Dual Effect of Acid pH on Purinergic P2X3 Receptors Depends on the Histidine 206 Residue
2007; Elsevier BV; Volume: 282; Issue: 47 Linguagem: Inglês
10.1074/jbc.m705840200
ISSN1083-351X
AutoresZoltán Gerevich, Zoltán Zádori, László Köles, Laurenz Kopp, Doreen Milius, Kerstin Wirkner, Klára Gyires, Péter Illés,
Tópico(s)Restless Legs Syndrome Research
ResumoWhole cell patch clamp investigations were carried out to clarify the pH sensitivity of native and recombinant P2X3 receptors. In HEK293 cells permanently transfected with human (h) P2X3 receptors (HEK293-hP2X3 cells), an acidic pH shifted the concentration-response curve for α,β-methylene ATP (α,β-meATP) to the right and increased its maximum. An alkalic pH did not alter the effect of α,β-meATP. Further, a low pH value increased the activation time constant (τon) of the α,β-meATP current; the fast and slow time constants of desensitization (τdes1, τdes2) were at the same time also increased. Finally, acidification accelerated the recovery of P2X3 receptors from the desensitized state. Replacement of histidine 206, but not histidine 45, by alanine abolished the pH-induced effects on hP2X3 receptors transiently expressed in HEK293 cells. Changes in the intracellular pH had no effect on the amplitude or time course of the α,β-meATP currents. The voltage sensitivity and reversal potential of the currents activated by α,β-meATP were unaffected by extracellular acidification. Similar effects were observed in a subpopulation of rat dorsal root ganglion neurons expressing homomeric P2X3 receptor channels. It is suggested that acidification may have a dual effect on P2X3 channels, by decreasing the current amplitude at low agonist concentrations (because of a decrease in the rate of activation) and increasing it at high concentrations (because of a decrease in the rate of desensitization). Thereby, a differential regulation of pain sensation during e.g. inflammation may occur at the C fiber terminals of small DRG neurons in peripheral tissues. Whole cell patch clamp investigations were carried out to clarify the pH sensitivity of native and recombinant P2X3 receptors. In HEK293 cells permanently transfected with human (h) P2X3 receptors (HEK293-hP2X3 cells), an acidic pH shifted the concentration-response curve for α,β-methylene ATP (α,β-meATP) to the right and increased its maximum. An alkalic pH did not alter the effect of α,β-meATP. Further, a low pH value increased the activation time constant (τon) of the α,β-meATP current; the fast and slow time constants of desensitization (τdes1, τdes2) were at the same time also increased. Finally, acidification accelerated the recovery of P2X3 receptors from the desensitized state. Replacement of histidine 206, but not histidine 45, by alanine abolished the pH-induced effects on hP2X3 receptors transiently expressed in HEK293 cells. Changes in the intracellular pH had no effect on the amplitude or time course of the α,β-meATP currents. The voltage sensitivity and reversal potential of the currents activated by α,β-meATP were unaffected by extracellular acidification. Similar effects were observed in a subpopulation of rat dorsal root ganglion neurons expressing homomeric P2X3 receptor channels. It is suggested that acidification may have a dual effect on P2X3 channels, by decreasing the current amplitude at low agonist concentrations (because of a decrease in the rate of activation) and increasing it at high concentrations (because of a decrease in the rate of desensitization). Thereby, a differential regulation of pain sensation during e.g. inflammation may occur at the C fiber terminals of small DRG neurons in peripheral tissues. High proton concentrations have been registered in inflamed tissue (down to pH 5.4), after surgical interventions (down to pH 5.5), in fracture-related hematomas (down to pH 4.7), in cardiac ischemia (down to pH 5.7), and in and around malignant tumors (1Häbler C. Klin. Wochenschr. 1929; : 1569-1572Crossref Scopus (47) Google Scholar, 2Revici E. Stoopen E. Frenk E. Ravich R.A. Bull. Inst. Appl. 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Thus, it has been proposed that local acidosis may play a major role in pain and hyperalgesia (7Reeh P.W. Steen K.H. Prog. Brain Res. 1996; 113: 143-151Crossref PubMed Scopus (230) Google Scholar). Hydrogen ions are able to excite dorsal root ganglion (DRG) 2The abbreviations used are: DRG, dorsal root ganglion; ASIC, acid-sensing ion channel; α,β-meATP, α,β-methylene ATP; WT, wild type. neurons via the activation and/or modulation of inward cation-selective currents, including the acid-sensing ion channels (ASICs) (14Wemmie J.A. Price M.P. Welsh M.J. Trends Neurosci. 2006; 29: 578-586Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar), the transient receptor potential vanilloid receptor 1 (TRPV1) (15Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7144) Google Scholar, 16Tominaga M. Caterina M.J. Malmberg A.B. Rosen T.A. Gilbert H. Skinner K. Raumann B.E. Basbaum A.I. Julius D. Neuron. 1998; 21: 531-543Abstract Full Text Full Text PDF PubMed Scopus (2597) Google Scholar), and P2X receptors (17King B.F. Wildman S.S. Ziganshina L.E. Pintor J. Burnstock G. Br. J. Pharmacol. 1997; 121: 1445-1453Crossref PubMed Scopus (134) Google Scholar, 18Stoop R. Surprenant A. North R.A. J. Neurophysiol. 1997; 78: 1837-1840Crossref PubMed Scopus (165) Google Scholar). P2X receptors represent a family of ligand-gated cationic channels that open in response to the binding of ATP, possess two transmembrane domains, intracellular N and C termini, a large extracellular loop, and assemble as homo- or heterotrimers (19North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2490) Google Scholar, 20Illes P. Ribeiro J.A. Eur. J. Pharmacol. 2004; 483: 5-17Crossref PubMed Scopus (139) Google Scholar, 21Vial C. Roberts J.A. Evans R.J. Trends Pharmacol. Sci. 2004; 25: 487-493Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In peripheral tissues, large quantities of ATP may leave the intracellular space in response to tissue trauma, tumors, inflammation, migraine, or visceral distension (22Burnstock G. Ciba Found. Symp. 1996; 198: 1-28PubMed Google Scholar). The resulting P2X receptor activation and the subsequent depolarization of the sensory cell membrane initiate action potentials that are perceived centrally as pain. Although sensory neurons express all known P2X subunits, the homomeric P2X3 and the heteromeric P2X2/3 receptors occur in these cells at the highest density (23Chizh B.A. Illes P. Pharmacol. Rev. 2001; 53: 553-568PubMed Google Scholar, 24North R.A. J. Physiol. 2004; 554: 301-308Crossref PubMed Scopus (168) Google Scholar). Whereas agonist-induced currents through recombinant P2X2 and P2X2/3 receptor channels (17King B.F. Wildman S.S. Ziganshina L.E. Pintor J. Burnstock G. Br. J. Pharmacol. 1997; 121: 1445-1453Crossref PubMed Scopus (134) Google Scholar, 18Stoop R. Surprenant A. North R.A. J. Neurophysiol. 1997; 78: 1837-1840Crossref PubMed Scopus (165) Google Scholar) were potentiated by acidification, the same change in pH depressed currents through P2X3 receptor channels (18Stoop R. Surprenant A. North R.A. J. Neurophysiol. 1997; 78: 1837-1840Crossref PubMed Scopus (165) Google Scholar). The failure to use more than a single concentration of α,β-meATP did not allow to construct concentration-response relationships for the P2X3 receptor subunit. Bullfrog DRG and rat nodose ganglion neurons are known to possess native P2X1, P2X2, and P2X3 receptors. Application of ATP onto these cells evoked non-desensitizing currents resembling P2X2 or P2X2/3 receptor activation (19North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2490) Google Scholar). These currents were enhanced by acidification, suggesting that extracellular protons can regulate the function of ATP-gated receptor channels in bullfrog DRG and rat nodose ganglion neurons (25Li C. Peoples R.W. Weight F.F. J. Neurophysiol. 1996; 76: 3048-3058Crossref PubMed Scopus (64) Google Scholar, 26Li C. Peoples R.W. Weight F.F. Pflugers Arch. 1997; 433: 446-454Crossref PubMed Scopus (40) Google Scholar). The aim of the present study was to investigate the effect of acidification on recombinant human P2X3 receptors (hP2X3R) transfected into human embryonic kidney (HEK) 293 cells. It was found that a change of pH from the normal 7.4 to 5.8 markedly decelerated both the kinetics of channel opening and the subsequent speed of desensitization, and thereby depressed currents caused by low, but facilitated currents caused by high agonist concentrations. Our findings demonstrate a dual, agonist concentration-dependent effect of acid pH at homomeric P2X3 receptors which may allow a differential modulation of pain sensation in response to small or large quantities of ATP released by noxious stimulation. Culturing of HEK293-hP2X3 Cells—The maintenance of HEK293 cells and their stable transfection with hP2X3R cDNA have been described previously (27Gerevich Z. Zadori Z. Müller C. Wirkner K. Schröder W. Rubini P. Illes P. Br. J. Pharmacol. 2007; 151: 226-236Crossref PubMed Scopus (52) Google Scholar). Cells were kept in Dulbecco's modified Eagle's medium also containing 25 mm HEPES, 110 μg/ml sodium pyruvate, 1 mg/ml d-glucose, 4 μg/ml pyridoxine (Invitrogen, Karlsruhe, Germany), 2 mm l-glutamine, 1% non-essential amino acids (NEAA) (all Sigma), 10% fetal bovine serum and 50 μg/ml geneticin (both from Invitrogen) at 37 °C and 10% CO2 in humidified air. They were plated on 35-mm plastic dishes (Sarstedt, Nürnberg, Germany) for electrophysiological recordings. Site-directed Mutagenesis and Transfection Procedures—The human P2X3 receptor cDNA (GenBank® accession number NM-00255) was subcloned per PstI and EcoRI restriction sites into pIRES2-EGFP vector from Clontech Laboratories (Mountain View, CA) for independent expression of P2X3 and EGFP, creating the pIR-P2 plasmid. All P2X3 receptor mutants were generated by introducing point mutations into the pIR-P2 construct, using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA) according to the instruction manual. HEK293 cells were plated in 35-mm plastic dishes 1 day before transient transfection. 0.5 μg of plasmid DNA per dish was combined with 10 μl of Polyfect reagent from Qiagen (Hilden, Germany) and 100 μl of Optimem (Invitrogen, Karlsruhe, Germany). After 10 min of incubation, the lipid-DNA complexes were introduced to the cells. Approximately 18-h post-transfection, the medium was replaced with Optimem to remove residual Plasmid DNA. Electrophysiological recordings took place 48–72 h after transient transfection during maximum protein expression. P2X3 receptor-positive cells were detected by viewing EGFP fluorescence with a fluorescence microscope. Preparation of DRG Neuronal Cultures—One-day-old Wistar rats (own breed) were used in the study. The animals were killed under CO2 and decapitated to obtain cell cultures of DRG neurons. The isolation and culturing procedures of thoracic and lumbar DRG cells have been described in detail previously (28Gerevich Z. Borvendeg S.J. Schröder W. Franke H. Wirkner K. Nörenberg W. Fürst S. Gillen C. Illes P. J. Neurosci. 2004; 24: 797-807Crossref PubMed Scopus (105) Google Scholar, 29Gerevich Z. Müller C. Illes P. Eur. J. Pharmacol. 2005; 521: 34-38Crossref PubMed Scopus (51) Google Scholar). DRG cells were plated at a density of 3 × 104 cells onto 35-mm plastic dishes coated by poly-l-lysine (25 μg/ml) (Sarstedt). They were kept in Dulbecco's modified Eagle's medium, 35 mm total glucose, 2.5 mm l-glutamine, 15 mm HEPES, 50 μg/ml gentamicin, 5% fetal bovine serum (Invitrogen), 30 ng/ml nerve growth factor, 10 μg/ml insulin, 5.5 μg/ml transferrin, and 5 ng/ml selenium (Sigma). Primary cultures of rat DRG neurons were maintained for 2–4 days in a humidified atmosphere (37 °C, 5% CO2) before experimentation. Whole Cell Patch Clamp Recordings—Whole cell patchclamp recordings were performed 2–6 days after the splitting of permanently transfected HEK293 cells, and 2–4 days after the plating of rat DRG neurons, at room temperature (20–22 °C), using an Axopatch 200B patch-clamp amplifier (Molecular Devices, Union City, CA). Patch pipettes (3–5 MΩ) for both HEK293 cells and DRG neurons were filled with intracellular solution of the following composition (in mm): 135 CsCl, 2 MgCl2, 20 HEPES, 11 EGTA, 1 CaCl2, 1.5 Mg-ATP, and 0.3 Li-GTP, pH was adjusted with CsOH. The external solution consisted of (in mm) 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 11 glucose, pH was adjusted with NaOH. The pH of all solutions was routinely checked before and during experiments. All recordings were made at a holding potential of –70 mV. Data were filtered at 2 kHz with the in-built filter of the Axopatch 200B, digitized at 5 kHz, and stored on a laboratory computer using a Digidata 1200 interface and pClamp 10.0 software (Molecular Devices). Drugs were dissolved in external solution and applied locally to single cells, using a rapid solution change system (SF-77B Perfusion Fast-Step, Warner Instruments, Hamden, CT; 10–90% rise time of the junction potential at an open pipette tip was 1–4 ms). Concentration-response curves for the P2X3 receptor currents at different pH values were constructed by applying every 5 min increasing concentrations of α,β-methylene ATP (α,β-meATP). To analyze P2X3 currents from the same cells at different pH values, α,β-meATP was applied at a given concentration six times with 5-min intervals. After the recording of two α,β-meATP-induced inward currents of the same size at pH 7.4, the pH was changed 2.5 min after the second α,β-meATP administration. Two α,β-meATP currents were recorded at the altered pH value and two further ones at pH 7.4 again. When recording from DRG cells, amiloride (200 μm) was given to the bath solution to block ASICs. This concentration of amiloride did not affect P2X3 currents either in HEK293-hP2X3 cells or in DRG neurons (see "Results"). Concentration-response curves established to determine the agonistic effects of α,β-meATP were fitted to mean data points using Equation 1, I=Imax×[A]nH/([A]nH+EC50nH)(Eq. 1) where I is the observed current, Imax the extrapolated maximal current, [A] the α,β-meATP concentration in μm, EC50 the concentration of α,β-meATP that produces 50% of Imax and nH the Hill coefficient (slope value) (SigmaPlot, SPSS, Erkrath, Germany). In experiments investigating the kinetics of P2X3 currents, decay phases of the curves were fitted by the following biexponential function in Equation 2, y=A1×e-t/τdes1+A2×e-t/τdes2+P(Eq. 2) using the in-built function of the pClamp 10.0 software (Molecular Devices), where A1 and A2 are the relative amplitudes of the first and second exponential, τdes1 and τdes2 are the desensitization time constants, and P is plateau. The onset time constants (τon(10–90%)) were calculated from the individual recordings, under the assumption that despite the relatively slow local application they give a rough approximation of the kinetics of channel opening. The effect of a pH change on the receptor activation and desensitization was found to be concentration-independent and was expressed as the mean ± S.E. of the time constant ratios at each concentration investigated. In experiments examining the recovery of P2X3 receptors from desensitization, HEK293 cells were stimulated repetitively with α,β-meATP at 3 μm (5-s pulses, each) with a progressive increase in the interpulse intervals. The recovery from desensitization was fitted using the following Equation 3 (30Sokolova E. Skorinkin A. Fabbretti E. Masten L. Nistri A. Giniatullin R. Br. J. Pharmacol. 2004; 141: 1048-1058Crossref PubMed Scopus (48) Google Scholar), I=Imax+Imax-Imin1+e(t50-t)/b(Eq. 3) where Imax and Imin are the start and finish levels during recovery, t is the time, t50 is the time to regain 50% of maximally recovered currents, and b is the slope factor giving the change in time per e-fold change in recovery. Materials and Drugs—α,β-Methylene ATP lithium salt (α,β-meATP) and amiloride hydrochloride were both purchased from Sigma. The drugs were prepared as a concentrated stock solution in distilled water and were diluted to the final concentration in external medium. Data Analysis—Data were analyzed off-line using pClamp 10.0 software (Molecular Devices). Figures show mean ± S.E. values of n experiments. Student's t test or one way ANOVA followed by Bonferroni's post hoc test were used for statistical analysis. A probability level of 0.05 or less was considered to reflect a statistically significant difference. Effect of Extracellular Protons on P2X3 Currents in HEK293-hP2X3 Cells—Application of various concentrations of α,β-meATP to HEK293 cells permanently transfected with hP2X3Rs (HEK293-hP2X3 cells) induced inward current responses (Fig. 1A). Acidification of the extracellular solution had 3-fold effects on the concentration-response curves for α,β-meATP. It enhanced the maximal current amplitudes (Imax), increased the EC50 values, and raised the Hill coefficient (nH; Fig. 1Ba and Table 1). In contrast, the alkalinization of the extracellular solution caused no major change in the Imax, the EC50 value or the nH (Fig. 1Bb; Table 1).TABLE 1Parameters of the α,β-meATP concentration-response relations and the t50 values of the recovery from α,β-meATP-induced desensitization for human P2X3 receptors permanently transfected into HEK293 cells at various pH levelspHImaxnHEC50t50pAμmmin8.63945 ± 4881.38 ± 0.291.13 ± 0.29NDaND, not determined.83935 ± 481.54 ± 0.050.66 ± 0.021.19 ± 0.057.43183 ± 1631.75 ± 0.240.77 ± 0.081.01 ± 0.076.45075 ± 703bp < 0.05, statistically significant difference from pH 7.4 (one way analysis of variance).1.55 ± 0.491.74 ± 0.5bp < 0.05, statistically significant difference from pH 7.4 (one way analysis of variance).0.74 ± 0.175.84302 ± 312.88 ± 0.09bp < 0.05, statistically significant difference from pH 7.4 (one way analysis of variance).1.67 ± 0.02bp < 0.05, statistically significant difference from pH 7.4 (one way analysis of variance).0.42 ± 0.03bp < 0.05, statistically significant difference from pH 7.4 (one way analysis of variance).a ND, not determined.b p < 0.05, statistically significant difference from pH 7.4 (one way analysis of variance). Open table in a new tab To further investigate the effect of protons on P2X3 receptors, we applied two selected agonist concentrations (1 and 4.64 μm) onto HEK293-hP2X3 cells and examined, whether a change in the pH affects P2X3 currents in the same cell. As shown in Fig. 2, the current amplitudes evoked by α,β-meATP at 1 μm (approximate EC50) were inhibited by shifting the pH from 7.4 to 5.8 by 43.7 ± 7.6% (n = 10, p < 0.05) whereas at 4.64 μm (high concentration of this agonist), the current amplitudes were facilitated by 28.9 ± 14.0% (n = 8, p < 0.05) (Fig. 2A). Both the increase and the decrease of the current responses to these α,β-meATP concentrations were reversible on returning to the original extracellular pH of 7.4. In contrast, a change of the pH from 7.4 to 8.0 did not modify the α,β-meATP currents at either concentration (1.4 ± 6.3%, n = 6 and 1.8 ± 8.6%, n = 6, at 1 and 4.64 μm, respectively) (Fig. 2B). The original tracings in Fig. 2, A and B show that acidification, but not alkalinization itself induces rapidly declining inward currents. These currents may be due to the activation of ASICs described to be present in HEK293 cells (31Lalo U. Pankratov Y. North R.A. Verkhratsky A. J. Cell Physiol. 2007; 212: 473-480Crossref PubMed Scopus (8) Google Scholar). As documented in Fig. 2, Aa and Ab, these currents completely desensitized within milliseconds, and therefore they are not expected to alter the currents evoked by α,β-meATP 2.5-min later. Nonetheless, to reliably exclude a possible interference of ASIC currents with α,β-meATP-induced currents, the same experiments as shown in Fig. 2A were performed in the presence of amiloride, a selective inhibitor of ASIC channels (14Wemmie J.A. Price M.P. Welsh M.J. Trends Neurosci. 2006; 29: 578-586Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). In fact, amiloride (200 μm) abolished the early current induced by a change of pH from 7.4 to 5.8 (96.5 ± 2.7% inhibition, n = 5, p < 0.05%), but had no effect on the response to α,β-meATP (1 μm) at a pH of 5.8 (40.3 ± 9.6% inhibition, n = 5, p > 0.05%; compare with Fig. 2Ac). It is noteworthy that the lower concentration (1 μm) of α,β-meATP evoked a current response at a pH of 5.8, which desensitized much slower than that observed at the control pH value of 7.4 (Fig. 2Aa; see below). Effect of Protons on P2X3 Receptor Activation and Desensitization—Next, we investigated whether different proton concentrations affect the activation and desensitization of P2X3 channels. The current decay in the presence of α,β-meATP, which represents the onset of desensitization, was fitted by a biexponential equation (see "Materials and Methods") and had a first fast and a second slow time constant (τdes1, τdes2). Of these time constants, τdes1 was found to be the more dominant one, because it constituted concentration-independently 84.9 ± 1.4% of the current decay, as calculated from the contribution of the relative amplitudes of the first and second exponential to the total current amplitude (A1/A1+A2, see Equation 2 under "Materials and Methods"; n = 39). The activation time constants (τon) and both desensitization time constants (τdes1, τdes2) inversely correlated with the α,β-meATP concentration (Fig. 3, Ca–Cc). These data are in accordance with earlier observations showing that the kinetics of activation, desensitization, and recovery from desensitization, but not deactivation of P2X receptors, inversely correlate with the agonist concentration (32Zemkova H. He M.L. Koshimizu T.A. Stojilkovic S.S. J. Neurosci. 2004; 24: 6968-6978Crossref PubMed Scopus (40) Google Scholar, 33Sokolova E. Skorinkin A. Moiseev I. Agrachev A. Nistri A. Giniatullin R. Mol. Pharmacol. 2006; 70: 373-382Crossref PubMed Scopus (59) Google Scholar, 34Yan Z. Liang Z. Obsil T. Stojilkovic S.S. J. Biol. Chem. 2006; 281: 32649-32659Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Lowering the pH to 5.8 increased both the activation time constant and the two desensitization time constants at all agonist concentrations applied (Fig. 3, A, B, and Ca–Cc). Increasing the pH value up to 8.6 did not change the activation and desensitization time constants appreciably. The normalized shift of concentration time constant curves at different pH values is shown in Fig. 3D. As shown in Fig. 3Cb, the pH-dependent amplification of τdes2 (but not τdes1; Fig. 3Ca) was attenuated at higher agonist concentrations. We calculated the contribution of τdes2 to the current decay at different pH and agonist concentrations and found that τdes2 was independent of these parameters and constituted only 16.3 ± 3.4% (n = 38) of the total current decay. It is interesting to note that the concentration versus τon curves were less steep than the concentration versus τdes1 curves. Thus at lower concentrations of α,β-meATP the current amplitude was determined mostly by the activation time constant, whereas at higher concentrations of α,β-meATP the current amplitude was determined mostly by the first time constant of desensitization (Fig. 3, Ca and Cc). Effect of Protons on the Recovery from Desensitization—After rapid desensitization, P2X3 receptors stay in the desensitized state for minutes and cannot fully open in response to further agonist application. We next examined the availability of the receptors after different time intervals i.e. how fast they recover from this desensitized state and, moreover, whether changing the proton concentration modulates this process. HEK293-hP2X3 cells were stimulated repetitively with α,β-meATP (10 μm, for 5 s), by progressively increasing the interpulse intervals (Fig. 4). During the application of α,β-meATP, P2X3 receptors completely desensitized. The recovery of P2X3 receptors from desensitization exhibited a sigmoidal time course and was best fitted with Equation 3 described under "Materials and Methods." The time course of recovery was expressed as t50, namely the time to regain 50% of the maximally recovered current amplitudes. Lowering the pH to 5.8 significantly accelerated the recovery (Table 1; analysis of variance, p < 0.05, n = 7–8), whereas increasing the pH to 8.0 had no effect on the speed of recovery (Table 1). The Role of Histidine Residues in pH Sensitivity—Protons modulated P2X3 receptors in the range between pH 7.4 and 5.8. The only amino acid with a pKa close to this range is histidine. The pKa of free histidine is 6.0 (35Tanokura M. Biochim. Biophys. Acta. 1983; 742: 576-585Crossref PubMed Scopus (138) Google Scholar), but in proteins its pKa can range from 5.0 to 8.0 (36Kao Y.H. Fitch C.A. Bhattacharya S. Sarkisian C.J. Lecomte J.T. Garcia-Moreno E.B. Biophys. J. 2000; 79: 1637-1654Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) making histidines apparent candidates for the proton-binding site. To test whether histidines are involved in the modulation of P2X3 receptors by protons, we substituted single histidine residues by alanines in the extracellular domain of the human P2X3 receptor (H45A and H206A). The wild-type (WT) P2X3 receptors transiently transfected into HEK293 cells responded to acidification in a manner similar to that reported for the permanently transfected receptor (compare Tables 1 and 2). In fact, lowering the pH from 7.4 to 5.8 shifted the concentration-response curve for α,β-meATP to the right and increased the Imax (Fig. 5, Aa and Ab). We investigated the effect of replacing two histidine residues in the extracellular loop of the P2X3 receptor by the uncharged amino acid alanine (H45A, H206A) on the pH sensitivity of the α,β-meATP effect (Fig. 5B and Table 2). The most important finding is that the H206A mutation abolished the increase of the maximal response, the shift of the concentration-response curve to the right, and the changes in all three kinetic parameters (τdes1, τdes2, and τon) by acidification to a pH of 5.8 (Fig. 5, Ba and C). In contrast, the single mutation H45A, accentuated the pH-sensitivity of the receptor; both the increase of the maximal response and the rightward shift of the concentration-response curve were more pronounced than in the case of the WT receptor (Fig. 5Bb). The effects of acidification on the τon, τdes1, and τdes2 were similar to that observed with the WT receptor (Fig. 5, Ca, Cb, Cc). The double mutant caused a mixture of these effects in that although a rightward shift of the concentration-response curve by the acidic pH was still present, the increase in Imax and a change in the kinetic parameters of desensitization were abolished (Fig. 5, Bc and C).TABLE 2Parameters of the α,β-meATP concentration-response relations for wild type and mutated human P2X3 receptors transiently transfected into HEK293 cells at various pH levelspHImaxnHEC50pAμmWT7.43399 ± 331.78 ± 0.091.89 ± 0.065.84985 ± 220ap < 0.05, statistically significant difference from pH 7.4 (Student's t-test).1.71 ± 0.358.58 ± 1.20ap < 0.05, statistically significant difference from pH 7.4 (Student's t-test).H206A7.41345 ± 371.98 ± 0.233.48 ± 0.245.81504 ± 481.64 ± 0.227.24 ± 0.70ap < 0.05, statistically significant difference from pH 7.4 (Student's t-test).H45A7.4948 ± 401.94 ± 0.300.46 ± 0.045.81633 ± 120ap < 0.05, statistically significant difference from pH 7.4 (Student's t-test).2.23 ± 0.504.50 ± 0.57ap < 0.05, statistically significant difference from pH 7.4 (Student's t-test).DMbDM, double mutant (H206A-H45A).7.4242 ± 131.55 ± 0.314.38 ± 0.725.8225 ± 42.20 ± 0.1915.41 ± 0.80ap < 0.05, statistically significant difference from pH 7.4 (Student's t-test).a p < 0.05, statistically significant difference from pH 7.4 (Student's t-test).b DM, double mutant (H206A-H45A). Open table in a new tab Effect of Intracellular pH—To study whether the site of proton action is extra- or intracellular, HEK293
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