…”

A shared mechanism for TNP-ATP recognition by members of the P2X receptor family

“…In addition, we evaluated the contribution of h3 F714, the only residue showing interaction with the TNP group in the hP2X3/TNP-ATP structure. Since it has been reported that the F 174 A mutant of hP2X3 does not alter the inhibition by TNP-ATP [49] , we designed a tryptophan mutant ( h3 F174W) to create a bulkier side chain. However, the inhibition by TNP-ATP remained unchanged (inhibition ratio = 0.810 ± 0.044 for h3 F174W, p > 0.05 vs h3 WT 0.764 ± 0.033, n = 8–12, unpaired t-test; Fig.…”