Differential Modulation by Copper and Zinc of P2X2 and P2X4 Receptor Function

Keming Xiong, Robert W. Peoples, Jennifer P. Montgomery, Yisheng Chiang, Randall R. Stewart, Forrest F. Weight, and Chaoying Li

Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892-8115


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Xiong, Keming, Robert W. Peoples, Jennifer P. Montgomery, Yisheng Chiang, Randall R. Stewart, Forrest F. Weight, and Chaoying Li. Differential Modulation by Copper and Zinc of P2X2 and P2X4 Receptor Function. The modulation by Cu2+ and Zn2+ of P2X2 and P2X4 receptors expressed in Xenopus oocytes was studied with the two-electrode, voltage-clamp technique. In oocytes expressing P2X2 receptors, both Cu2+ and Zn2+, in the concentration range 1-130 µM, reversibly potentiated current activated by submaximal concentrations of ATP. The Cu2+ and Zn2+ concentrations that produced 50% of maximal potentiation (EC50) of current activated by 50 µM ATP were 16.3 ± 0.9 (SE) µM and 19.6 ± 1.5 µM, respectively. Cu2+ and Zn2+ potentiation of ATP-activated current was independent of membrane potential between -80 and +20 mV and did not involve a shift in the reversal potential of the current. Like Zn2+, Cu2+ increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. However, Cu2+ did not enhance ATP-activated current in the presence of a maximally effective concentration of Zn2+, suggesting a common site or mechanism of action of Cu2+ and Zn2+ on P2X2 receptors. For the P2X4 receptor, Zn2+, from 0.5 to 20 µM enhanced current activated by 5 µM ATP with an EC50 value of 2.4 ± 0.2 µM. Zn2+ shifted the ATP concentration-response curve to the left in a parallel manner, and potentiation by Zn2+ was voltage independent. By contrast, Cu2+ in a similar concentration range did not affect ATP-activated current in oocytes expressing P2X4 receptors, and Cu2+ did not alter the potentiation of ATP-activated current produced by Zn2+. The results suggest that Cu2+ and Zn2+ differentially modulate the function of P2X2 and P2X4 receptors, perhaps because of differences in a shared site of action on both subunits or the absence of a site for Cu2+ action on the P2X4 receptor.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The P2X receptors are ligand-gated membrane ion channels that are activated by extracellular ATP. These receptor channels received recent attention because of their potential importance in the central and peripheral nervous systems. Activation of P2X receptors elicits excitatory postsynaptic currents or excitatory postsynaptic potentials in both central and peripheral neurons (Bardoni et al. 1997; Edwards et al. 1992, 1997; Evans et al. 1992; Galligan and Bertrand 1994; Gu and MacDermott 1997; Pankratov et al. 1998; Silinsky et al. 1992) and excitatory junction potentials in smooth muscle cells (Sneddon et al. 1982). Activation of P2X receptors also mediates excitatory responses in a variety of central and peripheral neurons (Bean 1990; Fieber and Adams 1991; Khakh et al. 1995; Krishtal et al. 1983; Li et al. 1993, 1997a; Shen and North 1993; Ueno et al. 1992). P2X receptors were found to be widely distributed in the CNS, including cerebral cortex, hippocampus, thalamus, hypothalamus, midbrain, cerebellum, and spinal cord, and in sensory and sympathetic ganglia in the peripheral nervous system (Collo et al. 1996).

Like other neurotransmitter-gated membrane ion channels, P2X receptors in neurons are sensitive to a number of endogenous agents, including Zn2+ (Cloues et al. 1993; Li et al. 1993, 1997a), Cu2+ (Li et al. 1996a), H+ (Li et al. 1996b), Mg2+, and Ca2+ (Krishtal and Marchenko 1984; Li et al. 1997b; Nakazawa and Hess 1993) as well as other neurotransmitters or neuromodulators, such as substance P (Hu and Li 1996; Wildman et al. 1997). Recent studies revealed that these substances can produce differential effects on P2X receptors in neurons. For instance, in rat nodose ganglion neurons, low micromolar concentrations of Zn2+ and Cu2+ enhance ATP-activated current in the majority of neurons but have no effect in a subset of neurons (Li et al. 1993, 1996a). On the other hand, in bullfrog dorsal root ganglion neurons, low micromolar concentrations of Zn2+ inhibit ATP-activated current (Li et al. 1997a). Extracellular protons markedly potentiate ATP-activated current in the majority of neurons from rat nodose ganglion but do not alter ATP-activated current in a subset of these neurons (Li et al. 1996a,b). Similarly, Mg2+ inhibits ATP-activated current in most but not all neurons from rat nodose ganglion (Li et al. 1997b). The molecular mechanisms underlying the diverse effects of these modulators, however, remain to be determined.

At least seven P2X receptor subunits, designated P2X1-P2X7, were cloned to date (Buell et al. 1996a). Each of these subunits can form ATP-selective homomeric cation channels when expressed in Xenopus oocytes or cell lines. Characterization of the properties of recombinant P2X receptor subunits should prove to be a useful first step in resolving the disparate effects of modulators on P2X receptors in neurons. In this regard, results of recent studies revealed a differential modulation of P2X receptor subunits by endogenous agents. For instance, extracellular Ca2+ strongly inhibits the P2X2 subunit but not the P2X1 subunit (Evans et al. 1996). In addition, low micromolar concentrations of Zn2+ potentiate P2X2 and P2X4 subunits (Brake et al. 1994; Garcia-Guzman et al. 1997; Séguéla et al. 1996; Wildman et al. 1998) but inhibit the P2X7 subunit (Virginio et al. 1997). Morever, extracellular protons inhibit P2X1, P2X3, P2X4, and P2X7 subunits but potentiate the P2X2 subunit as well as the P2X2 and P2X3 heteromeric receptor (Stoop et al. 1997). To characterize further the physiological regulation of P2X receptor subunits, we investigated the effects of Cu2+ and Zn2+ and their possible interactions on recombinant P2X2 and P2X4 receptors.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of cRNA and expression of receptors

cRNA was synthesized in vitro from a linearized cDNA template with T7 RNA polymerase in the presence of the cap analogue 7 mGpppG and was injected into Xenopus oocytes with a pressurized microinjection device (PV 800 Pneumatic Picopump, World Precision Instruments; Sarasota, FL). Mature X. laevis frogs were anesthetized by immersion in water containing 3-aminobenzoic acid ethyl ester (2 g/l). Oocytes were excised, mechanically isolated into clusters of four to five oocytes, and shaken in a water bath in two changes of 0.2% collagenase A in a solution containing (in mM) 83 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES, pH 7.4, for 1 h each. Each oocyte was injected with a total of 10 ng of RNA in 50 nl of diethylpyrocarbonate-treated water and was incubated at 17°C for 2-5 days in modified Barth's saline containing sodium pyruvate (2 mM), penicillin (10,000 U/l), streptomycin (10 mg/l), gentamycin (50 mg/l), and theophylline (0.5 mM).

The care and use of animals in this study was approved by the Animal Care and Use Committee of the National Institute on Alcohol Abuse and Alcoholism (protocol no. LMCN-SP-05) in accordance with National Institutes of Health guidelines.

Electrophysiological recording

Two-electrode, voltage-clamp recording was performed at room temperature with a Geneclamp (Axon Instruments; Foster City, CA) amplifier. Oocytes were placed in a recording chamber and impaled with two sharp electrodes filled with 3 M KCl. Electrode tip resistances were in the range 0.5-1.5 MOmega . Oocytes were usually voltage clamped at -70 mV, except as indicated. Currents were recorded on a pen recorder (Model RS3400, Gould; Valley View, OH). Oocytes were constantly superfused at the rate of ~2.5 ml/min with bathing medium containing (in mM) 95 NaCl, 2 KCl, 2 CaCl2, and 5 HEPES, pH 7.4. Solutions of ATP (as the sodium salt) and Cu2+ (as CuCl2) or Zn2+ (as ZnCl2) were prepared daily in extracellular medium. Solutions of ATP and Cu2+ or Zn2+ were administered via the bathing solution, which was applied by gravity flow from a 0.5-mm silica tube connected to a seven-barrel manifold. Solutions were changed via manually switched solenoid valves. At least 5 min was allowed to elapse between agonist applications.

Drugs and chemicals

All of the drugs and chemicals used in these experiments were purchased from Sigma Chemical (St. Louis, MO), except CuCl2, which was purchased from Aldrich Chemical (Milwaukee, WI), and the salts, which were purchased from Mallinckrodt (Paris, KY).

Estimation of Zn2+ concentration

Concentrations of free Zn2+ were estimated with the program "Bound and Determined" (Brooks and Storey 1992), which compensates for variation in temperature, pH, and ionic strength. Values for Mn2+ were used as estimates of Zn2+ concentrations because ATP has similar affinities for Mn2+ and Zn2+ (16 vs. 14 µM) (Sillen and Martell 1964), and the software does not directly calculate Zn2+ concentration. All concentrations of ATP, Zn2+, and Cu2+ given are total concentrations unless stated otherwise.

Data analysis

Current amplitudes reported are peak values, and average values are expressed as means ± SE, with n equal to the number of cells studied. Data were statistically compared with Student's t-test or ANOVA as noted. Statistical analysis of concentration-response data was performed with the nonlinear curve-fitting program ALLFIT (DeLean at al. 1978), which uses an ANOVA procedure. Values reported for concentrations yielding 50% of maximal effect (EC50) and slope factor (n) are those obtained by fitting the data to the equation
<IT>Y</IT><IT>=</IT><IT>E</IT><SUB><IT>max</IT></SUB><IT>&cjs0823;  </IT>[<IT>1+</IT>(<IT>EC<SUB>50</SUB>&cjs0823;  </IT><IT>X</IT>)<SUP><IT>n</IT></SUP>]
where X and Y are concentration and response, respectively, and Emax is the maximal response.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Modulation of P2X2 receptors by Cu2+ and Zn2+

ATP, at concentrations of <= 500 µM, did not evoke detectable ion current in uninjected oocytes (n = 6, data not shown). Figure 1 illustrates the ATP-activated inward current in oocytes expressing P2X2 receptors and the potentiation of that current by extracellular Cu2+ and Zn2+. As shown in Fig. 1A, the amplitude of inward current activated by 50 µM ATP was greatly enhanced by the application of 10 µM Cu2+. To compare the effect of Cu2+ with that of Zn2+ (Brake et al. 1994; Wildman et al. 1998), potentiation of ATP-activated current by Zn2+ was also tested. At the same concentration, Zn2+ produced enhancement of ATP-activated current that was comparable with that of Cu2+ in the same oocyte. On average, in the same oocytes, 10 µM Cu2+ or 10 µM Zn2+ increased the amplitude of current activated by 50 µM ATP by 240 ± 32% (n = 12) or 167 ± 24% (n = 14), respectively. The enhancement by both divalent cations was concentration dependent between 1 and 130 µM (Fig. 1B). The EC50 values for Cu2+ and Zn2+ enhancement of current activated by 50 µM ATP were 16.3 ± 0.9 µM and 19.6 ± 1.5 µM, the slope factors were 1.5 and 1.6, and the maximal effects were 845 ± 16% and 837 ± 26% of control, respectively. The EC50, slope factor, and Emax values obtained for Cu2+ did not differ significantly from those for Zn2+ (ANOVA, P > 0.1). Cu2+ or Zn2+ alone (1-130 µM) did not activate ion current in any oocytes tested (data not shown, n = 5).



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Fig. 1. Potentiation by Cu2+ and Zn2+ of ATP-activated current mediated by P2X2 receptors. A: records of current activated by 50 µM ATP in the absence and the presence of 10 µM Cu2+ or Zn2+. Records are sequential current traces (from left to right) obtained from a single oocyte. Solid bar above each record indicates time of agonist application in the absence or presence of Cu2+ or Zn2+, as labeled. B: concentration-response curves for Cu2+ (black-square) and Zn2+ () potentiation of current activated by 50 µM ATP. Each point is the average of 6-14 cells; error bars not visible are smaller than the size of the symbols. The curves shown are the best fit of the data to the equation described in METHODS. Fitting the data to this equation yielded EC50 values of 16.3 ± 0.9 µM and 19.6 ± 1.5 µM, slope factors of 1.5 and 1.6, and Emax values of 845 ± 16% and 837 ± 26% of control for Cu2+ and Zn2+, respectively. These values are not significantly different (ANOVA, P > 0.1).

Experiments performed to elucidate the mechanism by which Cu2+ augments ATP-activated current are shown in Fig. 2. As shown in Fig. 2A, the magnitude of Cu2+ potentiation decreased with increasing ATP concentration. On average, 5 µM Cu2+ increased the amplitude of the current activated by 10 and 100 µM ATP by 383 ± 28% (n = 6) and 15.3 ± 4% (n = 5), respectively. The graph in Fig. 2B shows the concentration-response curves for ATP-activated currents in the absence and presence of 5 µM Cu2+. As can be seen, Cu2+ shifted the ATP concentration-response curve to the left, reducing the EC50 for ATP from 51.7 ± 1.9 µM in the absence of Cu2+ to 15.5 ± 0.3 µM in the presence of 5 µM Cu2+ (ANOVA, P < 0.01) without significantly changing the slope or maximal value (ANOVA, P > 0.1). The lack of effect of Cu2+ on the maximal value of the ATP concentration-response curve was apparently not because of chelation of Cu2+ by high concentrations of ATP, as increasing the Cu2+ concentration threefold, which would yield a calculated concentration of free Cu2+ greater than that required to produce potentiation (results for Zn2+ potentiation of P2X4 receptors are described subsequently), did not potentiate current activated by 100 µM ATP (results not shown).



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Fig. 2. Effect of ATP concentration on Cu2+ potentiation of ATP-activated current mediated by P2X2 receptors. A: records showing currents activated by 10 µM ATP (top traces) and 100 µM ATP (bottom traces) before, during, and after application of 5 µM Cu2+ in a single oocyte. B: graph plotting the relative amplitude of ATP-activated current in the absence (open circle ) and presence () of 5 µM Cu2+ as a function of ATP concentration. Amplitude is normalized to the current activated by 300 µM ATP in the absence of Cu2+. Each data point is the average current from 5-8 cells. The curves shown are the best fits of the data to the equation described in METHODS. Cu2+ significantly decreased the EC50 for ATP from 51.7 ± 1.9 µM in the absence of Cu2+ to 15.5 ± 0.3 µM in the presence of 5 µM Cu2+ (ANOVA, P < 0.01).

The influence of membrane potential on the potentiation by Cu2+ and Zn2+ of ATP-activated current was evaluated by constructing current-voltage relationships for ATP-activated current. Figure 3A shows the current-voltage relationship for current activated by 50 µM ATP in the absence and presence of 5 µM Cu2+. Cu2+ produced a similar percentage enhancement of amplitude of current activated by ATP at membrane voltages between -80 and +20 mV and did not alter the reversal potential of ATP-activated current. In five of five cells tested, Cu2+ enhanced ATP-activated current in a voltage-independent manner (ANOVA, P > 0.25) and did not significantly change the reversal potential of ATP-activated current (Student's t-test, P > 0.25). Similarly, as shown in Fig. 3B, Zn2+ potentiation of ATP-activated current was voltage independent (ANOVA, P > 0.25, n = 4), and Zn2+ did not significantly change the reversal potential of ATP-activated current (Student's t-test, P > 0.25, n = 4).



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Fig. 3. Effect of membrane potential on potentiation by Cu2+ and Zn2+ of ATP-activated current mediated by P2X2 receptors. A: current-voltage relationship for currents activated by 50 µM ATP in the absence (open circle ) and the presence () of 5 µM Cu2+ at membrane potentials between -80 and +20 mV in a single cell. The average reversal potential of current activated by 50 µM ATP was -1 ± 4 mV in the absence and 1 ± 6 mV in the presence of 5 µM Cu2+; these values are not significantly different (Student's t-test, P > 0.25, n = 5). In addition, the percentage potentiation of ATP-activated current by Cu2+ was not significantly different at holding potentials from -80 to +20 mV (ANOVA, P > 0.25, n = 5). B: current-voltage relationship for currents activated by 50 µM ATP in the absence (open circle ) and the presence () of 5 µM Zn2+ at membrane potentials between -80 and +20 mV in a single oocyte. The average reversal potential of current activated by 50 µM ATP was -0 ± 5 mV in the absence and 0 ± 4 mV in the presence of 5 µM Zn2+; these values are not significantly different (Student's t-test, P > 0.25, n = 4). In addition, the percentage potentiation of ATP-activated current by Zn2+ was not significantly different at holding potentials from -80 to +20 mV (ANOVA, P > 0.25, n = 4). Data in A and B are from different cells. Membrane potential was held at each value for 1 min before application of ATP in both A and B.

Because Cu2+ and Zn2+ are closely related metals and have similar augmenting effects on ATP-activated current mediated by P2X2 receptors, we hypothesized that they might act at a common binding site. Results of an experiment designed to test this hypothesis are shown in Fig. 4. A near-threshold concentration of ATP was used to obtain a current in the presence of a maximally effective concentration of Zn2+ that was lower in amplitude than the maximal ATP-activated current. In the cell shown in Fig. 4A, a maximally effective concentration of Zn2+ (130 µM) potentiated current activated by 4 µM ATP by 2,867%, and 10 µM Cu2+ increased the ATP-activated current by 1,467%. However, concomitant application of 130 µM Zn2+ and 10 µM Cu2+ failed to produce enhancement of ATP-activated current greater than that produced by Zn2+ alone. On average, the potentiation of ATP-activated current produced by Cu2+ and Zn2+ applied together was not different from that produced by Zn2+ applied alone (Student's t-test, P > 0.25, n = 5; Fig. 4B).



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Fig. 4. Interaction of Zn2+ and Cu2+ on P2X2 receptors. A: records illustrating effect of 130 µM Zn2+ and 10 µM Cu2+ separately and in combination on ion current activated by 4 µM ATP. Note that 300 µM ATP-activated current was much greater in amplitude than that produced by 4 µM ATP in the presence of either 130 µM Zn2+ or Cu2+ and Zn2+ applied together. B: bar graph illustrating the average potentiation of 4 µM ATP-activated current (normalized to that activated by 300 µM ATP) by 130 µM Zn2+ and 10 µM Cu2+ separately and in combination. The average potentiation of ATP-activated current produced by Cu2+ and Zn2+ applied together was not different from that produced by Zn2+ applied alone (Student's t-test, P > 0.25, n = 5).

Modulation of P2X4 receptors by Zn2+ and Cu2+

The ATP-activated inward current in oocytes expressing P2X4 receptors and the modulation of that current by extracellular Zn2+ and Cu2+ are illustrated in Fig. 5. As shown in Fig. 5A, 10 µM Zn2+ markedly increased the amplitude of current activated by 5 µM ATP. By contrast, the same concentration of Cu2+ did not affect current activated by the same concentration of ATP. Zn2+ potentiation of ATP-activated current was concentration dependent between 0.5 and 20 µM. The EC50 value for Zn2+ potentiation of current activated by 5 µM ATP was 2.4 ± 0.2 µM, the slope factor was 1.8, and the maximal effect was 214 ± 12% of control (Fig. 5B). Zn2+ alone (0.5-20 µM) did not activate ion current in any oocytes tested (data not shown, n = 5). In contrast to the potentiation of ATP-activated current by Zn2+, Cu2+, in the same concentration range, did not significantly affect ATP-activated current (ANOVA, P > 0.25; Fig. 5B). In addition, Cu2+ at a concentration of 50 µM did not potentiate ATP-activated current in oocytes expressing P2X4 receptors (Student's t-test, P > 0.5, n = 7).



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Fig. 5. Potentiation by Zn2+ but not by Cu2+ of ATP-activated current mediated by P2X4 receptors. A: records of current activated by 5 µM ATP in the absence and the presence of 10 µM Zn2+ or Cu2+. Records are sequential current traces (from left to right) obtained from a single oocyte. B: concentration-response curves for Zn2+ () and Cu2+ (black-square) potentiation of current activated by 5 µM ATP. Each point is the average of 5-9 cells; error bars not visible are smaller than the size of the symbols. The sigmoid curve shown for Zn2+ is the best fit of the data to the equation described in METHODS. Fitting the data to this equation yielded an EC50 of 2.4 ± 0.2 µM, a slope factor of 1.8, and an Emax of 214 ± 12% of control. The data for Cu2+ could not be fitted to the equation in METHODS; the line shown for these data is a least-squares plot.

As the maximal potentiation by Cu2+ of ATP-activated current in P2X2 receptors occurred at the lowest ATP concentration, we tested whether Cu2+ would potentiate the current activated by a near-threshold concentration of ATP in P2X4 receptors. Results from one such experiment are illustrated in Fig. 6. In this experiment, 5 and 20 µM Cu2+ did not appreciably affect the current activated by 1.5 µM ATP. By contrast, 5 µM Zn2+ markedly enhanced ATP-activated current in the same cell. Similar results were obtained in five other experiments.



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Fig. 6. Effect of Cu2+ and Zn2+ on the current activated by a near-threshold concentration of ATP in P2X4 receptors. Records of current activated by 1.5 µM ATP in the absence and the presence of 5 and 20 µM Cu2+ and 5 µM Zn2+, respectively. Records are sequential current traces (from left to right) obtained from a single oocyte.

Figure 7A shows that Zn2+ shifted the ATP concentration-response curve to the left, reducing the EC50 value for ATP-activated current from 6.7 ± 1.3 µM in the absence of Zn2+ to 2.8 ± 0.2 µM in the presence of 5 µM Zn2+ (ANOVA, P < 0.01) without changing the slope or maximal value (ANOVA, P > 0.1). The lack of effect of Zn2+ on the maximal value of the ATP concentration-response curve did not appear to be due to chelation of Zn2+ by high concentrations of ATP, as addition of 10 µM Zn2+ yielded a calculated free Zn2+ concentration of 6.9 µM, but did not potentiate current activated by 100 µM ATP (results not shown). This calculated concentration of free Zn2+ is substantially greater than that produced by 5 µM Zn2+ in the presence of 5 µM ATP (4.4 µM), which produces marked potentiation of ATP-activated current. As shown in Fig. 7B, there was no difference in the percent potentiation by 5 µM Zn2+ of 5 µM ATP-activated current at membrane holding potentials between -80 and +20 mV (ANOVA, P > 0.1, n = 4). Furthermore, Zn2+ did not change the reversal potential of ATP-activated current (Student's t-test, P > 0.1, n = 4).



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Fig. 7. Investigation of the mechanism of Zn2+ potentiation of ATP-activated current mediated by P2X4 receptors. A: concentration-response for ATP-activated current in the absence (open circle ) and the presence () of 5 µM Zn2+. Amplitude is normalized to the current activated by 300 µM ATP in the absence of Zn2+. Each point is the average of 5-8 cells. The curves shown are the best fit of the data to the equation given in METHODS. Zn2+ significantly decreased the EC50 value for ATP from 6.7 ± 1.3 µM in the absence of Zn2+ to 2.8 ± 0.2 µM in the presence of 5 µM Zn2+ (ANOVA, P < 0.01). B: current-voltage relationship for currents activated by 5 µM ATP in the absence (open circle ) and the presence () of 5 µM Zn2+ at membrane potentials between -80 and +20 mV in a single oocyte. The average reversal potential of current activated by 5 µM ATP was -0 ± 4 mV in the absence and 1 ± 5 mV in the presence of 5 µM Zn2+; these values are not significantly different (Student's t-test, P > 0.1, n = 4). In addition, the percentage potentiation of ATP-activated current by Zn2+ was not significantly different at holding potentials from -80 to +20 mV (ANOVA, P > 0.1, n = 4). Membrane potential was held at each value for 1 min before application of ATP.

To evaluate a possible interaction of Cu2+ with the Zn2+ site on the P2X4 subunit, we examined whether Cu2+ could affect Zn2+ potentiation of ATP-activated current. As shown in Fig. 8A, Cu2+ did not alter either ATP-activated current or Zn2+ potentiation of ATP-activated current. The average potentiation of ATP-activated current produced by 10 µM Zn2+ was 211 ± 8% of control in the absence of Cu2+ and 212 ± 9% of control in the presence of 10 µM Cu2+; these values are not significantly different (Student's t-test, P > 0.5, n = 5; Fig. 8B).



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Fig. 8. Effect of Cu2+ on Zn2+ potentiation of ATP-activated current in P2X4 receptors. A: records illustrating similar enhancement by 10 µM Zn2+ of currents activated by 5 µM ATP in the absence and the presence of 10 µM Cu2+. Records are sequential current traces (from left to right) obtained from a single oocyte. B: bar graph illustrating the average potentiation of 5 µM ATP-activated current by 10 µM Zn2+ in the absence and the presence of 10 µM Cu2+. Cu2+ did not alter the average potentiation of ATP-activated current produced by Zn2+ (Student's t-test, P > 0.5; n = 5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Both Cu2+ and Zn2+ may be involved in the modulation of CNS function, as both ions were demonstrated to be widely distributed in brain (Barden 1971; Frederickson 1989; Kozma et al. 1981; Szerdahelyi and Kása 1986) and can be released on stimulation (Assaf and Chung 1984; Kardos et al. 1989). Previous studies revealed that these ions could produce similar modulation of the function of P2X receptors. For instance, micromolar concentrations of Cu2+ and Zn2+ potentiate ATP-activated current in rat nodose ganglion neurons (Li et al. 1993, 1996a). By contrast, micromolar concentrations of Cu2+ and Zn2+ inhibit ATP-activated current mediated by P2X7 receptors (Virginio et al. 1997). However, this study provides evidence that Cu2+ and Zn2+ differentially regulate the function of P2X2 and P2X4 receptors.

Low micromolar concentrations of Zn2+ were previously reported to potentiate ATP-activated current mediated by P2X2 receptors by increasing the apparent agonist affinity (Brake et al. 1994; Wildman et al. 1998). In the current study on P2X2 receptors, Zn2+ potentiated the current activated by 50 µM ATP with an EC50 value of 19.6 µM. Similarly, Cu2+ markedly potentiated current activated by 50 µM ATP in P2X2 receptors, with an EC50 value of 16.3 µM, which did not differ significantly from the EC50 value for Zn2+. Cu2+ shifted the ATP concentration-response curve to the left in a parallel manner, decreasing the EC50 value for ATP, as was found for Zn2+. These results suggest that Cu2+ and Zn2+ may facilitate the function of P2X2 receptors via a common mechanism, perhaps through a common binding site. If this is the case, when this site is saturated by Zn2+, Cu2+ should not be able to further enhance the function of the receptor, as was found in this study. The amplitude of ATP-activated current in the presence of a maximally effective concentration of Zn2+ was not increased further by addition of Cu2+. This was not due to a "ceiling effect," that is, the ATP-gated receptors were not already maximally activated in the presence of Zn2+ because 300 µM ATP activated a current of substantially greater amplitude. Thus Cu2+ and Zn2+ most probably act on a common site on the ATP-gated, receptor-channel complex. The location of the Cu2+-Zn2+ site cannot be precisely identified at present, but results of current-voltage experiments suggest that this site is beyond the influence of the membrane electrical field (Woodhull 1973) because the effects of both ions were not voltage dependent between -80 and +20 mV.

To date, P2X4 receptors were cloned from rat brain (Bo et al. 1995; Séguéla et al. 1996; Soto et al. 1996), rat superior cervical ganglion (Buell et al. 1996b), and human brain (Garcia-Guzman et al. 1997), and Zn2+ potentiation of ATP-activated current was reported for P2X4 receptors isolated from rat brain (Séguéla et al. 1996; Soto et al. 1996) and human brain (Garcia-Guzman et al. 1997). In this study, in oocytes expressing P2X4 receptors cloned from rat superior cervical ganglion (Buell et al. 1996b), low micromolar concentrations of Zn2+ potentiated ATP-activated current. Like P2X2 receptors, Zn2+ enhanced ATP receptor function by producing a parallel leftward shift in the ATP concentration-response curve. These results are consistent with previous studies in which Zn2+ was shown to induce a leftward shift of the concentration-response curve for ATP in P2X4 receptors isolated from human brain (Garcia-Guzman et al. 1997). In addition, the effect of Zn2+ on P2X4 receptors was not voltage dependent, suggesting that its site of action is not influenced by the membrane electrical field. In contrast to the effect of Zn2+ on P2X4 receptors, 0.5-50 µM Cu2+ did not significantly affect ATP-activated current in oocytes expressing P2X4 receptors. Furthermore, Cu2+ did not alter Zn2+ potentiation of ATP-activated current, suggesting that it does not interact with the Zn2+ site over this concentration range.

Cu2+ and Zn2+ at low micromolar concentrations were previously reported to differentially modulate glycine receptors in rat olfactory bulb neurons (Trombley and Shepherd 1996). The effects of Cu2+ and Zn2+ on glycine receptors are dependent on the state of the receptor; both Cu2+ and Zn2+ had no effect on the desensitized component of the current evoked by high concentrations of glycine, but Zn2+ dramatically potentiated and Cu2+ inhibited the current activated by nondesensitizing concentrations of glycine. Similarly, the results of this study suggest important differences in the modulatory sites of Cu2+ and Zn2+ on the P2X2 and P2X4 subunits. The observation that Cu2+ and Zn2+ interact with the site on the P2X2 receptor with similar affinity may indicate that the dimensions of this site, or the dimensions of the path of access to the site, are sufficient to accommodate both ions. The lack of effect of Cu2+ on the P2X4 subunit may thus indicate that the dimensions or path of access to the site are not sufficiently large to accommodate the larger Cu2+ ion. An alternative possibility is that on the P2X2 subunit there are separate sites for Cu2+ and Zn2+ but that both sites affect receptor function via a common mechanism (e.g., binding of either ion to its site produces the same conformational change in the receptor, increasing its affinity for ATP). If this is the case, then the inability of Cu2+ to enhance ATP-activated current mediated by P2X4 receptors may be due to the absence of the Cu2+ site on this subunit. Future studies may be able to distinguish between these two alternatives.


    ACKNOWLEDGMENTS

We thank Dr. Gary Buell for providing the cDNA for the P2X subunits.


    FOOTNOTES

Present address and address for reprint requests: C. Li, Dept. of Cell Biology, Astra Arcus USA, Inc., Three Biotech, One Innovation Drive, Worcester, MA 01605.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 18 October 1998; accepted in final form 29 January 1999.


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ABSTRACT
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society