Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892-8115
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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 M. 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
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RESULTS |
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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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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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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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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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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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We thank Dr. Gary Buell for providing the cDNA for the P2X subunits.
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FOOTNOTES |
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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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REFERENCES |
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