Two open states of P2X receptor channels

The occupancy of the ATP binding sites of P2X receptor (P2XR) channels causes the rapid opening of a small cation-permeable pore, followed by a gradual dilation that renders the pore permeable to large organic cations. Electrophysiologically, this phenomenon was shown using whole-cell current recording on P2X2R-, P2X2/X5R-, P2X4R- and P2X7R-expressing cells that were bathed in N-methyl-D-glucamine-containing buffers in the presence and/or absence of small monovalent and divalent cations. The pore dilation of P2X4R and P2X7R caused a secondary current growth, whereas that of P2X2R showed a sustained kinetic coupling of dilation and desensitization, leading to receptor channel closure. The pore size of the P2X7R open and dilated states was estimated to be approximately 0.85 nm and greater than 1 nm, respectively. The P2XR pore dilation was also observed in intact cells by measurement of fluorescent dye uptake/release, application of polyethylene glycols of different sizes, and atomic force microscopy. However, pore dilation was not observed at the single channel level. Structural data describing the dilated state are not available, and the relevance of orthosteric and allosteric ligand interactions to pore dilation was not studied

Ivermectin rescues the I_max of low-functioning mutants.

<p>(A) Acute effect of 3 µM ivermectin (IVM) applied for 10 s (gray areas) during ongoing stimulation with 100 µM ATP for 30 s (horizontal bars) in cells expressing the WT, the DF mutants (R203A, N204A, I205A, and L214A), or the LF mutants (D280A, R282A, P290A and N293A). Recordings are examples of traces similar to 3–5 traces per mutant and 30 per WT receptor. (B) Summary data showing the potentiating effect of IVM preapplication (for 4–6 min) on I_max in WT and alanine mutant receptors. The I_max values were derived from measurements taken in the absence (open bars) or in the presence (filled bars) of IVM. Values are presented as the mean ± SEM from 5–8 measurements per mutant and 15 measurements per WT. IVM treatment rescued the I_max of all low-functioning receptors, except in the case of N293A, which is an ATP binding mutant. The statistical significance was determined by an ANOVA comparing the WT I_max and the I_max of mutant receptors in the presence of IVM. **, p<0.01.</p

Multiple roles of the extracellular vestibule amino acid residues in the function of the rat P2X4 receptor.

The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening

The relative responsiveness of the wild type (WT) and selected rP2X4R mutants to P2XR agonist analogs.

<p>ATP and agonists were applied in 100 µM concentrations for 2 s with a washing interval of 60 s. The data are the mean ± SEM, relative to ATP efficacy (100%), from 26 to 37 measurements for the WT receptor and from 3 to 18 measurements per mutant. <i>Group I:</i> Mutants that exhibited changes in ATP potency/efficacy, deactivation kinetics, and changes in the relative responsiveness to orthosteric analog agonists. <i>Group II:</i> Mutants that exhibited changes in ATP potency/efficacy and deactivation, but no changes in the relative responsiveness to analog agonists. <i>Group III:</i> Mutants that showed no significant changes in ATP potency/efficacy. The statistical significance was determined by an ANOVA comparing the responsiveness to agonists between WT and mutant receptors: **, p<0.01, *, p<0.05. DF, Dorsal Fin; LF, Left Flipper.</p><p>The relative responsiveness of the wild type (WT) and selected rP2X4R mutants to P2XR agonist analogs.</p

Identification of Functionally Important Residues of the Rat P2X4 Receptor by Alanine Scanning Mutagenesis of the Dorsal Fin and Left Flipper Domains

<div><p>Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. Here, we focused on the role of these domains in receptor activation, responsiveness to orthosteric ATP analogue agonists, and desensitization. Alanine scanning mutagenesis of the R203-L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor showed that ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists 2-(methylthio)adenosine 5′-triphosphate, adenosine 5′-(γ-thio)triphosphate, 2′(3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate, and α,β-methyleneadenosine 5′-triphosphate. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. Among these mutants, D280A, T281A, R282A, H286A, G291A, and Y292A also exhibited increased time-constant of the desensitizing current response. These experiments, together with homology modeling, indicate that residues located in the upper part of the dorsal fin and left flipper domains, relative to distance from the channel pore, contribute to the organization of the ATP binding pocket and to the initiation of signal transmission towards residues in the lower part of both domains. The R203 and N204 residues, deeply buried in the protein, may integrate the output signal from these two domains towards the gate. In addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state.</p></div

Structural and tridimensional organization of the DF and LF domains.

<p>(A) Alignment of amino acid sequences from R203-L214 (DF domain) and D280-N293 (LF domain) using seven rP2X and zfP2X subunits. Structurally, these regions are composed of random coils that terminate with short α-helix and β-sheet structures (indicated by arrows). Conserved amino acid residues are shown in boxes. (B) The models of the zfP2X4.1R are shown in the open (4DW1) or closed (4DW0) state, and the Debye-Waller factor (B-factor) indicates the degree to which the electron density is spread (miniatures). The model shows the elevated B-factor values within the region of intersubunit interaction (magnified segments). Higher B-factors are indicated with thicker cylinders and a red-shifted color, while the lowest B-factors are represented with the thinnest cylinders and a blue-shifted color.</p

The structure of the ATP binding site in a rP2X4R homology model.

<p>Two panels show the position of affected residues (rotated 180°) at the interface between the LF and DF domains. The low-response residues without defined EC₅₀ values are grey spheres. The amino acid residues presented as green spheres demonstrate the topology of mutants with changes in ATP potency and/or efficacy (EC₅₀ and I_max), and agonist profile (<i>Group I</i> from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112902#pone-0112902-t002" target="_blank">Table 2</a>). The amino acids presented in red spheres illustrate the position of residues whose mutation has affected ATP potency and/or efficacy without changing the action of ATP analogs (<i>Group II</i> from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112902#pone-0112902-t002" target="_blank">Table 2</a>). In both panels, the ATP molecule is situated between two adjacent P2X4R subunits (blue and gray). The ATP molecule is shown in a wireframe model.</p

Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel

P2X2 receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating

Summary of the changes in estimated EC₅₀ values for ATP and changes in desensitization at the DF and LF alanine/cysteine mutants of P2X1-4R residues from published data.

<p>n. f., non-functional mutants; ↑ mutant with significant increased EC₅₀ in comparison to WT (values represent fold increase); ≈ close to WT receptor; D, affected time-constant of the desensitizing current response; -, non-investigated position; n.i., non-investigated P2X4R mutants that were analyzed in this study.</p><p>Summary of the changes in estimated EC₅₀ values for ATP and changes in desensitization at the DF and LF alanine/cysteine mutants of P2X1-4R residues from published data.</p

DF and LF mutants exhibit a rightward shift in EC₅₀.

<p>(A, B) Example records of ATP-induced currents from cells expressing the WT receptor and I205A, T210A, and L214A DF mutants (A) and D280A, R282A, H286A, and G291A LF mutants (B). Currents were stimulated by a short (2–5 s) application of different concentrations of ATP (1–1000 µM), indicated by horizontal bars above the traces. Experiments were performed on naïve receptors, and traces from different cells are shown. (C, D) Concentration response curves for WT, I205A, T210A, and L214A DF mutants (C) and D280A, R282A, H286A, and G291A LF mutants (D). Data points are presented as the mean ± SEM from 7–35 measurements per mutant, per concentration and 78 measurements for WT.</p