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.

Department of Cellular and Molecular Neuroendocrinology Institute of Physiology of the Academy of Sciences of the Czech Republic Prague Czech Republic

Zobrazit více v PubMed

Fountain SJ, Parkinson K, Young MT, Cao L, Thompson CR, et al. (2007) An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum. Nature. 448: 200–3. PubMed PMC

Burnstock G (2006) Pathophysiology and therapeutic potential of purinergic signaling. Pharmacol Rev. 58: 58–86. PubMed

Khakh BS, North RA (2012) Neuromodulation by extracellular ATP and P2X receptors in the CNS. Neuron. 76: 51–69. PubMed PMC

Stojilkovic SS (2009) Purinergic regulation of hypothalamopituitary functions. Trends Endocrinol Metab. 20: 460–8. PubMed PMC

Surprenant A, North RA (2009) Signaling at purinergic P2X receptors. Annu Rev Physiol. 71: 333–59. PubMed

Virginio C, MacKenzie A, Rassendren FA, North RA, Surprenant A (1999) Pore dilation of neuronal P2X receptor channels. Nat Neurosci. 2: 315–21. PubMed

Vavra V, Bhattacharya A, Zemkova H (2011) Facilitation of glutamate and GABA release by P2X receptor activation in supraoptic neurons from freshly isolated rat brain slices. Neuroscience. 188: 1–12. PubMed

Inoue K, Tsuda M, Koizumi S (2004) ATP- and adenosine-mediated signaling in the central nervous system: chronic pain and microglia: involvement of the ATP receptor P2X4. J Pharmacol Sci. 94: 112–4. PubMed

Lorca RA, Rozas C, Loyola S, Moreira-Ramos S, Zeise ML, et al. (2011) Zinc enhances long-term potentiation through P2X receptor modulation in the hippocampal CA1 region. Eur J Neurosci. 33: 1175–85. PubMed PMC

Li C, Xiong K, Weight FF (2000) Ethanol inhibition of adenosine 5′-triphosphate-activated current in freshly isolated adult rat hippocampal CA1 neurons. Neurosci Lett. 295: 77–80. PubMed

North RA (2002) Molecular physiology of P2X receptors. Physiol Rev. 82: 1013–67. PubMed

Kaczmarek-Hajek K, Lorinczi E, Hausmann R, Nicke A (2012) Molecular and functional properties of P2X receptors–recent progress and persisting challenges. Purinergic Signal. 8: 375–417. PubMed PMC

Nicke A, Baumert HG, Rettinger J, Eichele A, Lambrecht G, et al. (1998) P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. Embo J. 17: 3016–28. PubMed PMC

Marquez-Klaka B, Rettinger J, Bhargava Y, Eisele T, Nicke A (2007) Identification of an intersubunit cross-link between substituted cysteine residues located in the putative ATP binding site of the P2X1 receptor. J Neurosci. 27: 1456–66. PubMed PMC

Coddou C, Yan Z, Obsil T, Huidobro-Toro JP, Stojilkovic SS (2011) Activation and regulation of purinergic P2X receptor channels. Pharmacol Rev. 63: 641–83. PubMed PMC

Roberts JA, Evans RJ (2004) ATP binding at human P2X1 receptors. Contribution of aromatic and basic amino acids revealed using mutagenesis and partial agonists. J Biol Chem. 279: 9043–55. PubMed

Khakh BS, Bao XR, Labarca C, Lester HA (1999) Neuronal P2X transmitter-gated cation channels change their ion selectivity in seconds. Nat Neurosci. 2: 322–30. PubMed

Buell G, Lewis C, Collo G, North RA, Surprenant A (1996) An antagonist-insensitive P2X receptor expressed in epithelia and brain. Embo J. 15: 55–62. PubMed PMC

Khakh BS, Proctor WR, Dunwiddie TV, Labarca C, Lester HA (1999) Allosteric control of gating and kinetics at P2X(4) receptor channels. J Neurosci. 19: 7289–99. PubMed PMC

Stojilkovic SS, Yan Z, Obsil T, Zemkova H (2010) Structural Insights into the Function of P2X4: An ATP-Gated Cation Channel of Neuroendocrine Cells. Cell Mol Neurobiol. 30: 1251–8. PubMed PMC

Jelinkova I, Yan Z, Liang Z, Moonat S, Teisinger J, et al. (2006) Identification of P2X(4) receptor-specific residues contributing to the ivermectin effects on channel deactivation. Biochem Biophys Res Commun. 349: 619–25. PubMed

Priel A, Silberberg SD (2004) Mechanism of ivermectin facilitation of human P2X4 receptor channels. J Gen Physiol. 123: 281–93. PubMed PMC

Bobanovic LK, Royle SJ, Murrell-Lagnado RD (2002) P2X receptor trafficking in neurons is subunit specific. J Neurosci. 22: 4814–24. PubMed PMC

Qureshi OS, Paramasivam A, Yu JC, Murrell-Lagnado RD (2007) Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis. J Cell Sci. 120: 3838–49. PubMed

Royle SJ, Bobanovic LK, Murrell-Lagnado RD (2002) Identification of a non-canonical tyrosine-based endocytic motif in an ionotropic receptor. J Biol Chem. 277: 35378–85. PubMed

Hattori M, Gouaux E (2012) Molecular mechanism of ATP binding and ion channel activation in P2X receptors. Nature. 485: 207–12. PubMed PMC

Kawate T, Michel JC, Birdsong WT, Gouaux E (2009) Crystal structure of the ATP-gated P2X(4) ion channel in the closed state. Nature. 460: 592–8. PubMed PMC

Samways DS, Khakh BS, Egan TM (2012) Allosteric Modulation of Ca2+ flux in Ligand-gated Cation Channel (P2X4) by Actions on Lateral Portals. J Biol Chem. 287: 7594–602. PubMed PMC

Samways DS, Khakh BS, Dutertre S, Egan TM (2011) Preferential use of unobstructed lateral portals as the access route to the pore of human ATP-gated ion channels (P2X receptors). Proc Natl Acad Sci U S A. 108: 13800–5. PubMed PMC

Kracun S, Chaptal V, Abramson J, Khakh BS (2010) Gated access to the pore of a P2X receptor: Structural implications for closed-open transitions. J Biol Chem. 285: 10110–21. PubMed PMC

Kawate T, Robertson JL, Li M, Silberberg SD, Swartz KJ (2011) Ion access pathway to the transmembrane pore in P2X receptor channels. J Gen Physiol. 137: 579–90. PubMed PMC

Allsopp RC, El Ajouz S, Schmid R, Evans RJ (2011) Cysteine scanning mutagenesis (residues Glu52-Gly96) of the human P2X1 receptor for ATP: mapping agonist binding and channel gating. J Biol Chem. 286: 29207–17. PubMed PMC

Jiang LH, Rassendren F, Surprenant A, North RA (2000) Identification of amino acid residues contributing to the ATP-binding site of a purinergic P2X receptor. J Biol Chem. 275: 34190–6. PubMed

Roberts JA, Evans RJ (2006) Contribution of conserved polar glutamine, asparagine and threonine residues and glycosylation to agonist action at human P2X1 receptors for ATP. J Neurochem. 96: 843–52. PubMed

Yan Z, Liang Z, Obsil T, Stojilkovic SS (2006) Participation of the Lys313-Ile333 sequence of the purinergic P2X4 receptor in agonist binding and transduction of signals to the channel gate. J Biol Chem. 281: 32649–59. PubMed

Coddou C, Morales B, Gonzalez J, Grauso M, Gordillo F, et al. (2003) Histidine 140 plays a key role in the inhibitory modulation of the P2X4 nucleotide receptor by copper but not zinc. J Biol Chem. 278: 36777–85. PubMed

Friday SC, Hume RI (2008) Contribution of extracellular negatively charged residues to ATP action and zinc modulation of rat P2X2 receptors. J Neurochem. 105: 1264–75. PubMed PMC

Khakh BS, Egan TM (2005) Contribution of transmembrane regions to ATP-gated P2X2 channel permeability dynamics. J Biol Chem. 280: 6118–29. PubMed

Roberts JA, Evans RJ (2007) Cysteine substitution mutants give structural insight and identify ATP binding and activation sites at P2X receptors. J Neurosci. 27: 4072–82. PubMed PMC

Samways DS, Migita K, Li Z, Egan TM (2008) On the role of the first transmembrane domain in cation permeability and flux of the ATP-gated P2X2 receptor. J Biol Chem. 283: 5110–7. PubMed

Cao L, Broomhead HE, Young MT, North RA (2009) Polar residues in the second transmembrane domain of the rat P2X2 receptor that affect spontaneous gating, unitary conductance, and rectification. J Neurosci. 29: 14257–64. PubMed PMC

Jiang LH, Rassendren F, Spelta V, Surprenant A, North RA (2001) Amino acid residues involved in gating identified in the first membrane-spanning domain of the rat P2X(2) receptor. J Biol Chem. 276: 14902–8. PubMed

Li Z, Migita K, Samways DS, Voigt MM, Egan TM (2004) Gain and loss of channel function by alanine substitutions in the transmembrane segments of the rat ATP-gated P2X2 receptor. J Neurosci. 24: 7378–86. PubMed PMC

Rassendren F, Buell G, Newbolt A, North RA, Surprenant A (1997) Identification of amino acid residues contributing to the pore of a P2X receptor. Embo J. 16: 3446–54. PubMed PMC

Egan TM, Khakh BS (2004) Contribution of calcium ions to P2X channel responses. J Neurosci. 24: 3413–20. PubMed PMC

Samways DS, Egan TM (2007) Acidic amino acids impart enhanced Ca2+ permeability and flux in two members of the ATP-gated P2X receptor family. J Gen Physiol. 129: 245–56. PubMed PMC

Silberberg SD, Chang TH, Swartz KJ (2005) Secondary Structure and Gating Rearrangements of Transmembrane Segments in Rat P2X4 Receptor Channels. J Gen Physiol. 125: 347–59. PubMed PMC

Du J, Dong H, Zhou HX (2012) Gating mechanism of a P2X4 receptor developed from normal mode analysis and molecular dynamics simulations. Proc Natl Acad Sci U S A. 109: 4140–5. PubMed PMC

Jindrichova M, Vavra V, Obsil T, Stojilkovic SS, Zemkova H (2009) Functional relevance of aromatic residues in the first transmembrane domain of P2X receptors. J Neurochem. 109: 923–34. PubMed PMC

Das M, Basu G (2012) Glycine Rescue of beta-Sheets from cis-Proline. J Am Chem Soc. 134: 16536–9. PubMed

Digby HR, Roberts JA, Sutcliffe MJ, Evans RJ (2005) Contribution of conserved glycine residues to ATP action at human P2X1 receptors: mutagenesis indicates that the glycine at position 250 is important for channel function. J Neurochem. 95: 1746–54. PubMed

Popova M, Asatryan L, Ostrovskaya O, Wyatt LR, Li K, et al. (2010) A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors. J Neurochem. 112: 307–17. PubMed PMC

Role of Conserved Residues and F322 in the Extracellular Vestibule of the Rat P2X7 Receptor in Its Expression, Function and Dye Uptake Ability

Allosteric regulation of the P2X4 receptor channel pore dilation

Identification of functionally important residues of the rat P2X4 receptor by alanine scanning mutagenesis of the dorsal fin and left flipper domains

Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating