 |
INTRODUCTION |
ATP plays a role of cotransmitter of noradrenaline, acetylcholine, and substance P in prostaglandinic sympathetic, postganglionic parasympathetic, and sensory neurons, respectively (Burnstock 1986
; von Kügelgen and Starke 1991
). Released ATP interacts with postsynaptic P2 purinoceptors, of which various subtypes have been recognized (Dalziel and Westfall 1994
; Fredholm et al. 1994
; Kennedy and Leff 1995
). Among these, P2X, P2Y, and P2U (Webb et al. 1993
) receptors have been cloned. P2X receptors are thereby further classified as P2X1 (Valera et al. 1994
, 1995
), P2X2 (Brake et al. 1994
; Lewis et al. 1995
), P2X2-1 (Housley et al. 1995
), P2X3 (Chen et al. 1995
; Lewis et al. 1995
), P2X4 (Bo et al. 1995
; Buell et al. 1996
), P2X5 (Collo et al. 1996
), and P2X6 (Collo et al. 1996
). The P2Y receptor is also subclassified into P2Y1 through P2Y7 (Abbracchio and Burnstock 1994
). More recently, the P2Z receptor has been cloned as the P2X7 receptor (Surprenant et al. 1996
).
We reported previously that ATP induces a nonselective cation current and increases intracellular Ca2+ concentration in NG108-15 cells via P2Z receptors (Kaiho et al. 1996
). During the course of the experiment, we observed an increase in the ATP-induced current when external Cl
was replaced with methanesulfonic acid (MS
). In the present study we investigated the effects of various external anions on the ATP-induced nonselective cation current with the patch-clamp technique in NG108-15 cells.
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METHODS |
Cell culture
Neuroblastoma × glioma hybrid NG108-15 cells were grown in high-glucose Dulbecco's modified Eagle's medium supplemented with 7% fetal bovine serum, 100 mM hypoxanthine, 0.4 mM aminopterin, and 16 mM thimidine. Cells were grown in 12-well plates in a humidified atmosphere of 10% CO2-90% air at 37°C.
Solutions
The external control solution was a Tyrode solution containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 0.33 NaH2PO4,5 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES),and 5.5 D-glucose, pH 7.4. In F
, Br
, or I
solution, 140 mM of each anion (Na+ salt) was used instead of NaCl in Tyrode solution and KCl, CaCl2, MgCl2, and D-glucose were omitted. To replace Cl
with aspartic acid (Asp
) and MS
, each anion was mixed with 140 mM NaOH and pH was adjusted to 7.4 with Asp
or MS
, respectively. The final concentrations of Asp
and MS
were 133.7 and 132.6 mM, respectively. To reduce Cl
concentration in the experiments shown in Figs. 2 and 3, Cl
was replaced by either MS
or Asp
, and solutions of various extracellular Na+ anion concentration ([Na+ anion]o) were made up by replacing Na+ anion with the equiosmolar concentration of D-mannitol. The pipette solution contained (in mM) 110 CsOH, 30 CsCl, 75 Asp
, 5 ATP (Mg2+ salt), 5 potassium creatine phosphate, 3 MgCl2, 10 ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid, and 20 HEPES, pH 7.2.

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| FIG. 2.
Effects of various levels ofextracellular sodium methanesulfonateconcentration ([NaMS]o) on the current. Representative I-V curves from same cell. External Cl , Ca2+, and Mg2+ were absent. Mannitol was added to keep osmolarity constant. a, c, e, and g: controls before application of ATP. b, d, f, and h: peak steady-state response in presence of 125 µM ATP. I-V curves are shown without LJP compensation.
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| FIG. 3.
Effect of [Cl ]o on current induced by 125 µM ATP at different levels of [Na+]o. A: concentration-response curves for [Na+]o and current density at various levels of [Cl ]o. Current density was calculated at 50 mV. B: double reciprocal plot of A. Values in parentheses: number of data.
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Membrane current recording
Single NG108-15 cells were dispersed in a bath on the stage of an inverted microscope (Nikon), and were continuously perfused with the external solution. The temperature of the external solution was kept constant at 36 ± 1°C (mean ± SE). Currents were recorded by a patch-clamp amplifier (EPC-7, List Electronic, Darmstadt, Germany) in the whole cell mode (Hamill et al. 1981
). Briefly, a gigaohm seal was formed with a patch pipette having a tip diameter of 1-1.5 µm and an input resistance of 2-4 M
. The current-voltage (I-V) relation was obtained with the use of ramp pulses from the holding potential of
10 mV to 60 mV, then to
110 mV and back to the holding potential at a speed of ~0.7 V/s with a function generator (FG-121B, NF, Yokohama, Japan) (for details see Miura and Kimura 1989
). The ramp pulses were applied at a 3-s interval. The current density was calculated by dividing the current amplitude by the membrane capacitance of the cell. The membrane capacitance was calculated from the capacitative current, which was a half-value of the difference between the descending limb and ascending limb currents in response to ramp pulses. The data were acquired on-line with the use of a NEC PC-9801RX computer. The liquid junction potentials (LJPs) between the 140 mM KCl-indifferent electrode and the bath solution were measured by filling a patch pipette with 3 M KCl, adjusting the zero current voltage to 0 mV in Tyrode solution under the current-clamp mode, and then measuring the potential in all the anionic solutions tested, including the partial replacement of anions. LJP was
10.7 mV in Asp
,
7.9 mV in MS
,
4.2 mV in F
,
0.2 mV in Br
, 0 mV in Cl
, and 0.1 mV in I
solution with the total replacement. All I-V curves shown in the figures are without LJP compensation. However, all the reversal potentials (Erevs) of the current described in the text are after LJP compensation.
Calculation of maximum current density and median effective concentration
Each set of data of the concentration-response relation in different anions (Fig. 5) was fitted by the following equation with the Hill coefficient of 2 with the use of a nonlinear Marquardt method by a computer program (Kotaro, Sankaido, Tokyo)
where i is current density, Imax is maximum current density, n is the Hill coefficient, EC50 is the half-maximum value of ATP, and [ATP] is the concentration of ATP. In the Marquardt methods, the data fitting depends on the initial values inserted, and does not necessarily provide satisfactory results. Thus we first ran the program with roughly estimated values for Imax and EC50 and, with the use of the values obtained from the first run, the second run was made. With these dual trials, the correlation coefficients were improved to be 0.87-0.99. We adopted the values obtained from the second run as final data (Table 1).

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| FIG. 5.
Concentration-response curves between ATP and net ATP-induced current density. Each current was calculated at 50 mV in various anionic solutions as indicated. Mean values of current densities are plotted. Values in parentheses: number of data. Curves were fitted by the Marquardt method with a Hill coefficient of 2. Maximum current densities in aspartic acid (Asp ), MS , and F are not significantly different, nor are those in Br and I . Single asterisk: P > 0.05. Double asterisk: P < 0.05.
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Drugs
The following chemicals were used: high-glucose Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY); fetal bovine serum (Moregate, Melbourne, Australia); hypoxanthine, aminopterin, thyimidine, ATP, DL-aspartic acid, MS
, D-mannitol, and sodium salts of F
, Cl
, Br
, and I
(Wako Pure Chemicals, Tokyo); and potassium creatine phosphate (Calbiochem, La Jolla, CA). All the chemicals and drugs were of reagent grade or the highest quality available.
Statistics
The numerical values of the data were described as the mean ±SE followed by the number of data in parentheses. Statistical significance of the values was evaluated by Student's t-test.
 |
RESULTS |
Comparison of the ATP-induced current in Cl
and MS
In our previous study, we identified that the ATP-induced current was a nonselective cation current in NG108-15 cells (Kaiho et al. 1996
). To check whether or not anions can also carry the current, we compared the ATP-induced currents in Cl
and MS
, a larger anion than Cl
, in the external solution in the presence of Ca2+ and Mg2+. In the whole cell clamp mode, ATP at 1 mM induced a nonselective cation current that showed an inwardly rectifying property with a mean Erev of 12.5 ± 0.8 mV (n = 10) in Cl
solution (Fig. 1). In the same cell, the external Cl
was totally changed to MS
and 1 mM ATP was applied again. The nonselective cation current was again activated, and crossed with the control at 18.2 ± 1.4 mV (n = 9). This Erev value as well as all the following Erev values are after LJP compensation, whereas the I-V curves, including that in Fig. 1, are shown without LJP compensation. The current magnitude was larger but the inward rectification was less prominent in MS
than that in Cl
solution (Fig. 1). To examine the effect of MS
more systematically, we tested ATP (125 µM) at four different MS
concentrations by replacing Cl
with MS
at 140 mM Na+ under Ca2+- and Mg2+-free conditions. The magnitude of the current became larger as [[Cl
]o was progressively lowered and MS
was elevated in place of Cl
(not shown). Erevs of the current were13.5 ± 0.6 mV (n = 8), 9.8 ± 0.7 mV (n = 7), 12.9 ± 0.3 mV (n = 8), and 11.8 ± 0.9 mV (n = 5) at 0, 35, 70, and 140 mM Cl
, respectively (or at 140, 105, 70, and 0 mM MS
, respectively), in Ca2+- and Mg2+-free solutions. All the values are after LJP compensation. Thus there is no significant difference in Erev at different concentrations of Cl
. This result indicates that there is no difference in Cl
and MS
permeability, even if the channels are permeable to these anions.

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| FIG. 1.
Comparison of ATP-induced membrane currents in Cl and methanesulfonic acid (MS ) external solutions. External Ca2+ and Mg2+ were present. Holding potential: 10 mV; ramp pulses were given every 3 s. Top: current recording. Cell was exposed to 1 mM ATP twice for periods indicated by bars above current trace. Bottom: current-voltage (I-V) relations obtained at each control (a and c) and maximum response (b and d) during the 2 ATP applications (left, in Cl solution; right, in MS solution). I-V curves are shown without liquid junction potential (LJP) compensation.
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We performed another set of experiments to measure Erev at different anion concentrations to check the anionic permeability. The external concentration of sodium methanesulfonate (NaMS) was varied from 140 mM to 105, 70, and 35 mM while the total osmolarity was kept constant by adding mannitol, an uncharged molecule. The external solution did not contain Ca2+ and Mg2+. Under these conditions, if cation was a major current carrier, Erev should shift in the negative direction as the external cation progressively decreased. By contrast, if anion carried the current, Erev should shift in the positive direction. As shown in Fig. 2, as the NaMS concentration decreased, the current induced by 125 µM ATP became progressively smaller and Erev shifted in the negative direction. The average Erevs were
12.4 ± 1.8 mV (n = 7), 1.4 ± 1.2 mV (n = 6), 6.9 ± 0.3 mV (n = 8), and 12.4 ± 0.6 mV (n = 14) at 35, 70, 105, and 140 mM NaMS, respectively, after LJP compensation. The plot of Erev against the Na+ concentration (not shown) produced a line that could be fitted by the equation Erev = 41 log(PNa[Na+]o/PCs[Cs+]i), where PNa and PCs are the permeability of Na+ and Cs+, respectively. Because the slope of 41 was significantly smaller than 61, the value predicted from an ionic pathway purely selective for cations, this result indicates that the major carrier of the ATP-induced current is cations, but anions may also carry the current as a minor component.
Relation between external Cl
and Na+
We next examined whether external Cl
interacts with external Na+ that passes through the ATP-activated cation channels. We obtained the concentration-response relation between [Na+]o and the ATP-induced current density at two different levels of [Cl
]o (0 and 35 mM) to check EC50 of external Na+ and the Imax values. With the use of the same protocol as described above, [Na+ anion]o (Asp
or MS
+ 35 mM Cl
) was reduced from 140 mM to 105, 70, and to 35 mM by replacing the rest of the Na+ anion equiosmotically with 70, 140, and 210 mM mannitol, respectively. ATP (125 µM) was applied to induce the current at every [Na+ anion]o in the Ca2+- and Mg2+-free external solution. The concentration-response curves were plotted by measuring the current magnitude at
50 mV from the data represented in Fig. 2 (in MS
solution, 0 mM Cl
) and those obtained in Asp
(0 mM Cl
) and in MS
(35 mM Cl
) solution (Fig. 3A). From these concentration-response curves the double reciprocal plot (Lineweaver-Burk plot) was performed (Fig. 3B). All three lines of the reciprocal plot crossed each other on the X-axis. This result indicates that external Cl
is a noncompetitive inhibitor with respect to external Na+ in the ATP-activated nonselective cation channel in NG108-15 cells.
Effects of various anions on ATP-induced current
To further examine the effects of anions on the ATP-activated current, we tested various anions in the external solution by replacing 140 mM Cl
with Asp
, MS
, F
, Br
, and I
. It is possible that the anions might affect the interaction between ATP and divalent cations such as Mg2+ and Ca2+, and thereby change the ATP4
concentration, which is the most effective form of ATP for stimulating the P2Z receptor (Kaiho et al. 1996
). Thus Ca2+ and Mg2+ were omitted from the external solution to check this possibility. The representative I-V relations in various anionic solutions are shown in Fig. 4. The control currents in the absence of ATP are not significantly different among the different anions. Even in the absence of Mg2+ and Ca2+, the magnitude of the ATP-induced current varied considerably in the different anions. This result indicates that external anions affected the ATP-induced current not by changing the interaction between the divalent cations and ATP4
. The average Erevs of the ATP-induced currents at 125 µM ATP in Ca2+- and Mg2+-free external solutions were 11.2 ± 0.3 mV (n = 6) in Asp
, 12.4 ± 0.6 mV (n = 7) in MS
, 13.4 ± 0.8 mV (n = 3) in F
, 10.5 ± 0.9 mV (n = 5) in Cl
, 21 ± 0.8 mV (n = 3) in Br
, and 5.6 ± 2.2 mV (n = 3) in I
.

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| FIG. 4.
Effects of various anions on ATP-induced current. Representative I-V curves in various anions. a, c, e, g, i, and k: controls before addition of ATP at 250 or 500 µM. b, d, f, h, j, and l: peak steady-state responses in the presence of ATP. External Ca2+ and Mg2+ were absent. I-V curves are shown without LJP compensation.
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We then varied ATP concentrations to obtain the concentration-response curves for ATP in each anionic solution under the Ca2+- and Mg2+-free conditions. The current density was calculated by measuring the magnitude of the net ATP-induced current at
50 mV and dividing the value with the capacitance of each cell. The concentration-response relation is plotted in Fig. 5. When the Hill plot was performed for the data in each anion, the Hill coefficient was ~2 for all the cases. The Imax and EC50 values obtained by fitting the data as described in METHODS are summarized in Table 1. Both Imax and EC50 values were affected by anions, indicating that anions are a mixed-type inhibitor with respectto ATP.
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DISCUSSION |
We found that the magnitude of the ATP-induced current varied in different external anionic solutions. There are the following possibilities that anions affect the ATP-induced current. 1) Anions pass through the ATP-activated ionic channel and this current component is large enough to affect overall amplitude of the ATP-activated current. 2) Anions influence the reaction between ATP and divalent cations such as Mg2+ and Ca2+, and change the concentration of ATP4
, which is the most effective form of ATP for stimulating the P2Z receptor. 3) Anions alter the activity of external cations that carries the ATP-induced current. 4) Anions interfere with the binding of ATP4
to the receptor by competing with this negatively charged agonist or through an allosteric mechanism.
For the first possibility, Thomas and Hume (1990)
demonstrated a class of ATP-activated channels permeable to both cation and anion in the developing skeletal muscle. In our cells, cation is a major carrier of the ATP-induced current and anions appear to be a minor component, if permeable at all, from the results shown in Fig. 2. Erev in Ca2+- and Mg2+-containing solution was 12.5 ± 0.8 mV in Cl
, whereas it was 18.2 ± 1.4 mV in MS
solution. These values are statistically significant. In the absence of Ca2+ and Mg2+, however, Erevs of 10.5 ± 0.9 mV in Cl
and 12.4 ± 0.6 mV in MS
are not significantly different. Therefore anion permeability may not be large enough to affect overall ATP-activated current and thus may not be the cause of the difference in the current magnitude. Erevs are more positive in the presence of Ca2+ and Mg2+ than in the absence of the divalent cations. This effect of the divalent cations can be explained by their influence on surface charge or their permeation through the channel in addition to monovalent cations (Kaiho et al. 1996
).
The second possibility is unlikely, because the effect was obtained both in Mg2+- and Ca2+-containing and -free solutions. For the third possibility, if the anions alter the activity of cations in the external solution, Erev should be changed. Erev of the current showed minor shifts in the various anions in the following order: I
(5.6 ± 2.2 mV) < Cl
(10.5 ± 0.9 mV) < Asp
(11.2 ± 0.3 mV) < MS
(12.4 ± 0.6mV) < F
(13.4 ± 0.8 mV) < Br
(21 ± 0.8 mV). Statistical significance was seen only for Erevs between Br
and I
, but not for those among Cl
, Asp
, MS
, and F
. This order does not correspond to that of Imax. Thus the change in the external cationic activity, if it occurs, cannot account for the change in the current magnitude observed.
To investigate the fourth possibility, we analyzed the concentration-response curves for ATP and the current in different anions. As shown in Fig. 5 and Table 1, both the maximum current and the EC50 values were varied by the different anions. This result indicates that anions act as a mixed-type inhibitor with respect to ATP. The sequence of anions corresponding to the magnitude of Imax was Asp
>MS
> F
> Cl
> Br
I
. The EC50 value of ATP was shifted to the right in the order of Asp
< F
<Ms
< Br
< Cl
< I
. The shift of the EC50 values by different anions indicates that anions compete with ATP in the P2Z receptor, or that an allosteric modulation by anions affects the binding of ATP. The decrease in Imax by the anions may indicate that there is an additional noncompetitive site for the anions in the P2Z receptor to diminish the current magnitude. The sequence of anions for the maximum current and that for the EC50 values are slightly different, which may also support this view.
The molecular weight of the anions used was Asp
(133.1) > I
(126.9) > MS
(96.1) > Br
(79.9) > Cl
(35.5) > F
(19.0). The ionic radius of halides is I
(2.16 Å) > Br
(1.95 Å) > Cl
(1.81 Å) > F
(1.36 Å) (Araki et al. 1961
). These sequences are similar, but do not precisely correspond to either of those for the current magnitude or for the EC50 values. In any case, the inhibitory actions of the anions tend to decrease with a large molecular weight or ionic radius. Large anions like Asp
and MS
may not fit to the site where smaller halide ions bind and block the effect of ATP.
The sequence of the halide anions for Imax was F
>Cl
> I
Br
, and that for the EC50 values was F
< Br
< Cl
< I
. These sequences are similar to that of hydration energies of the anions, which are F
(121 kcal/mol) > Cl
(90 kcal/mol) > Br
(82 kcal/mol) > I
(71 kcal/mol) (Bittar 1970
). Thus the order of inhibition of the halides can be explained in terms of hydration energies of each ion. Ions are hydrated in the solution and the hydration shell must be removed before binding to the site for the inhibition.
We also examined the effect of external Cl
on external Na+ passing through the channel by constructing the concentration-response curves for [Na+]o and the current at two different Cl
concentrations, i.e., 35 and 0 mM (Fig. 3A). External Cl
was replaced with Asp
or MS
. The three lines of the double reciprocal plots of the concentration-response curves cross each other on the X-axis (Fig. 3B). Therefore external Cl
is a noncompetitive inhibitor with respect to external Na+ in the ATP-induced cation channel. The anions may not compete with Na+ permeating through the ATP-activated channels.
All the concentration-response curves in Fig. 5 were fitted well with the Hill coefficient of 2. The lack of changes in the Hill coefficient indicates that the anions do not affect factors that may contribute to the slope of the concentration-response relationship, such as the cooperativity of the purinoceptors or the number of ATP molecules required for the channel activation. Akaike et al. (1989)
found that replacement of external Cl
to Br
shifted the EC50 value of
-aminobutyric acid to the right in the
-aminobutyric-acid-activated anionic current in bullfrog sensory neurons. This shift is similar to our result. Thus there is a possibility that the receptors for negatively charged ligands might be more or less affected by external anions.
There have been several reports that ATP activates Cl
channels in aortic endothelial cells (Hosoki and Iijima 1994
) and ventricular myocytes (Kaneda et al. 1994
). These Cl
channels are known to be blocked by N-phenylanthranilic acid (Mandel et al. 1986
; Ueda et al. 1990
) or 4,4
-diisothiocyanatostilbene-2,2
-disulphonic acid (de Lisle and Hopfer 1986
). We studied whether the Cl
channel antagonists affect ATP-induced nonselective cation current. N-phenylanthranilic acid and anthracene-9-carboxylic acid, another Cl
channel antagonist (Cuppoletti and Sachs 1984
), at 1 mM did not affect the ATP-induced current (not shown). Thus the results in this report may not be related to the ATP-activated Cl
channels.
In conclusion, the ATP-induced nonselective cation current associated with P2Z purinoceptors is inhibited by various external anions in NG108-15 cells. The inhibition may at least partly be explained by the reduction by the anions of the affinity of ATP to the receptors. The order of the blocking potency was similar to that of the molecular size and the hydration energy of the anions. On the basis of our results, it is speculated that the ATP binding to the P2Z receptors is inhibited by Cl
ions approximately by half under physiological conditions. However, we do not know whether this inhibition by Cl
has any physiological role. Our results may give an important insight for considering the structure of the P2Z receptor. Whether this anionic effect is also true for the other types of P2 receptors requires further investigation.