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 beta-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

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 effects of the Y54 and Q55 mutations on rP2X4R function.

<p>(A) The ATP-concentration dependence curves for the WT and Y54 substitution mutants. (B) The I_max values for the WT and Y54 substitution mutants in the presence (gray bars) and absence (white bars) of IVM. (C) The ATP concentration response curve for the Q55 mutants. (D) The I_max values of the Q55 mutants measured in the absence (white bars) and in the presence (gray bars) of IVM. The data are shown as the mean ± SEM values from 5–26 cells per mutant; **p<0.01 between control (−IVM) and ivermectin-treated (+IVM) receptors.</p

The effects of the F324 and G325 mutations on rP2X4R function.

<p>(A) The ATP-concentration response curves for the single point mutants at position F324. (B) The I_max values of the F324 mutants measured in the absence (white bars) and presence (gray bars) of IVM. (C) The concentration-response curve for the rP2X4-G325P (symbols) and WT receptor (dotted line). (D) The I_max values of G325 mutants measured in the absence (white bars) and in the presence (gray bars) of IVM. The mean ± SEM values from 7–23 cells per mutant are shown; **p<0.01 between IVM-untreated (-IVM) and -treated (+IVM) receptors.</p

Effect of alanine and cysteine point mutations on the maximum current amplitude.

<p>(A) Alanine and cysteine scanning mutagenesis of residues V47–V61 and F324–N338 that contain the upper parts of the TM1 and TM2 helices and the β-sheets in the open state. The maximum amplitude of currents (I_max) induced by 100 µM ATP in the wild type (WT; white bars) and cysteine (dark bars) and alanine (gray bars) mutant receptors. The receptors most affected had mutations at positions V49, Y54, Q55 (blue), F324 and G325 (red). The data are expressed as the mean ± SEM from 10–96 cells; **p<0.01 between WT and alanine or cysteine mutants. (B) The pattern of the ATP-induced currents by the WT and low-responsive rP2X4R alanine mutants. The horizontal bars indicate the duration of the application of 100 µM ATP (60 s). Notice the variable Y-scales for the WT and mutant receptors. (C) The topology of low-active residue mutants in the zfP2X4R in the apo-closed state (left) and ATP-bound open state (right); the mutated regions containing the upper parts of the TM1 and TM2 are shown in blue and red, respectively; affected mutated residues (rP2X4 numbering) are shown in red and blue spheres. Identity between rat and zebrafish P2X4R in amino acid sequences V47–V61 and F324–N338 is 67% and all functionally important residues are identical in both receptors.</p

Alanine- and cysteine-scanning mutagenesis of the F324–N338 rP2X4R segement.

<p>The I_max data shown are expressed as the mean ± SEM from 10–38 measurement per mutant.</p>**<p>p<0.01, between mutant and WT (used for I_max measurements);</p>*<p>p<0.05 between mutant and WT (used for EC₅₀ estimation).</p

The membrane expression of the low-responsive alanine mutants and functional characterization of the V49-rP2X4R mutants.

<p>(A) Western blots showing the expression pattern of the rP2X4R-WT and V49A, Y54A, Q55A, F324A and G325A mutants. (B) Densitometry quantification of the membrane/total ratio for five low-active mutants. The data are expressed as the mean ± SEM of 4 Western blot images, **p<0.01 between WT and alanine mutants. (C) The ATP concentration response curves of the WT and V49 mutant receptors. All concentration-response curves shown in this and following figures were generated using Hill coefficient of 1.3 obtained for WT receptor (dashed line). (D) The augmentation of the maximum current amplitude of the WT and V49 mutants by ivermectin (IVM). The I_max was determined prior to the IVM application (white bars) and 2–6 min after application of 3 µM IVM (gray bars). The mean ± SEM from 5–18 cells per mutant is shown; **p<0.01 between control (−IVM) and ivermectin-treated (+IVM) receptors.</p

The possible interactions of Y54 and Q55 with other residues in the same P2X4R subunit.

<p>Residue Y54 can form stacking interactions with F330 or F48, Q55 can form H-bonds with N262 (zfP2X4 has aspartic acid in corresponding position) or D264. The structure shows the zfP2X4R in the apo-closed state, numbering is rP2X4R.</p

The effect of hydrophobicity and the size at position 324 on ATP potency and the localization of the F324 and G325 residues in the rP2X4R molecule.

<p>(A and B) The correlation between the EC₅₀ values with the hydrophobic effect (A) and the change in side chain residue volume (B). (C) Both the F324 and G325 residues are within the β-sheet (in green) connecting the ATP binding site and the pore in the zfP2X4 apo-closed state (left) and outside the β-sheet in the ATP-bound open state (right); rP2X4 numbering. Notice the stable position of Y54 and Q55, and conserved protein fold above TM1 (arrowhead) both in the closed and open state.</p

Alanine- and cysteine-scanning mutagenesis of the V47–V61 rP2X4R segment.

<p>In this and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059411#pone-0059411-t002" target="_blank">Table 2</a>, the potency is expressed as the ATP concentration producing 50% of the maximal response (EC₅₀), and the efficacy as the maximum induced current (I_max) in response to 100 µM ATP. The I_max data are expressed as the mean ± SEM from 12–40 measurements per mutant and 96 measurements from the wild type (WT) receptor. n.d., not determined; n.f., nonfunctional;</p>**<p>p<0.01, between mutant and WT (used for I_max measurements);</p>*<p>p<0.05 between mutant and WT (used for EC₅₀ estimation).</p