P2X receptors are ATP-gated cation channels consisting of three subunits that are mutually intertwined and form an upper, central, and extracellular vestibule with three lateral portals and the channel pore. Here we used cysteine and alanine scanning mutagenesis of the rat P2X4R receptor V47-V61 and K326-N338 sequences to study structural and functional properties of extracellular vestibule during gating. Cysteine mutants were used to test the accessibility of these residue side chains to cadmium during closed-open-desensitized transitions, whereas alanine mutants served as controls. This study revealed the accessibility of residues E51, T57, S59, V61, K326, and M336 to cadmium in channels undergoing a transition from a closed-to-open state and the accessibility of residues V47, G53, D331, I332, I333, T335, I337, and N338 in channels undergoing a transition from an open-to-desensitized state; residues E56 and K329 were accessible during both transitions. The effect of cadmium on channel gating was stimulatory in all reactive V47-V61 mutants and inhibitory in the majority of reactive K326-N338 mutants. The rat P2X4 receptor homology model suggests that residues affected by cadmium in the closed-to-open transition were located within the lumen of the extracellular vestibule and toward the central vestibule; however, the residues affected by cadmium in the open-to-desensitized state were located at the bottom of the vestibule near the pore. Analysis of the model assumed that there is ion access to extracellular and central vestibules through lateral ports when the channel is closed, with residues above the first transmembrane domain being predominantly responsible for ion uptake. Upon receptor activation, there is passage of ions toward the residues located on the upper region of the second transmembrane domain, followed by permeation through the gate region.

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

Department of Cellular and Molecular Neuroendocrinology Institute of Physiology of the Academy of Sciences of the Czech Republic Prague Czech Republic ; Section on Cellular Signaling Program in Developmental Neuroscience The Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health Bethesda MD USA

Section on Cellular Signaling Program in Developmental Neuroscience The Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health Bethesda MD USA

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Acuna-Castillo C., Coddou C., Bull P., Brito J., Huidobro-Toro J. P. (2007). Differential role of extracellular histidines in copper, zinc, magnesium and proton modulation of the P2X7 purinergic receptor. J. Neurochem. 101 17–26 10.1111/j.1471-4159.2006.04343.x PubMed DOI

Allsopp R. C., El Ajouz S., Schmid R., Evans R. J. (2011). Cysteine scanning mutagenesis (residues Glu52-Gly96) of the human P2X1 receptor for ATP: mapping agonist binding and channel gating. J. Biol. Chem. 286 29207–29217 10.1074/jbc.M111.260364 PubMed DOI PMC

Benkert P., Tosatto S. C., Schomburg D. (2008). QMEAN: a comprehensive scoring function for model quality assessment. Proteins 71 261–277 10.1002/prot.21715 PubMed DOI

Bobanovic L. K., Royle S. J., Murrell-Lagnado R. D. (2002). P2X receptor trafficking in neurons is subunit specific. J. Neurosci. 22 4814–4824 PubMed PMC

Brake A. J., Wagenbach M. J., Julius D. (1994). New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature 371 519–523 10.1038/371519a0 PubMed DOI

Coddou C., Lorca R. A., Acuna-Castillo C., Grauso M., Rassendren F., Huidobro-Toro J. P. (2005). Heavy metals modulate the activity of the purinergic P2X4 receptor. Toxicol. Appl. Pharmacol. 202 121–131 10.1016/j.taap.2004.06.015 PubMed DOI

Coddou C., Yan Z., Obsil T., Huidobro-Toro J. P., Stojilkovic S. S. (2011). Activation and regulation of purinergic P2X receptor channels. Pharmacol. Rev. 63 641–683 10.1124/pr.110.003129 PubMed DOI PMC

Egan T. M., Haines W. R., Voigt M. M. (1998). A domain contributing to the ion channel of ATP-gated P2X2 receptors identified by the substituted cysteine accessibility method. J. Neurosci. 18 2350–2359 PubMed PMC

Egan T. M., Khakh B. S. (2004). Contribution of calcium ions to P2X channel responses. J. Neurosci. 24 3413–3420 10.1523/JNEUROSCI.5429-03.2004 PubMed DOI PMC

Friday S. C., Hume R. I. (2008). Contribution of extracellular negatively charged residues to ATP action and zinc modulation of rat P2X2 receptors. J. Neurochem. 105 1264–1275 10.1111/j.1471-4159.2008.05228.x PubMed DOI PMC

Haines W. R., Voigt M. M., Migita K., Torres G. E., Egan T. M. (2001). On the contribution of the first transmembrane domain to whole-cell current through an ATP-gated ionotropic P2X receptor. J. Neurosci. 21 5885–5892 PubMed PMC

Hattori M., Gouaux E. (2012). Molecular mechanism of ATP binding and ion channel activation in P2X receptors. Nature 485 207–212 10.1038/nature11010 PubMed DOI PMC

Heymann G., Dai J., Li M., Silberberg S. D., Zhou H. X., Swartz K. J. (2013). Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels. Proc. Natl. Acad. Sci. U.S.A. 110 E4045–E4054 10.1073/pnas.1311071110 PubMed DOI PMC

Huber T., Freisinger E. (2013). Sulfide ions as modulators of metal-thiolate cluster size in a plant metallothionein. Dalton Trans. 42 8878–8889 10.1039/c3dt32438a PubMed DOI

Jiang L. H., Rassendren F., Spelta V., Surprenant A., North R. A. (2001). Amino acid residues involved in gating identified in the first membrane-spanning domain of the rat P2X(2) receptor. J. Biol. Chem. 276 14902–14908 10.1074/jbc.M011327200 PubMed DOI

Jiang R., Martz A., Gonin S., Taly A., Prado De Carvalho L., Grutter T. (2010). A putative extracellular salt bridge at the subunit interface contributes to the ion channel function of the ATP-gated P2X2 receptor. J. Biol. Chem. 285 15805–15815 10.1074/jbc.M110.101980 PubMed DOI PMC

Jindrichova M., Khafizov K., Skorinkin A., Fayuk D., Bart G., Zemkova H., et al. (2011). Highly conserved tyrosine 37 stabilizes desensitized states and restricts calcium permeability of ATP-gated P2X3 receptor. J. Neurochem. 119 676–685 10.1111/j.1471-4159.2011.07463.x PubMed DOI

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 10.1007/s11302-012-9314-7 PubMed DOI PMC

Kawate T., Michel J. C., Birdsong W. T., Gouaux E. (2009). Crystal structure of the ATP-gated P2X(4) ion channel in the closed state. Nature 460 592–598 10.1038/nature08198 PubMed DOI PMC

Kawate T., Robertson J. L., Li M., Silberberg S. D., Swartz K. J. (2011). Ion access pathway to the transmembrane pore in P2X receptor channels. J. Gen. Physiol. 137 579–590 10.1085/jgp.201010593 PubMed DOI PMC

Khakh B. S., Lester H. A. (1999). Dynamic selectivity filters in ion channels. Neuron 23 653–658 10.1016/S0896-6273(01)80025-8 PubMed DOI

Kielland J. (1937). Individual activity coefficients of ions in aqueous solutions. J. Am. Chem. Soc. 59 1675–1678 10.1021/ja01288a032 DOI

Kracun S., Chaptal V., Abramson J., Khakh B. S. (2010). Gated access to the pore of a P2X receptor: structural implications for closed-open transitions. J. Biol. Chem. 285 10110–10121 10.1074/jbc.M109.089185 PubMed DOI PMC

Kunihiro S., Saito T., Matsuda T., Inoue M., Kuramata M., Taguchi-Shiobara F., et al. (2013). Rice DEP1, encoding a highly cysteine-rich G protein gamma subunit, confers cadmium tolerance on yeast cells and plants. J. Exp. Bot. 64 4517–4527 10.1093/jxb/ert267 PubMed DOI PMC

Li M., Chang T. H., Silberberg S. D., Swartz K. J. (2008). Gating the pore of P2X receptor channels. Nat. Neurosci. 11 883–887 10.1038/nn.2151 PubMed DOI PMC

Li Z., Migita K., Samways D. S., Voigt M. M., Egan T. M. (2004). Gain and loss of channel function by alanine substitutions in the transmembrane segments of the rat ATP-gated P2X2 receptor. J. Neurosci. 24 7378–7386 10.1523/JNEUROSCI.1423-04.2004 PubMed DOI PMC

Lorca R. A., Coddou C., Gazitua M. C., Bull P., Arredondo C., Huidobro-Toro J. P. (2005). Extracellular histidine residues identify common structural determinants in the copper/zinc P2X2 receptor modulation. J. Neurochem. 95 499–512 10.1111/j.1471-4159.2005.03387.x PubMed DOI

Martin S. R., Linse S., Bayley P. M., Forsen S. (1986). Kinetics of cadmium and terbium dissociation from calmodulin and its tryptic fragments. Eur. J. Biochem. 161 595–601 10.1111/j.1432-1033.1986.tb10483.x PubMed DOI

Nicke A., Baumert H. G., Rettinger J., Eichele A., Lambrecht G., Mutschler E., et al. (1998). P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. EMBO J. 17 3016–3028 10.1093/emboj/17.11.3016 PubMed DOI PMC

Popova M., Asatryan L., Ostrovskaya O., Wyatt L. R., Li K., Alkana R. L., et al. (2010). A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors. J. Neurochem. 112 307–317 10.1111/j.1471-4159.2009.06460.x PubMed DOI PMC

Rajagopal S., Vishveshwara S. (2005). Short hydrogen bonds in proteins. FEBS J. 272 1819–1832 10.1111/j.1742-4658.2005.04604.x PubMed DOI

Rassendren F., Buell G., Newbolt A., North R. A., Surprenant A. (1997). Identification of amino acid residues contributing to the pore of a P2X receptor. EMBO J. 16 3446–3454 10.1093/emboj/16.12.3446 PubMed DOI PMC

Roberts J. A., Valente M., Allsopp R. C., Watt D., Evans R. J. (2009). Contribution of the region Glu181 to Val200 of the extracellular loop of the human P2X1 receptor to agonist binding and gating revealed using cysteine scanning mutagenesis. J. Neurochem. 109 1042–1052 10.1111/j.1471-4159.2009.06035.x PubMed DOI PMC

Rokic M. B., Stojilkovic S. S., Vavra V., Kuzyk P., Tvrdonova V., Zemkova H. (2013). Multiple roles of the extracellular vestibule amino acid residues in the function of the rat P2X4 receptor. PLoS ONE 8:e59411 10.1371/journal.pone.0059411 PubMed DOI PMC

Rothwell S. W., Stansfeld P. J., Bragg L., Verkhratsky A., North R. A. (2014). Direct gating of ATP-activated ion channels (P2X2 receptors) by lipophilic attachment at the outer end of the second transmembrane domain. J. Biol. Chem. 289 618–626 10.1074/jbc.M113.529099 PubMed DOI PMC

Royle S. J., Bobanovic L. K., Murrell-Lagnado R. D. (2002). Identification of a non-canonical tyrosine-based endocytic motif in an ionotropic receptor. J. Biol. Chem. 277 35378–35385 10.1074/jbc.M204844200 PubMed DOI

Royle S. J., Qureshi O. S., Bobanovic L. K., Evans P. R., Owen D. J., Murrell-Lagnado R. D. (2005). Non-canonical YXXGPhi endocytic motifs: recognition by AP2 and preferential utilization in P2X4 receptors. J. Cell Sci. 118 3073–3080 10.1242/jcs.02451 PubMed DOI

Samways D. S., Egan T. M. (2007). Acidic amino acids impart enhanced Ca2+ permeability and flux in two members of the ATP-gated P2X receptor family. J. Gen. Physiol. 129 245–256 10.1085/jgp.200609677 PubMed DOI PMC

Samways D. S., Khakh B. S., Dutertre S., Egan T. M. (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 13800–13805 10.1073/pnas.1017550108 PubMed DOI PMC

Schwede T., Kopp J., Guex N., Peitsch M. C. (2003). SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31 3381–3385 10.1093/nar/gkg520 PubMed DOI PMC

Shannon R. D. (1976). Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenide. Acta Crystallogr. A 32 751–767 10.1107/S0567739476001551 DOI

Stoop R., Thomas S., Rassendren F., Kawashima E., Buell G., Surprenant A., et al. (1999). Contribution of individual subunits to the multimeric P2X(2) receptor: estimates based on methanethiosulfonate block at T336C. Mol. Pharmacol. 56 973–981 10.1124/mol.56.5.973 PubMed DOI

Valera S., Hussy N., Evans R. J., Adami N., North R. A., Surprenant A., et al. (1994). A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP. Nature 371 516–519 10.1038/371516a0 PubMed DOI

Yan Z., Liang Z., Obsil T., Stojilkovic S. S. (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 32649–32659 10.1074/jbc.M512791200 PubMed DOI