Dissecting the Facilitator and Inhibitor Allosteric Metal Sites of the P2X4 Receptor Channel
2007; Elsevier BV; Volume: 282; Issue: 51 Linguagem: Inglês
10.1074/jbc.m706925200
ISSN1083-351X
AutoresClaudio Coddou, Claudio Acuña‐Castillo, Paulina Bull, J. Pablo Huidobro‐Toro,
Tópico(s)Pharmacological Receptor Mechanisms and Effects
ResumoZinc and copper are atypical modulators of ligand-gated ionic channels in the central nervous system. We sought to identify the amino acids of the rat P2X4 receptor involved in trace metal interaction, specifically in the immediate linear vicinity of His140, a residue previously identified as being critical for copper-induced inhibition of the ATP-evoked currents. Site-directed mutagenesis replaced conspicuous amino acids located within the extracellular domain region between Thr123 and Thr146 for alanines. cDNAs for the wild-type and the receptor mutants were expressed in Xenopus laevis oocytes and examined by the two-electrode technique. Cys132, but not Cys126, proved crucial for zinc-induced potentiation of the receptor activity, but not for copper-induced inhibition. Zinc inhibited in a concentration-dependent manner the ATP-gated currents of the C132A mutant. Likewise, Asp138, but not Asp131 was critical for copper and zinc inhibition; moreover, mutant D138A was 20-fold more reactive to zinc potentiation than wild-type receptors. Asp129, Asp131, and Thr133 had minor roles in metal modulation. We conclude that this region of the P2X4 receptor has a pocket for trace metal coordination with two distinct and separate facilitator and inhibitor metal allosteric sites. In addition, Cys132 does not seem to participate exclusively as a structural receptor channel folding motif but plays a role as a ligand for zinc modulation highlighting the role of trace metals in neuronal excitability. Zinc and copper are atypical modulators of ligand-gated ionic channels in the central nervous system. We sought to identify the amino acids of the rat P2X4 receptor involved in trace metal interaction, specifically in the immediate linear vicinity of His140, a residue previously identified as being critical for copper-induced inhibition of the ATP-evoked currents. Site-directed mutagenesis replaced conspicuous amino acids located within the extracellular domain region between Thr123 and Thr146 for alanines. cDNAs for the wild-type and the receptor mutants were expressed in Xenopus laevis oocytes and examined by the two-electrode technique. Cys132, but not Cys126, proved crucial for zinc-induced potentiation of the receptor activity, but not for copper-induced inhibition. Zinc inhibited in a concentration-dependent manner the ATP-gated currents of the C132A mutant. Likewise, Asp138, but not Asp131 was critical for copper and zinc inhibition; moreover, mutant D138A was 20-fold more reactive to zinc potentiation than wild-type receptors. Asp129, Asp131, and Thr133 had minor roles in metal modulation. We conclude that this region of the P2X4 receptor has a pocket for trace metal coordination with two distinct and separate facilitator and inhibitor metal allosteric sites. In addition, Cys132 does not seem to participate exclusively as a structural receptor channel folding motif but plays a role as a ligand for zinc modulation highlighting the role of trace metals in neuronal excitability. Extracellular ATP and structurally related nucleotides act as novel cell messengers through the activation of P2X receptors, which belong to the family of ligand-gated ionic channels. In addition, nucleotides, and particularly pyrimidine nucleotides, such as UTP and UDP, act on metabotropic P2Y receptors, members of the G protein-coupled receptor family. Seven subtypes of P2X channels have been identified and have been shown to be involved in a variety of neuronal pathways including pain transmission, the urination reflex, vas deferens contraction favoring sperm migration, etc. (1Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). These receptors are unique among ligand-gated ionic channels because each receptor subunit has only two transmembrane domains, with both the C and N termini facing the cytosol (2Vial C. Roberts J.A. Evans R.J. Trends Pharmacol Sci. 2004; 25: 487-493Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 3Khakh B.S. North R.A. Nature. 2006; 442: 527-532Crossref PubMed Scopus (717) Google Scholar). Moreover, recent studies using atomic force microscopy (4Barrera N.P. Ormond S.J. Henderson R.M. Murrell-Lagnado R.D. Edwardson J.M. J. Biol. Chem. 2005; 280: 10759-10765Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) provided topological evidence of the channel conformation and established that the functional P2X receptor channels are trimers, composed of either homo or heterotrimeric subunits (4Barrera N.P. Ormond S.J. Henderson R.M. Murrell-Lagnado R.D. Edwardson J.M. J. Biol. Chem. 2005; 280: 10759-10765Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 5Nicke A. Kerschensteiner D. Soto F. J. Neurochem. 2005; 92: 925-933Crossref PubMed Scopus (83) Google Scholar, 6Torres G.E. Egan T.M. Voigt M.M. J. Biol. Chem. 1999; 274: 6653-6659Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Site-directed mutagenesis has provided pivotal information about specific P2X properties: the channel pore, agonist binding residues, receptor desensitization and allosteric modulation (7Rassendren F. Buell G. Newbolt A. North R.A. Surprenant A. EMBO J. 1997; 16: 3446-3454Crossref PubMed Scopus (181) Google Scholar, 8Jiang L.H. Rassendren F. Surprenant A. North R.A. J. Biol. Chem. 2000; 275: 34190-34196Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 9Yan Z. Liang Z. Tomic M. Obsil T. Stojilkovic S.S. Mol. Pharmacol. 2005; 67: 1078-1088Crossref PubMed Scopus (55) Google Scholar, 10Ennion S. Hagan S. Evans R.J. J. Biol. Chem. 2000; 275: 29361-29367Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 11Silberberg S.D. Li M. Swartz K.J. Neuron. 2007; 54: 263-274Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 12Clyne J.D. LaPointe L.D. Hume R.I. J. Physiol. 2002; 539: 347-359Crossref PubMed Scopus (79) Google Scholar, 13Coddou C. Morales B. González J. Grauso M. Gordillo F. Bull P. Rassendren F. Huidobro-Toro J.P. J. Biol. Chem. 2003; 278: 36777-36785Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 14Lorca R.A. Coddou C. Gazitua M.C. Bull P. Arredondo C. Huidobro-Toro J.P. J. Neurochem. 2005; 95: 499-512Crossref PubMed Scopus (34) Google Scholar, 15Acuña-Castillo C. Coddou C. Bull P. Brito J. Huidobro-Toro J.P. J. Neurochem. 2007; 101: 17-26Crossref PubMed Scopus (66) Google Scholar, 16Acuña-Castillo C. Morales B. Huidobro-Toro J.P. J. Neurochem. 2000; 74: 1529-1537Crossref PubMed Scopus (91) Google Scholar). As with other ligand-gated ionic channels, the P2X receptors are modulated by divalent metals including trace metals although the nature of the modulation and the magnitude of these effects vary among the different P2X subunits (12Clyne J.D. LaPointe L.D. Hume R.I. J. Physiol. 2002; 539: 347-359Crossref PubMed Scopus (79) Google Scholar, 13Coddou C. Morales B. González J. Grauso M. Gordillo F. Bull P. Rassendren F. Huidobro-Toro J.P. J. Biol. Chem. 2003; 278: 36777-36785Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 14Lorca R.A. Coddou C. Gazitua M.C. Bull P. Arredondo C. Huidobro-Toro J.P. J. Neurochem. 2005; 95: 499-512Crossref PubMed Scopus (34) Google Scholar, 15Acuña-Castillo C. Coddou C. Bull P. Brito J. Huidobro-Toro J.P. J. Neurochem. 2007; 101: 17-26Crossref PubMed Scopus (66) Google Scholar, 16Acuña-Castillo C. Morales B. Huidobro-Toro J.P. J. Neurochem. 2000; 74: 1529-1537Crossref PubMed Scopus (91) Google Scholar, 17Xiong K. Peoples R.W. Montgomery J.P. Chiang Y. Stewart R.R. Weight F.F. Li C. J. Neurophysiol. 1999; 81: 2088-2094Crossref PubMed Scopus (89) Google Scholar). The role of divalent trace metals as neuromodulators is of interest as zinc and copper are both novel and atypical brain transmitters (18Barañano D.E. Ferris C.D. Snyder S.H. Trends Neurosci. 2001; 24: 99-106Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 19Hershfinkel M. Moran A. Grossman N. Sekler I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11749-11754Crossref PubMed Scopus (208) Google Scholar) and novel intracellular second messengers (20Yamasaki S. Sakata-Sogawa K. Hasegawa A. Suzuki T. Kabu K. Sato E. Kurosaki T. Yamashita S. Tokunaga M. Nishida K. Hirano T. J. Cell Biol. 2007; 177: 637-645Crossref PubMed Scopus (460) Google Scholar). The notion that zinc and copper are stored in neurons and are released upon electrical depolarization further highlights their importance in brain excitability with ample physiological and pharmacological implications (21Assaf S. Chung S.H. Nature. 1984; 208: 734-736Crossref Scopus (1028) Google Scholar, 22Kardos J. Kovacs I. Hajos F. Kalman M. Simonyi M. Neurosci. Lett. 1989; 103: 139-144Crossref PubMed Scopus (280) Google Scholar). The P2X4 receptor is an interesting model of an ionic channel differentially modulated by divalent trace metals. In a series of studies, Acuña-Castillo et al. (16Acuña-Castillo C. Morales B. Huidobro-Toro J.P. J. Neurochem. 2000; 74: 1529-1537Crossref PubMed Scopus (91) Google Scholar) and Coddou et al. (23Coddou C. Villalobos C. Gonzalez J. Acuña-Castillo C. Loeb B. Huidobro-Toro J.P. J. Neurochem. 2002; 80: 626-633Crossref PubMed Scopus (20) Google Scholar, 24Coddou C. Morales B. Huidobro-Toro J.P. Eur. J. Pharmacol. 2003; 472: 49-56Crossref PubMed Scopus (19) Google Scholar) reported that zinc potentiated the ATP-evoked currents, whereas copper exerts an inhibitory effect on the activity of this receptor. Furthermore, single site-directed mutagenesis of each of the three extracellular histidine residues of the P2X4 receptor revealed that only histidine 140 plays a key role in the inhibitory modulation by copper and high zinc concentrations (13Coddou C. Morales B. González J. Grauso M. Gordillo F. Bull P. Rassendren F. Huidobro-Toro J.P. J. Biol. Chem. 2003; 278: 36777-36785Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The replacement of His140 by an alanine (H140A mutant) was not only resistant to the copper-induced inhibition of the ATP-gated receptor activity but evidenced a dramatic increase in the zinc-induced potentiation. Zinc potentiated more than 20-fold the ATP-evoked currents in the H140A mutant; the metal evidenced in this mutant a sigmoid concentration-response dependence instead of the bell-shaped zinc curve described in the wild-type receptor. This finding brought forth the hypothesis that the P2X4 receptor channel must have a separate and distinct trace metal binding site to account for the facilitator action of zinc. The present research aimed at identifying structural determinants for the binding of zinc and to further assess the hypothesis that zinc and copper modulate the P2X4 receptor by interacting with separate and apparently independent metal binding sites. For this purpose, we mutated several amino acids in the vicinity of His140 and identified residues in the ectodomain of the P2X4 receptor that participate in the facilitator and inhibitor allosteric trace metal sites. We demonstrate that Cys132, but not Cys126, is critical for the modulator role of zinc but not copper, identifying the first critical amino acid residue necessary for zinc potentiation in this receptor channel. The C132A mutant proved not only resistant to the zinc-induced potentiation, but this metal inhibited ATP-gated currents, in full support that large zinc concentrations may also interact with the copper inhibitory site (13Coddou C. Morales B. González J. Grauso M. Gordillo F. Bull P. Rassendren F. Huidobro-Toro J.P. J. Biol. Chem. 2003; 278: 36777-36785Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In addition, we determined the key role of the carboxylic acid group of Asp138 as a second residue critically involved in copper inhibition, establishing a possible metal coordination binding pocket in the extracellular receptor domain region surrounding His140. The finding that the sulfhydryl group of Cys132 is critical for the modulator action of zinc suggests that this residue does not form a disulfide bridge with other cysteines as has been suggested for other P2X subtypes. In our view, Cys132 plays a relevant role in zinc modulation, and contributes to the molecular basis of trace metal modulation of the P2X4 receptor channel. ATP tri-sodium salt, hydrogen, penicillin-streptomycin and ivermectin were purchased from Sigma-Aldrich. Copper, zinc, and mercury chloride were obtained from Merck (Darmstadt, Germany). (2-(trimethylammonium)-ethyl)Methanethiosulfonate bromide (MTSET) 4The abbreviations used are:MTSET(2-(trimethylammonium)-ethyl)methanethiosulfonate bromidewtwild-typeEC50median effective concentrationnHHill coefficientIC50median inhibitory concentrationImaxmaximal current. was obtained from Toronto Research Chemical Inc. (Ontario, Canada). The salts used to prepare the incubation media were purchased from Sigma-Aldrich or Merck. Samples of the triple-distilled water used in buffer preparation, were analyzed for electro conductivity; metal contamination was assessed by inductively coupled plasma optical emission spectrometry using a Optima 2000 DV ICP-Emission Spectrometer (PerkinElmer Life Sciences). Trace metal contamination was less than 0.01 μm. (2-(trimethylammonium)-ethyl)methanethiosulfonate bromide wild-type median effective concentration Hill coefficient median inhibitory concentration maximal current. Site-directed Mutagenesis—The mutants were generated by PCR using the proofreading Pfu polymerase (Promega) followed by DpnI digestion of the methylated parental plasmid. Primers designed for the rat P2X4 receptor mutants were as follows: T123A sense: 5′-GTCCAGAGATTCCTGATAAGGCCAGCATTTGTAA-3′; T123A antisense: 5′-CTTATCAGGAATCTCTGGACAGGTGCTCTG-3′; S124A sense: 5′-CAGAGATTCCTGATAAGACCGCCATTTGTAATTC-3′; S124A antisense: 5′-GGTCTTATCAGGAATCTCTGGACAGGTGCT-3′; C126A sense: 5′-AAGACCAGCATTGCAAATTCAGACG-3′; C126A antisense: 5′-CGTCTGAATTTGCAATGCTGGTCTT-3′; D129A sense: 5′-AGACCAGCATTTGTAATTCAGCCGCCGACTGACC-3′; D129A antisense: 5′-TGAATTACAAATGCTGGTCTTATCAGGAAT-3′; D131A sense: 5′-GCATTTGTAATTCAGACGCCGCCTGCACTCCTGG-3′; D131A antisense: 5′-GGCGTCTGAATTACAAATGCTGGTCTTATC-3′; C132A sense: 5′-TTTGTAATTCAGACGCCGACGCCACTCCTGGCTC-3′; C132A antisense: 5′-GTCGGCGTCTGAATTACAAATGCTGGTCTT-3′; T133A sense: 5′-GTAATTCAGACGCCGACTGCGCCCCTGGCTCCGT-3′; T133A antisense: 5′-GCAGTCGGCGTCTGAATTACAAATGCTGGT-3′; D138A sense: 5′-ACTGCACTCCTGGCTCCGTGGCCACCCACAGCAG-3′; D138A antisense: 5′-CACGGAGCCAGGAGTGCAGTCGGCGTCTGA-3′; D138N sense: 5′-ACTGCACTCCTGGCTCCGTGAACACCCACAGCAG-3′; D138N antisense: 5′-CACGGAGCCAGGAGTGCAGTCGGCGTCTGA-3′; T146A sense: 5′-CCCACAGCAGTGGAGTTGCGGCCGGAAGATGTGT-3′; T146A antisense: 5′-CGCAACTCCACTGCTGTGGGTGTCCACGGA-3′. The double mutants T123A/T146A and S124A/T146A were generated in two steps with the same primers used for individual mutations. The triple mutant T123A/S124A/T146A was generated in two steps: first the T123A/S124A double mutant was generated using a single primer (5′-GTCCAGAGATTCCTGATAAGGCCGCCATTTGTAA-3′), then the T146A mutation was generated with their respective primers. To circumvent unwanted mutations, a region surrounding the targeted amino acid and presenting unique restriction sites was subcloned in the parental cDNA; the exact synthesis of each mutation was verified by automated sequencing. Oocyte Harvesting, Microinjection, ATP Receptor Expression, and Quantification of ATP-evoked Currents—A segment of the ovary was surgically removed under anesthesia from the frog, Xenopus laevis. Oocytes were manually defolliculated and incubated with collagenase as previously detailed by Acuña-Castillo et al. (16Acuña-Castillo C. Morales B. Huidobro-Toro J.P. J. Neurochem. 2000; 74: 1529-1537Crossref PubMed Scopus (91) Google Scholar). Oocytes were injected intranuclearly with 3–5 ng of cDNA coding for the rat P2X4 wild-type and mutated receptors. After a 36–48 h incubation in Barth's solution (NaCl, 88 mm; KCl, 1 mm; NaHCO3, 2.4 mm; HEPES, 10 mm; MgSO4, 0.82 mm; Ca(NO3)2, 0.33 mm; CaCl2, 0.91 mm; pH 7.5) supplemented with 10 international units/liter penicillin/10 mg streptomycin and 2 mm pyruvate, oocytes were clamped at –70 mV using the two-electrode voltage-clamp configuration with an OC-725C clamper (Warner Instruments Corp., Hamden, CT). ATP-gated currents were recorded following regular 10-s ATP applications repeated every 10 min for up to 10 μm ATP; for higher ATP concentrations, the pulses were spaced out to 25 min to avoid receptor desensitization. After metal incubation the recovery of control currents was always assessed. Non-injected oocytes did not evoke currents when exogenous ATP was applied (16Acuña-Castillo C. Morales B. Huidobro-Toro J.P. J. Neurochem. 2000; 74: 1529-1537Crossref PubMed Scopus (91) Google Scholar). ATP, metal chloride salts, and amino acids were dissolved in Barth's media and perfused using a pump operating at a constant flow of 2 ml/min. ATP concentration-response curves were performed by applying for 10 s, increasing concentrations of the nucleotide ranging between 1 and 1000 μm. Curves were normalized against the concentration of ATP that evoked the maximal response. For metal modulation experiments, at least 5 control ATP applications were performed; the average of all the control currents was used as the standard response (100%). This procedure allowed us to determine the variation between the control responses, which never exceeded 10%. The ATP median effective concentration (EC50) was interpolated from each concentration-response curve. Likewise, the maximal ATP current (Imax) was obtained from each ATP concentration-response curve. Each protocol was performed in at least two separate batches of oocytes from different frogs; each experiment was repeated at least in four separate oocytes. Special care was taken to complete each protocol in a single oocyte; with incomplete protocols being discarded, to favor correct statistical analysis. ATP and metals solutions were prepared daily before usage. Metal Characterization Protocols—These protocols describe the general outline of the experiments performed in wild-type and mutant receptors. Care was exercised to run the whole protocol in a single oocyte allowing each oocyte as its own internal control, particularly when concentration-response curves were performed. Metal Selectivity—We examined the modulator role of copper and zinc chloride salts on the ATP-gated currents of the P2X4 wild-type and mutant receptors. Reversal of the metal effect was mandatory prior to testing other metal concentrations, or examining the effect of another metal in the same oocyte. The recovery of the ATP-evoked currents following additions of the metals was controlled in all cases by the sequential application of ATP challenges until the full original current was attained. Metal and Ligand Concentration Studies—The metal concentration dependence was assessed by quantifying the ATP-gated currents in the absence and later in the presence of 0.1–300 μm copper and zinc. In these assays, each oocyte served as its own control; reversal of the metal action was carefully controlled as mentioned above. To examine the action of zinc, experiments used a concentration of the nucleotide that elicits only a 5% of the maximal ATP response (EC5), because this protocol favored the facilitator action of the metal (16Acuña-Castillo C. Morales B. Huidobro-Toro J.P. J. Neurochem. 2000; 74: 1529-1537Crossref PubMed Scopus (91) Google Scholar). For example, the ATP EC5 for the wild-type P2X4 receptor is 1 μm while for the C132A and D138A mutants this value is 3 μm. To assess the copper-induced inhibition, these protocols were systematically performed using the EC50 for each receptor (see Table 1). These ATP concentrations were established previously as optimal to test the effect of each metal (16Acuña-Castillo C. Morales B. Huidobro-Toro J.P. J. Neurochem. 2000; 74: 1529-1537Crossref PubMed Scopus (91) Google Scholar).TABLE 1EC50, nH and Imax values for wild-type and mutated P2X4 receptors expressed in X. laevis oocytesReceptorEC50nHImaxμmμAP2X4 wild-type11.4 ± 2.8 (12)1.4 ± 0.24.9 ± 0.8 (18)T123A22.3 ± 6.4 (8)1.2 ± 0.13.3 ± 0.6 (12)S124A24.6 ± 4.8 (5)1.0 ± 0.23.3 ± 0.7 (10)C126A15.4 ± 4.6 (6)1.9 ± 0.29.0 ± 2.8 (8)D129A33.3 ± 8.6 (6)ap < 0.05, compared with the values obtained with the wild-type P2X4 receptor.1.4 ± 0.23.4 ± 0.9 (8)D131A17.5 ± 6.4 (6)1.2 ± 0.16.4 ± 1.7 (9)C132A36.1 ± 13.2 (6)1.3 ± 0.23.4 ± 1.4 (6)T133A13.3 ± 3.7 (4)0.9 ± 0.44.1 ± 1.3 (7)D138A36.8 ± 6.5 (5)bp < 0.01, compared with the values obtained with the wild-type P2X4 receptor.0.9 ± 0.16.2 ± 1.8 (9)D138N19.1 ± 8.7 (5)1.1 ± 0.35.6 ± 2.2 (2)H140A61.2 ± 9.5 (8)bp < 0.01, compared with the values obtained with the wild-type P2X4 receptor.1.4 ± 0.24.3 ± 0.6 (11)T146A29.0 ± 7.0 (5)1.7 ± 0.62.8 ± 1.1 (6)T123/T14613.7 ± 2.3 (4)1.4 ± 0.34.7 ± 0.8 (6)S124/T14619.2 ± 13.2 (3)0.8 ± 0.42.7 ± 1.0 (5)T123A/S124A/T146A24.6 ± 4.1 (4)0.9 ± 0.24.5 ± 0.9 (7)a p < 0.05, compared with the values obtained with the wild-type P2X4 receptor.b p < 0.01, compared with the values obtained with the wild-type P2X4 receptor. Open table in a new tab To investigate how copper or zinc modified the ATP concentration-response curve, ATP concentration-response curves were performed in the absence, and later, in the presence of either 10 μm copper or 10 μm zinc. For these protocols, the metals were pre-applied for 1-min and next co-applied with ATP. Sets of at least 4–6 oocytes from separate batches were studied; complete ATP concentration-response protocols were performed per oocyte. MTSET Studies—Consistent with the role of free thiol groups in the extracellular receptor domain, we used a non-permeable SH reactive agent to chemically modify SH groups in the external surface of the receptor domain. Trimethyl ammoniummethylmethane thiosulfonate (MTSET) was used as a prototype alkylthiosulfonate, an agent that covalently alkylates free thiol groups of cysteine residues. Oocytes were treated with 1 mm MTSET, applied for 3-min period. The effect of zinc, copper, and ivermectin were tested before and after the application of MTSET, in the same oocyte. These protocols were repeated in 4–5 different oocytes from separate oocyte batches. Statistical Data Analysis—Curve fitting was performed with GraphPad software (San Diego, CA). ATP and zinc EC50 values, ATP Hill coefficient (nH), copper median inhibitory concentration (IC50), and the Imax were obtained from each concentration-response curve; values were derived after adjusting experimental values to a sigmoid curve generated using Graph Pad software (San Diego, CA). Statistical studies included the Mann-Whitney test; we had previously determined the convenience of non-parametric analysis procedures in our statistical evaluations (16Acuña-Castillo C. Morales B. Huidobro-Toro J.P. J. Neurochem. 2000; 74: 1529-1537Crossref PubMed Scopus (91) Google Scholar). Site-directed Mutagenesis of Critical Amino Acid Residues in the Thr123-Thr146 Region of the P2X4 Receptor—To examine the role of key amino acid residues in the vicinity of His140, an amino acid previously identified to play a critical role in copper-induced modulation (13Coddou C. Morales B. González J. Grauso M. Gordillo F. Bull P. Rassendren F. Huidobro-Toro J.P. J. Biol. Chem. 2003; 278: 36777-36785Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), we systematically mutated for alanines selected amino acids localized in the Thr123-Thr146 region (Fig. 1A). As candidates for mutagenesis we choose the following amino acids: aspartic acids, Asp129, Asp131, and Asp138, commonly described in protein metal-binding motifs and two cysteines, Cys126 and Cys132, which through their sulfhydryl groups could interact as metals ligands (25Aitken A. Mol. Biotechnol. 1999; 12: 241-253Crossref PubMed Google Scholar), particularly zinc. In addition, we also mutated 3 threonines and a serine; based on a theoretical computational model developed by P. Bull. All these P2X4 receptor mutants were functional; their half-maximal ATP potency (EC50) and maximal ATP-gated currents (Imax) were, within experimental error, similar to the wild-type P2X4 receptor. Table 1 summarizes the main parameters examined in the single mutants tested, along with several double mutations and a triple mutant. Modulator Effect of Zinc and Copper in Wild-type and the Receptor Mutants—Each mutant was examined independently to evaluate the modulatory activity of 10 μm zinc and 10 μm copper; a summary of these results is shown in Fig. 1, B and C. Zinc-induced potentiation was completely abolished in the mutant C132A, demonstrating the critical role of this residue for the modulator action of zinc but not copper. The zinc potentiation was also significantly reduced, although not abolished, in the mutant T133A (Fig. 1B, p < 0.01). Additionally, the modulator activity of 10 μm zinc was almost 2-fold larger in mutants D131A and D138A, mimicking the observation previously reported for the H140A mutant (Fig. 1B). In the rest of the mutants examined, the modulator action of zinc was within the experimental variation, not manifesting statistical differences to the wild-type receptor. With regard to the modulator activity of copper, mutant D138A was resistant to the 10 μm copper-induced inhibition of the ATP-gated currents; as indicated above, the modulator action of zinc was significantly augmented 2-fold (Fig. 1B). Mutant D129A demonstrated a significant 50% reduction in copper-induced inhibition (p < 0.05, Fig. 1C), while the modulator activity of zinc was conserved (Fig. 1B). The other mutants examined did not evidence significant deviations from the wild-type phenotype. The C132A Mutant—In view of the novelty of the results derived from the C132A mutant, and to further confirm and study the nature of its resistance to zinc, we investigated in further detail the interaction of this receptor mutant with trace metals. The zinc concentration-response curve in C132A was dramatically modified from a biphasic curve in the wild-type receptor phenotype (Fig. 2B), to a flat curve with negative slope and an estimated IC50 of 18.2 ± 10.1 μm, demonstrating the inhibitory action of zinc. Representative recordings shown in Fig. 2A evidence the zinc-induced reversible inhibition of the ATP-gated currents in oocytes expressing C132A mutant instead of the potentiation observed in the wild-type receptor. A 1-min pre-application of 10 μm zinc in the C132A mutant inhibited the 3 μm ATP-evoked currents; the inhibitor action of zinc was concentration-dependent (Fig. 2B). In further proof of the two independent sites of metal action in the P2X4 receptor, the C132A mutant showed a copper inhibition curve identical to the wild-type phenotype (Fig. 2C). Consonant with these results, 10 μm zinc did not modify the ATP concentration-response curve, while 10 μm copper inhibited non-competitively the ATP curve, much as in the wild-type receptors (Fig. 2D). Interestingly, the C126A mutant showed a wild-type phenotype to the modulation by zinc, evidencing the classical biphasic zinc interaction curve (Fig. 2B) or the non-competitive copper-induced inhibition (data not shown). Taken together, these results allow the conclusion that Cys132 is critical for zinc-induced potentiation, but not for copper inhibition. Based on the notion that Cys132 is part of the zinc-facilitator site, we reasoned that the facilitator action of zinc would be eliminated, at least in part, by reagents such as MTSET, which alkylate free sulfhydryl groups. Oocyte treatment with MTSET halved significantly the maximal zinc-induced potentiation from 5.9 ± 0.4- to 3.1 ± 0.2-fold (p < 0.01) in wild-type P2X4 receptors without altering the magnitude of the ATP-evoked currents (Fig. 3A). The ATP EC50 and maximal response after MTSET-treatment were 19.8 ± 4.0 μm and 6.4 ± 1.9 μA respectively (n = 5, data not shown). MTSET treatment was irreversible; 45 min after the treatment we consistently observed the reduction of the zinc-induced potentiation. As a further control for this set of experiments, MTSET treatment did not modify the ivermectin-induced potentiation (Fig. 3B) or copper inhibition (Fig. 3C). 3 μm IVM potentiated 3.7 ± 0.6, indicating that this agent has a smaller potentiation than that of zinc. After MTSET treatment, the IVM potentiation was 4.4 ± 1.4-fold, a value that did not differ from the non-treated oocytes. These results indicate that the chemical modification of the sulfhydryl of extracellular cysteines affected exclusively the zinc-induced modulation, but not the copper-induced modulation. The D138A Mutant—This mutant was copper-resistant, no significant inhibition was observed with metal concentrations up to 100 μm (Fig. 4B); in contrast, 10 μm copper elicited 75% current inhibition in the wild-type receptors. Further increasing the copper concentration to 300 μm, inhibited the currents ∼40%. Representative tracings illustrate the inhibitory modulation of 10 μm copper in oocytes transfected with either wild-type or the Asp138 mutant (Fig. 4A). Consonant with the finding reported for the H140A mutant (13Coddou C. Morales B. González J. Grauso M. Gordillo F. Bull P. Rassendren F. Huidobro-Toro J.P. J. Biol. Chem. 2003; 278: 36777-36785Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), which was also shown to be resistant to the action of copper, the zinc concentration-response curve was dramatically modified from a biphasic curve in wild-type receptors, to a sigmoid, in the D138A mutant (Fig. 4C). The maximal zinc-evoked potentiation in the mutant was at least 3-fold larger than in wild-type receptors (19.0 ± 2.1-versus 6.3 ± 0.7-fold increase, p < 0.01, n = 6). Moreover, when the ATP concentration-response curve was examined in the presence of 10 μm copper, the curve in the D138A mutant
Referência(s)