Histidine 140 Plays a Key Role in the Inhibitory Modulation of the P2X4 Nucleotide Receptor by Copper but Not Zinc
2003; Elsevier BV; Volume: 278; Issue: 38 Linguagem: Inglês
10.1074/jbc.m305177200
ISSN1083-351X
AutoresClaudio Coddou, Bernardo Morales, Jorge González, Marta Grauso, Felipe Gordillo, Paulina Bull, François Rassendren, J. Pablo Huidobro‐Toro,
Tópico(s)Pharmacological Effects and Toxicity Studies
ResumoTo elucidate the role of extracellular histidines in the modulation of the rat P2X4 receptor by trace metals, we generated single, double, and triple histidine mutants for residues 140, 241, and 286, replacing them with alanines. cDNAs for the wild-type and receptor mutants were expressed in Xenopus laevis oocytes and in human embryonic kidney 293 cells and examined by the two electrode and patch clamp techniques, respectively. Whereas copper inhibited concentration-dependently the ATP-gated currents in the wild-type and in the single or double H241A and H286A receptor mutants, all receptors containing H140A were insensitive to copper in both cell systems. The characteristic bell-shaped concentration-response curve of zinc observed in the wild-type receptor became sigmoid in both oocytes and human embryonic kidney cells expressing the H140A mutant; in these mutants, the zinc potentiation was 2.5–4-fold larger than in the wild-type. Results with the H140T and H140R mutants further support the importance of a histidine residue at this position. We conclude that His-140 is critical for the action of copper, indicating that this histidine residue, but not His-241 or His-286, forms part of the inhibitory allosteric metal-binding site of the P2X4 receptor, which is distinct from the putative zinc facilitator binding site. To elucidate the role of extracellular histidines in the modulation of the rat P2X4 receptor by trace metals, we generated single, double, and triple histidine mutants for residues 140, 241, and 286, replacing them with alanines. cDNAs for the wild-type and receptor mutants were expressed in Xenopus laevis oocytes and in human embryonic kidney 293 cells and examined by the two electrode and patch clamp techniques, respectively. Whereas copper inhibited concentration-dependently the ATP-gated currents in the wild-type and in the single or double H241A and H286A receptor mutants, all receptors containing H140A were insensitive to copper in both cell systems. The characteristic bell-shaped concentration-response curve of zinc observed in the wild-type receptor became sigmoid in both oocytes and human embryonic kidney cells expressing the H140A mutant; in these mutants, the zinc potentiation was 2.5–4-fold larger than in the wild-type. Results with the H140T and H140R mutants further support the importance of a histidine residue at this position. We conclude that His-140 is critical for the action of copper, indicating that this histidine residue, but not His-241 or His-286, forms part of the inhibitory allosteric metal-binding site of the P2X4 receptor, which is distinct from the putative zinc facilitator binding site. The notion that trace metals such as zinc or copper are atypical brain messengers has attracted much attention in view of their emerging role in the modulation of brain excitability (1Barañano D.E. Ferris C.D. Snyder S.H. Trends Neurosci. 2001; 24: 99-106Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The importance of trace metals in synaptic activity is highlighted by the description that both copper and zinc are stored in synaptic vesicles from where they are released by electrical depolarization, reaching a high micromolar concentration at the synaptic cleft (2Assaf S. Chung S.H. Nature. 1984; 208: 734-736Crossref Scopus (1028) Google Scholar, 3Howell G.A. Welch M.G. Fredericksson C.J. Nature. 1984; 208: 737-738Google Scholar, 4Kardos J. Kovacs I. Hajos F. Kalman M. Simonyi M. Neurosci. Lett. 1989; 103: 139-144Crossref PubMed Scopus (280) Google Scholar). Trace metals are known to modulate a wide variety of brain ionotropic receptors such as glycine, N-methyl-d-aspartate, γ-aminobutyric acid, nicotinic, and the novel nucleotide receptors (P2X) family (5Bloomenthal A.B. Goldwater E. Pritchett D.B. Harrison N.L. Mol. Pharmacol. 1994; 46: 1156-1159PubMed Google Scholar, 6Soto F. García-Guzmán M. Gómez-Hernández J.M. Hollmann M. Karschin C. Stühmer W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3684-3688Crossref PubMed Scopus (307) Google Scholar, 7Trombley P.Q. Shepherd G.M. J. Neurophysiol. 1996; 76: 2536-2546Crossref PubMed Scopus (114) Google Scholar, 8Yan Ma J. Narahashi T. Brain Res. 1993; 607: 222-232Crossref PubMed Scopus (76) Google Scholar, 9Palma E. Maggi L. Miledi R. Eusebi F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10246-10250Crossref PubMed Scopus (63) Google Scholar). The P2X purinoceptors are homomeric or heteromeric membrane channels gated by extracellular ATP and related synthetic nucleotides; North (10North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2490) Google Scholar) recently reviewed the principles of the molecular physiology of this family of receptors. Within the P2X receptor family, the P2X4 is the most widely distributed in the central nervous system, including the cerebellum and the CA1 region of the hippocampus, where it has been proposed to play a role in glutamatergic synapses (11Rubio M.E. Soto F. J. Neurosci. 2001; 21: 641-653Crossref PubMed Google Scholar). The P2X4 receptor is an interesting model of an ionic channel differentially modulated by trace metals. Acuña-Castillo et al. (12Acuña-Castillo C. Morales B. Huidobro-Toro J.P. J. Neurochem. 2000; 74: 1529-1537Crossref PubMed Scopus (91) Google Scholar) and Coddou et al. (13Coddou C. Villalobos C. González J. Acuña-Castillo C. Loeb B. Huidobro-Toro J.P. J. Neurochem. 2002; 80: 626-633Crossref PubMed Scopus (20) Google Scholar) reported that zinc potentiates the ATP-evoked currents whereas copper has an inhibitory effect on the activity of this receptor. Based on these findings, Acuña-Castillo et al. (12Acuña-Castillo C. Morales B. Huidobro-Toro J.P. J. Neurochem. 2000; 74: 1529-1537Crossref PubMed Scopus (91) Google Scholar) proposed that trace metals modulate the activity of the P2X4 receptor via two separate metal-binding sites. One of these sites has a preferential selectivity for copper and is characterized by a non-competitive inhibition of the ATP-gated channel activity. The second site shows preference for zinc and apparently leads to an increase in affinity for the binding of ATP. Copper and zinc also modulate other members of the P2X family of receptors, but their effects depend on the receptor subtype. For example, both zinc and copper facilitate the ATP-evoked responses on the P2X2 receptor (14Xiong 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, 15Lorca R. González J. Huidobro-Toro J.P. Biol. Res. 2001; 34 (Abstr. R-92): 105Google Scholar) whereas in the P2X7 receptor, copper inhibits the ATP-gated currents, whereas zinc is virtually without effect (13Coddou C. Villalobos C. González J. Acuña-Castillo C. Loeb B. Huidobro-Toro J.P. J. Neurochem. 2002; 80: 626-633Crossref PubMed Scopus (20) Google Scholar). These findings further support the notion that the P2X purinoceptors may have separate metal-binding sites to modulate the channel activity. This research was aimed at identifying structural determinants for the binding of copper and to assess the hypothesis that copper and zinc bind to separate, independent metal-binding sites in the receptor. Based on the notion that histidine residues are commonly found in consensus copper-binding motifs (16Aitken A. Mol. Biotechnol. 1999; 12: 241-253Crossref PubMed Google Scholar), as extensively studied in superoxide dismutase, where the imidazole ring is essential to coordinate copper (17Richardson J. Thomas K.A. Rubin B.H. Richardson D.C. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1349-1353Crossref PubMed Scopus (468) Google Scholar), and both histidines and cysteines have been defined in zinc-binding motifs (18Vallee B.L. Auld D.S. Biochemistry. 1990; 29: 5647-5659Crossref PubMed Scopus (1536) Google Scholar), we were challenged to clarify the role of the three histidines of the rat P2X4 receptor in the modulatory action of trace metals. Interestingly, the three rat P2X4 histidine residues are located extracellularly. To tentatively identify putative residues involved in the copper inhibitory modulation, we preliminarily used chemicals to selectively alkylate histidine and cysteine residues. Next, we performed site-directed mutagenesis to create single, double, and triple mutants where the histidine residues (His-140, His-241, and His-286) of the receptor were replaced by alanines. The cDNAs coding for the wild-type and mutated receptors were expressed in two cell systems; the modulation by copper or zinc was examined in either Xenopus laevis oocytes and HEK 1The abbreviations used are: HEK, human embryonic kidney; DEPC, diethyl pyrocarbonate; NEM, N-ethylmaleimide; EC50, median effective concentration; n H, Hill coefficient; IC50, median inhibitory concentration; Imax, maximal current. 293 cells. The present results allow us to conclude that only His-140 plays a key role in the modulator action of copper, because this particular amino acid cannot be replaced by alanine, threonine, or arginine and still conserve the inhibitory modulation by copper. Materials—ATP trisodium salt and penicillin-streptomycin were purchased from Sigma. Copper and zinc chlorides were obtained from Merck. The salts used to prepare the incubation media were purchased from Sigma-Aldrich or Merck. Diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM) were purchased from Sigma. Dulbecco's modified Eagle's medium, Opti-MEM, and LipofectAMINE were purchased from Invitrogen. Triple-distilled water with minimal electroconductivity was used; the copper or zinc contamination was less than 0.1 μm. Single Mutations—The rat P2X4 cDNA (GenBank™ accession number NM_031594) was cloned in a pcDNA3 vector (Invitrogen). P2X4 single mutants were generated by PCR using the proof-reading Pfu polymerase (Promega) followed by DpnI digestion of the methylated parental plasmid. Three 27-mer couples of primers were as follows: H140A (forward), 5′-CCGTGGACACCGCCAGCAGTGGAGTTG-3′; H140A (reverse), 5′-CAACTCCACTGCTGGCGGTGTCCACGG-3′; H241A (forward), 5′-GGGGACGCGGGAGCTAGCTTCCAGGAG-3′; H241A (reverse), 5′-CTCCTGGAAGCTAGCTCCCGCGTCCCC-3′; H286A (forward), 5′-CCGGGACCTGGAAGCCAATGTGTCTCC-3′; H286A (reverse), 5′-GGAGACACATTGGCTTCCAGGTCCCGG-3′. The primers were used to generate the H140A, H241A, and H286A single mutant clones, respectively. To circumvent unwanted mutations, a region surrounding the targeted amino acid and presenting unique restriction sites was subcloned in the parental cDNA and then verified by automated sequencing. Double and Triple Histidine Mutants—For the rat P2X4 receptor (GenBank™ accession number NM_031594) double and triple histidine mutants were made using the GeneTailor™ site-directed mutagenesis system (Invitrogen), using the single mutants and a double mutant as template, respectively. Oligonucleotides were designed following the manufacturer's instructions, and the sequences were as follows: H241A (forward), 5′-TCGTGGGGGACGCGGGAGCCAGCTTCCAGG-3′; H241A (reverse), 5′-TCCCGCGTCCCCCACGATTGTGCCAAGACG-3′; H286A (forward), 5′-TGGACACCCGGGACCTGGAAGCCAATGTGTCTCC-3′; H286A (reverse), 5′-TTCCAGGTCCCGGGTGTCCAGGCGCCGGAAGG-3′. Plasmid DNA was isolated using the Qiagen plasmid DNA purification kit according to the manufacturer's instructions. All the mutations were confirmed by sequencing using an Applied Biosystems ABI PRISM 310 DNA sequencer. Substitution of Histidine 140 by Other Amino Acid Residues—The H140T and H140R mutants were made using the overlap extension method (19Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar) with the following oligonucleotides: H140T (forward), 5′-CCGTGGACACCACCAGCAGTGGAGTTGCG-3′; H140T (reverse), 5′-CCAGTCGCAACTCCACTGCTGGTGGTGTCCACGG-3′; H140R (forward), 5′-CCGTGGACACCCGGAGCAGTGGAGTTGCG-3′; H140R (reverse), 5′-CCAGTCGCAACTCCACTGCTCCGGGTGTCCACGG-3′. A segment of the ovary was surgically removed under anesthesia from X. laevis frogs; oocytes were manually defolliculated and next incubated with collagenase as detailed previously (12Acuña-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 either the wild-type or H140A, H241A, and H286A P2X4 receptor proteins. After 36–48 h of incubation at 15 °C 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) containing 10 IU/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). ATP-gated currents were recorded following a 15-s ATP application dissolved in the regular perfusion buffer. ATP and metal chloride salts dissolved in Barth's medium were perfused using a peristaltic pump operating at a constant flow of 2 ml/min. Non-injected oocytes did not evoke currents when exogenous ATP was applied. Each experimental protocol was performed in at least two separate batches of oocytes from different frogs; each experiment was replicated in at least four separate oocytes. The relative ATP potency was estimated from single concentration-response curves; the median effective concentration (EC50) and the Hill coefficient (n H) was graphically derived using Graph Pad software. The maximal current (Imax, nanoAmpere) was obtained from each oocyte challenged with a saturating ATP concentration. To ascertain whether certain amino acids are essential for trace metal modulation, we first assessed whether alkylation of histidine residues with DEPC altered the inhibitory action of copper or the facilitator action of zinc. After routine applications of 10 μm ATP and testing of the inhibitory action of 10 μm copper or the facilitator action of 10 μm zinc, oocytes were treated for 5 min with 500 μm DEPC to modify the histidine residues. After this treatment, oocytes were perfused for 20–30 min to wash out the remaining DEPC prior to challenges with ATP or the metals. ATP concentration-response protocols (1–1000 μm) of ATP alone or ATP plus copper (1 min pre-incubated) were performed in separate oocytes. The ATP concentration-response curve was normalized against 500 μm ATP, a value close to the maximal current. The current gated by 500 μm ATP was obtained prior to the treatment with DEPC. Likewise, similar protocols were performed in separate oocytes using 300 μm NEM to selectively alkylate cysteine residues. As in the case of the DEPC-treated oocytes, oocytes were washed for 20–30 min from the residual chemical prior to testing with ATP and the trace metals. Results were normalized, as in the case of the DEPC treatment, against a standard of ATP obtained prior to applying the alkylating agent. To examine the effect of copper, 0.1–600 μm of the metal was pre-applied for 1 min before a 15-s ATP pulse, at a concentration close to the receptor EC50, which in the wild-type receptor was 10 μm ATP. Care was taken to achieve full reversal of the copper effect prior to a larger copper application. The median inhibitory concentration of copper (IC50) was interpolated from each metal concentration-response curve. A single oocyte was tested with at least four increasing concentrations of copper. If the relative potency of ATP was modified in the mutant receptors, the ATP concentration was adjusted to a value close to its EC50;60 μm ATP was used to evaluate the H140A mutant receptor, and 30 μm ATP was used for the His-286 mutant. This protocol was also used to evaluate the potency of copper in the double or triple histidine mutants or the mutants that substituted His-140 by either threonine or arginine. Parallel experiments were performed to assess whether the histidine mutants affected the facilitator action of zinc. In these protocols, the metal was applied 1 min prior to a challenge with 1 μm ATP in the wild-type P2X4 receptor or the H241A mutant. 3 μm ATP was used to evaluate the facilitator action of zinc in the H140A and H286A mutants. These concentrations of ATP were chosen based on previous findings (12Acuña-Castillo C. Morales B. Huidobro-Toro J.P. J. Neurochem. 2000; 74: 1529-1537Crossref PubMed Scopus (91) Google Scholar) when we reported that the largest zinc potentiation was attained with low ATP concentrations, which induce 5% of the maximal nucleotide-evoked current (EC5). In all cases, ATP applications were spaced at regular 10-min intervals, minimizing desensitization and after attaining full reversibility of the zinc effect. The effect of zinc in the double or triple mutants was assessed likewise. To study with more detail the mechanism of trace metal modulation, we next assessed the effect of the pre-application of copper or zinc in the ATP concentration-response curves in the wild-type and mutant P2X4 receptors. Protocols were performed applying increasing concentrations of ATP (0.1–3,000 μm) for 15 s. Curves were normalized against 500 μm ATP for the wild-type receptor and H241A mutant receptor and against 1 mm ATP for the H140A and H286A mutants. The ATP EC50 and its Imax were derived from each ATP concentration-response curve. HEK 293 cells were transiently transfected with 1 μg of cDNA coding for the wild-type, H140A, H241A, or H286A P2X4 receptor protein. Cells grown to 50% confluence on 35-mm culture dishes were incubated with cDNA mixed with 8 μl of LipofectAMINE in 1 ml of serum-free medium (Opti-MEM). After 3 h at 37 °C, the medium was replaced with Dulbecco's modified Eagle's medium, and 48 h later the cells were transferred to the experimental chamber containing recording buffer, which contained the following (in mm): 150 NaCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, pH 7.3. Whole-cell ATP-dependent currents were obtained from single HEK 293 cells using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Patch pipettes (2–4 megohm) were filled with the following (in mm): 150 CsCl, 10 tetraethyl ammonium chloride, 10 EGTA, 10 HEPES, pH 7.3, and 275–285 mOsm; pH was adjusted with CsOH. The junction potential (typically <5 mV) was compensated. Only cells with membrane potential more negative than –55 mV, access resistance 100 megohm were tested. The cells were discarded if the input or access resistance changed >0%. The ATP-dependent currents were recorded at a holding potential of –80 mV to augment the driving force of the ions that mediate the ATP-evoked currents and to avoid contamination of voltage-dependent currents. Responses were digitized at a frequency of 10 KHz and analyzed using the pCLAMP 8 system (Axon Instruments, Foster City, CA). All the experiments were carried out at room temperature. ATP-dependent currents were evoked with 5-s ATP pulses applied by superfusion, using a peristaltic pump (Masterflex Pump; Cole Palmer Instruments). ATP concentration-response protocols were performed to derive the EC50, by applying 0.1 to 500 μm pulses of ATP for 5 s; between ATP applications, the cells were washed for 5 min with drug-free buffer. To minimize desensitization because of the larger concentrations of ATP (30 μm or more), these applications were spaced 10 min. The ATP EC50, Imax, and n H were obtained from each ATP concentration-response plot by fitting the data using the Hill equation. Full concentration-response curves were derived from single cells; protocols were repeated using several batches of cells. To study the modulation of the ATP-evoked currents by the metals, separate cell batches expressing the wild-type or the mutant receptors were pre-incubated with 1–300 μm copper or 1–100 μm zinc for 1 min before the addition of a challenge concentration of 10 μm ATP, a value in the order of the nucleotide EC50. ATP applications were regularly spaced at 10-min intervals, the optimal time previously determined to attain full recovery of the ATP-evoked current, an indication of the reversibility of the metal effects. Metal concentration-response plots were fitted using Graph Pad software; the copper IC50 was interpolated from each plot. Curve fitting was performed using the sigmoidal equations proper of concentration-response curves (GraphPad Prism 3.0; San Diego, CA). ATP and zinc EC50,Imax, and n H, as well as the copper IC50 were derived per oocyte in each experimental protocol (13Coddou C. Villalobos C. González J. Acuña-Castillo C. Loeb B. Huidobro-Toro J.P. J. Neurochem. 2002; 80: 626-633Crossref PubMed Scopus (20) Google Scholar). Maximal amplitudes were also derived. Non-parametric analysis and parametric tests were performed as appropriate (Kruskal-Wallis, Friedman, and Quade (see Ref. 20Theodorsson-Norheim E. Comput. Biol. Med. 1987; 17: 85-99Crossref PubMed Scopus (206) Google Scholar for review) and Dunnetts's tests for multiple comparisons with a single control); significance was set at p < 0.05. Copper non-competitively inhibited the currents evoked by ATP in wild-type receptors expressed in oocytes (Fig. 1A). DEPC treatment, which is expected to alkylate-exposed histidine residues, decreased the ATP Imax (44.5 ± 7.8%, p < 0.01) and abolished the inhibitory action of copper (Fig. 1B). In contrast, NEM treatment, which preferentially modifies the SH group of cysteines, significantly increased the ATP Imax (166.3 ± 24.5%, p < 0.001). Moreover, NEM treatment did not abolish the non-competitive copper inhibition (Fig. 1C), suggesting that histidines, rather than cysteines, are primarily involved in the action of copper. Both in the wild-type and the receptor mutants, the 15-s ATP pulse application resulted in currents with a distinct rapid peak and a slower component. The P2X4 receptor mutants resulted in functional channels; their ATP EC50 and Imax values are summarized in Table I. Compared with the wild-type, the ATP EC50 of the H140A and H286A mutants was 6- and 4-fold larger than the wild-type receptor; mutant H241A conserved the wild-type EC50 (Table I). On the other hand, the H286A mutant Imax was reduced by 67% (p < 0.01), whereas that of the H140A and H241A mutants was unaltered. The double and the triple histidine mutants with the H140A and H286A substitution also had a lower Imax and a larger EC50 than the wild-type (Table I).Table IEC50 and Imax values for wild-type and mutated P2X4 receptors expressed in X. laevis oocytes and HEK 293 cellsOocytesHEK cellsEC50ImaxnHEC50ImaxnHμ mnAμ mnAWild-type8.9 ± 2.1 (11)4676 ± 663 (20)1.3 ± 0.25.2 ± 1.0 (6)1.1 ± 0.2 (8)1.8 ± 0.4H140A61.2 ± 9.5ap < 0.01, as compared with the values obtained with the wild-type P2X4 receptor. (8)4266 ± 594 (11)1.4 ± 0.25.4 ± 1.2 (3)1.1 ± 0.2 (4)2.0 ± 0.6H241A8.4 ± 0.8 (8)4939 ± 907 (11)1.2 ± 0.35.1 ± 3.3 (5)1.3 ± 0.2 (11)1.0 ± 0.2H286A40.2 ± 8.3bp < 0.05, as compared with the values obtained with the wild-type P2X4 receptor. (11)1540 ± 510ap < 0.01, as compared with the values obtained with the wild-type P2X4 receptor. (16)1.3 ± 0.49.3 ± 4.7 (4)0.3 ± 0.1bp < 0.05, as compared with the values obtained with the wild-type P2X4 receptor. (5)0.6 ± 0.3H140A/H241A40.8 ± 10.6bp < 0.05, as compared with the values obtained with the wild-type P2X4 receptor. (4)795 ± 301ap < 0.01, as compared with the values obtained with the wild-type P2X4 receptor. (4)1.7 ± 0.4H140A/H286A103.1 ± 22.8ap < 0.01, as compared with the values obtained with the wild-type P2X4 receptor. (5)402 ± 125ap < 0.01, as compared with the values obtained with the wild-type P2X4 receptor. (4)1.7 ± 0.3H241A/H286A25.5 ± 6.5 (4)1112 ± 600ap < 0.01, as compared with the values obtained with the wild-type P2X4 receptor. (4)0.9 ± 0.1H140A/H241A/H286A114.5 ± 17.2ap < 0.01, as compared with the values obtained with the wild-type P2X4 receptor. (4)560 ± 135ap < 0.01, as compared with the values obtained with the wild-type P2X4 receptor. (5)2.5 ± 1.7H140T59.6 ± 9.2ap < 0.01, as compared with the values obtained with the wild-type P2X4 receptor. (4)3358 ± 724 (5)1.7 ± 0.4H140R32.2 ± 7.4bp < 0.05, as compared with the values obtained with the wild-type P2X4 receptor. (3)43 ± 9ap < 0.01, as compared with the values obtained with the wild-type P2X4 receptor. (5)0.5 ± 0.2bp < 0.05, as compared with the values obtained with the wild-type P2X4 receptor.a p < 0.01, as compared with the values obtained with the wild-type P2X4 receptor.b p < 0.05, as compared with the values obtained with the wild-type P2X4 receptor. Open table in a new tab Copper Modulation—Copper decreased in a reversible and concentration-dependent manner the current elicited by a concentration of ATP close to the EC50 of each receptor (Fig. 2). The copper IC50 derived from these experiments was 8.9 ± 1.5 μm (n = 7) for the wild-type receptor, a value that was not significantly different from 8.7 ± 2.4 (n = 4) and 7.1 ± 2.7 μm (n = 6) for the H241A and H286A mutants, respectively. However, the H140A mutant was largely resistant to the modulator action of copper; 600 μm copper inhibited only 33.8 ± 8.5% (n = 4; see Fig. 2), suggesting that His-140 is essential for the copper-induced inhibition. Zinc Modulation—Zinc facilitated in a biphasic manner the ATP-gated currents; a maximum was reached at 10 μm zinc, which amounted to a 7.1 ± 0.7-fold (n = 7) increase over the current elicited by an ATP EC5, the preferred concentration used to evaluate the zinc modulation. Larger metal concentrations elicited smaller effects, resulting in a bell-shaped curve (recordings and inset in Fig. 3). The H241A and H286A mutants also showed a similar biphasic zinc curve; the maximal potentiation of the currents was 8.9 ± 0.6- and 8.0 ± 2.4-fold for the H241A and H286A mutants, respectively (n = 4; see Fig. 3). In contrast, the zinc curve for the H140A mutant was sigmoid, reaching a maximal potentiation at 30 μm zinc, which resulted in a 27.4 ± 7.7-fold potentiation (p < 0.001, n = 4; see Fig. 3). This potentiation was 4-fold larger than for the wild-type receptor. Mechanisms of Trace Metal Modulation: ATP Concentration-response Curves—In contrast to the wild-type receptor, 10 μm copper did not modify the ATP concentration-response curve in the H140A mutant, whereas it non-competitively inhibited the H241A and the H286A receptor mutants (see Fig. 4 and Table II). 10 μm zinc displaced the ATP-concentration-response curve of the H140A mutant to the left (Fig. 4) and additionally increased the Imax from 104 ± 8.3% in the wild-type to 205 ± 21.8% (p < 0.001; see Table II). Zinc also increased the ATP Imax of the H286A mutant to 238.2 ± 58.2% (p < 0.001; see Table II and Fig. 4). The effect of zinc in the H241A mutant was identical to that of the wild-type receptor.Table IIEffect of a 1-min pre-application of 10 μm copper or 10 μm zinc on the ATP EC50 and Imax values for the wild-type and mutant P2X4 receptors expressed in X. laevis oocytesEC50ImaxControl+ Cu2++ Zn2+Control+ Cu2++ Zn2+μ m%Wild-type8.9 ± 2.120.8 ± 6.62.6 ± 1.2ap < 0.05, as compared with the values obtained in control conditions.103.1 ± 5.946.2 ± 4.1bp < 0.01, as compared with the values obtained in control conditions.107.2 ± 7.7H140A61.2 ± 9.587.5 ± 16.728.4 ± 11.6ap < 0.05, as compared with the values obtained in control conditions.104.0 ± 8.3108.6 ± 7.1205.5 ± 21.8cp < 0.01, as compared with the values obtained in control conditions.H241A8.4 ± 0.816.0 ± 3.73.6 ± 1.1ap < 0.05, as compared with the values obtained in control conditions.102.3 ± 5.555.1 ± 2.6bp < 0.01, as compared with the values obtained in control conditions.112.2 ± 6.9H286A40.2 ± 8.356.4 ± 26.033.1 ± 25.295.3 ± 5.541.7 ± 9.3cp < 0.01, as compared with the values obtained in control conditions.238.2 ± 58.2cp < 0.01, as compared with the values obtained in control conditions.a p < 0.05, as compared with the values obtained in control conditions.b p < 0.01, as compared with the values obtained in control conditions.c p < 0.01, as compared with the values obtained in control conditions. Open table in a new tab Trace Metal Modulation in the Double and Triple Histidine Mutants—The mutants containing the H140A substitution were resistant to the inhibitory action of up to 100 μm copper. In contrast, the mutant containing the other two histidines conserved the inhibition by copper and behaved similar to that of the wild-type receptor (Fig 5A). Zinc potentiated the magnitude of the ATP current in the H140A/H241A/H286A receptor, reaching a plateau at 100 μm; the maximal potentiation was 32.9 ± 5.5-fold, a value significantly larger than that observed in the wild-type receptor (p < 0.01; see Fig. 5B). In the double receptor mutants H140A/H241A and H140A/H286A, the maximal zinc potentiation amounted to 12.0 ± 6.0% and 11.7 ± 4.7-fold, respectively (p < 0.01 each, n = 5 each), values which are two-three times smaller than those attained with the H140A mutant. In contrast, the H241A/H286A mutant had a biphasic zinc curve, similar to that observed in the wild-type receptor (Fig. 5B); the maximal potentiation reached only 6.2 ± 1.6-fold and served as a negative control, because the maximal potentiation in the wild-type receptor reached 7.1 ± 0.7-fold. Substitution of His-140 by Other Amino Acids—The copper IC50 in the H140T mutant was 78.5 ± 31.2 μm (n = 4), a value 8-fold higher than in the wild-type receptor (n = 6, p < 0.01; see Fig. 6). The zinc curve was biphasic; its maximal effect reached 17.3 ± 2.9-fold, a value 2.5-fold higher than the wild-type receptor (p < 0.01), but slightly smaller than the maximal potentiation obtained in the H140A mutant (27.4 ± 7.7-fold increase; see Fig. 6). The currents elicited by the H140R mutant were 100-fold smaller than the wild-type and were long lasting, even after ATP removal (Fig. 6, tracings). The estimated ATP EC50 was 32.2 ± 7.4 μm (n = 3; see Table I); furthermo
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