Molecular basis of selective antagonism of the P2X1 receptor for ATP by NF449 and suramin: contribution of basic amino acids in the cysteine‐rich loop

Abstract

BACKGROUND AND PURPOSEThe cysteine-rich head region, which is adjacent to the proposed ATP-binding pocket in the extracellular ligand-binding loop of P2X receptors for ATP, is absent in the antagonist-insensitive Dictyostelium receptors. In this study we have determined the contribution of the head region to the antagonist action of NF449 and suramin at the human P2X1 receptor.EXPERIMENTAL APPROACHChimeras and point mutations in the cysteine-rich head region were made between human P2X1 and P2X2 receptors. Mutant receptors were expressed in Xenopus oocytes and P2X receptor currents characterized using two-electrode voltage clamp.KEY RESULTSThe chimera replacing the region between the third and fourth conserved cysteine residues of the P2X1 receptor with the corresponding part of P2X2 reduced NF449 sensitivity a thousand fold from an IC50 of ∼1 nM at the P2X1 receptor to that of the P2X2 receptor (IC50∼1 µM). A similar decrease in sensitivity resulted from mutation of four positively charged P2X1 receptor residues in this region that are absent from the P2X2 receptor. These chimeras and mutations were also involved in determining sensitivity to the antagonist suramin. Reciprocal chimeras and mutations in the P2X2 receptor produced modest increases in antagonist sensitivity.CONCLUSIONS AND IMPLICATIONSThese data indicate that a cluster of positively charged residues at the base of the cysteine-rich head region can account for the highly selective antagonism of the P2X1 receptor by the suramin derivative NF449.

Time-Resolved Fluorescence Resonance Energy Transfer Assay for Discovery of Small-Molecule Inhibitors of Methyl-CpG Binding Domain Protein 2

“…The P2X family form homo or heterotrimeric complexes on the surface of many cell types, forming ATP-gated cation channels 27 that have been implicated in multiple cellular processes 28 . The mechanism of inhibition features ionic interactions between the 8 sulfonic acid groups on NF449 with positively charged basic amino acids at the base of a cysteine-rich loop on P2X family members 26 . The selectivity of NF449 to P2X1 compared to other P2X and P2Y members is thought to arise through a second favorable contact between the compound and lysine 138 near the ATP binding pocket 26 .…”

“…The mechanism of inhibition features ionic interactions between the 8 sulfonic acid groups on NF449 with positively charged basic amino acids at the base of a cysteine-rich loop on P2X family members 26 . The selectivity of NF449 to P2X1 compared to other P2X and P2Y members is thought to arise through a second favorable contact between the compound and lysine 138 near the ATP binding pocket 26 . NF449 has also been shown to inhibit other proteins, including the G protein alpha subunit 29 and FGFR3 30 .…”

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

“…The architecture of head domain of the zfP2X4 receptor was determined by X-ray diffraction and showed a high similarity in folding pattern with rat P2X4 (rP2X4) resolved by nuclear magnetic resonance, suggesting the conservation of the P2X4 head domain in different species [40] . Deletion of 42 residues in the head domain of P2X1 resulted in the loss of channel function without interfering with membrane trafficking [43] , suggesting that the head domain is an integrant domain of channel gating. Using molecular dynamic (MD) simulations and normal mode analysis, previous studies revealed a spontaneous downward motion of the head domain, probably resulting from its inherent dynamics [16,44,45] ( Figure 2B and 2C).…”

Purinergic P2X Receptors: Structural and Functional Features Depicted by X-Ray and Molecular Modelling Studies

“…In hP2X1, a cluster of positively charged residues at the base of the head domain is proposed to be involved in binding the competitive antagonist, suramin and its derivative NF449 [89]. A structure of a P2X receptor in complex with an antagonist such as PPADS or suramin would aid our understanding of how these molecules block the receptors, as well as why some receptors are sensitive and some resistant to antagonism.…”

“…The same model and set of mutants was later used to analyse agonist binding and channel gating [30]; in this work both ATP and BzATP were docked into the model using GOLD [88], allowing comparison of the docking with experimental data. A third publication using the model analysed the effect of deletion and mutation of the head region of P2X1 on channel function, suramin and NF449 sensitivity [89]. Lorinczi et al constructed a homology model of rP2X1 using…”