Partial agonists and antagonists reveal a second permeability
state of human lymphocyte P2Z/P2X7
channel
J. S.
Wiley1,
C. E.
Gargett2,
W.
Zhang1,
M. B.
Snook2, and
G. P.
Jamieson2
1 Sydney University Department
of Medicine, Nepean Hospital, Penrith, New South Wales 2750; and
2 Department of Haematology,
Austin and Repatriation Medical Centre, Heidelberg, Victoria 3084, Australia
 |
ABSTRACT |
Extracellular ATP is known to trigger apoptosis of thymocytes
and lymphocytes through a P2Z receptor at which ATP is a partial agonist, giving only 70% of the maximum response of
3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (BzATP), a full agonist. This cytolytic receptor and its associated ion channel are
Ca2+ (and
Ba2+) selective but also pass
molecules up to the size of ethidium cation (314 Da).
RT-PCR showed identity between lymphocyte P2Z and the
hP2X7 gene recently cloned from
human monocytes. When human leukemic B lymphocytes were incubated with
ATP and
133Ba2+,
an immediate influx of isotope occurred. It was augmented by 45% when
ATP was added 10 min before isotope. Time-resolved flow cytometry was
used to examine kinetics of ethidium uptake in cells incubated with
BzATP or the partial agonists ATP, 2-methylthioadenosine 5'-triphosphate, or adenosine
5'-O-(3-thiotriphosphate).
Maximally effective concentrations of BzATP (50 µM) induced immediate
uptake of ethidium at a rate linear with time. In contrast, a delay was observed (30 s) before ethidium uptake commenced after addition of
maximally effective ATP concentrations (500 µM) at 37°C, and the
delay was longer at 24°C. ATP addition 2-10 min before
ethidium abolished the delay. The delay was longer with other partial
agonists and inversely related to maximal flux produced by agonist. A
delay was also observed for submaximal BzATP concentrations (10-20
µM). P2Z/P2X7 inhibitors, KN-62
and
5-(N,N-hexamethylene)-amiloride, reduced the rate of agonist-induced ethidium uptake and lengthened the
delay. The results support a model in which agonists for
P2Z/P2X7 receptor mediate an
immediate channel opening allowing passage of small inorganic cations,
followed by a slow further permeability increase allowing passage of
larger permeant cations like ethidium. The rate of the second step
depends on time and temperature and the efficacy and concentration of
agonist and is slowed by antagonists, suggesting it depends on the
fraction of P2Z/P2X7 channels held in the initial open
state.
extracellular adenosine 5'-triphosphate; chronic lymphocytic
leukemia; B lymphocytes
 |
INTRODUCTION |
EXTRACELLULAR ATP MEDIATES many biological responses
ranging from smooth muscle contraction to synaptic transmission in the autonomic and central nervous systems (4, 6). The biological effects of
ATP are less well defined in hemopoietic cells, although extracellular
ATP has been shown to stimulate granule exocytosis from mast cells and
to induce apoptosis in thymocytes (5, 41). These diverse actions of ATP
are mediated by the interactions of this nucleotide with P2 purinergic
receptors, which can be broadly classified into those coupled via G
proteins to phospholipase C (the P2Y class) and those for which the
receptor is a ligand-gated ion channel (the P2X and P2Z classes). Many
studies of the P2Z receptor subtype have been in hemopoietic cells such
as macrophages and mast cells, which coexpress P2Z and either
P2Y2 or
P2Y1 receptors (1, 6, 8, 14,
31). To distinguish between effects mediated by these two
P2 receptors, an agonist specific for the P2Z subtype such as
3'-O-(4-benzoyl)benzoyl-adenosine
5'-triphosphate (BzATP) is generally used, although this agonist
is a weak agonist for P2Y2
receptors (2) and a partial agonist for some P2X receptors (31).
Activation of the P2Z receptor of human lymphocytes produces multiple
membrane effects, including ionic fluxes, stimulation of phospholipase
D, and activation of a membrane protease. In both normal and leukemic
lymphocytes, the P2Z purinoceptor is part of or closely coupled to an
ionic channel that shows strong selectivity for the divalent cations
Ca2+ and
Ba2+, and, because divalent cation
fluxes are enhanced in KCl or other Na+-free media, it is likely that
Na+ exerts a weak inhibitory
effect on the channel as well as being a permeant ion (3, 23, 26,
38-40). Anion permeability of this channel has been reported to be
negligible (22). The P2Z purinoceptor shows an absolute specificity for
the ATP4
species, since
addition of Mg2+ in molar excess
over nucleotide leads to closure of the channel in <1 s (39). Suramin
is the best known inhibitor of P2 receptors but is only weakly active
against the P2Z receptor, with an
IC50 value of 60 µM (39). In
contrast, isoquinoline sulfonamides such as KN-62 are potent inhibitors
of the human P2Z receptor, with an
IC50 of 13 nM (11), whereas
amiloride analogs such as 5-(N, N-hexamethylene)-amiloride
(HMA) have intermediate antagonist potency (40).
Recently the genes for both the rat and human P2Z receptors were cloned
and expressed in HEK-293 cells (27, 32). Both have homology to the six
described members of the P2X family of receptors, and it is proposed
that P2Z be termed P2X7. All
members of this family have protein structures with two putative
transmembrane segments linked by a long extracellular loop and
intracellular NH2- and
COOH-termini. However, the number of
P2Z/P2X7 monomers required to form
an oligomeric channel through the membrane is unknown. The
P2Z/P2X7 receptor has a long
COOH-terminal tail that confers the property of permeation by large
fluorescent cationic dyes as well as smaller cations through this
channel (27, 32). A recent study of
Xenopus oocytes injected with
macrophage mRNA and stimulated with BzATP showed two conductance
states. There was a small, rapidly activated inward current carried by
monovalent cations, which was followed 1 min later by a large current
carried by organic cations (N-methyl-D-glucamine
or Tris) in addition to the monovalent cations (25). The detailed
kinetics of this transition have not been examined.
Cells often coexpress several P2 receptor subtypes, so multiple
pathways may be available for ATP-stimulated fluxes. However, competitive experiments with BzATP and ATP in lymphocytes from patients
with chronic lymphocytic leukemia show that the permeability responses
are dominated by the P2Z/P2X7 receptor (10). At this receptor, the natural ligand ATP is a partial agonist, producing only
70% of the maximal response in ethidium cation influx observed for the
full agonist BzATP. Several other ATP analogs such as 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) and adenosine 5'-O-(3-thiotriphosphate)
(ATP
S) are partial agonists that stimulate P2Z-mediated responses in
lymphocytes but with even lower maximal activities than BzATP or ATP,
such that the rank order of agonist efficacy is BzATP > ATP > 2-MeS-ATP > ATP
S (10). In this study, the increase in permeability
of the P2Z channel was measured using time-resolved flow cytometry with
ethidium as the permeant ion and compared with the uptake of isotopic
133Ba2+.
The results suggest that partial agonists, including ATP, produce a
biphasic opening of the P2Z channel for which the second increment in
permeability develops slowly over 0.5-1.5 min.
 |
METHODS |
Materials.
Ficoll-Paque (density 1.077) was obtained from Pharmacia (Uppsala,
Sweden). ATP, BzATP, ethidium bromide,
BaCl2, phorbol 12-myristate 13-acetate, and BSA were from Sigma Chemical (St. Louis, MO). The
amiloride analog HMA and KN-62
{1-[N,O-bis(5-isoquinoline sulfonyl)N-methyl-L-tyrosyl]-4-phenylpiperazine}
were from Research Biochemicals (Natick, MA), and
133Ba2+
(0.5 mCi/ml, 3 mCi/mg) was from DuPont (Boston, MA). HMA was kept
dessicated at room temperature in the dark, since traces of moisture
lead to loss of activity. Stock solutions of HMA and KN-62 were
prepared in dry DMSO. HMA solutions were prepared immediately before
use, whereas stocks of KN-62 were stored at 4°C for up to 1 mo.
Di-n-butyl phthalate and
di-iso-octyl phthalate were from
British Drug Houses (Poole, UK). HEPES was from Boehringer Mannheim
(Mannheim, Germany). Streptolysin O was from Wellcome Reagents.
YO-PRO-1 iodide was from Molecular Probes (Eugene, OR).
Source of cells.
Peripheral blood lymphocytes were obtained from six
patients with B-cell chronic lymphocytic leukemia whose
cells showed permeability responses in our previous studies
(38-40). Liver tissue was freshly dissected from diagnostic
samples taken intraoperatively, whereas peripheral blood monocytes were
obtained from peripheral blood drawn from normal subjects.
Lymphocyte preparation.
Venous blood from patients was diluted with an equal volume of
HEPES-buffered saline (pH 7.4; 10 mM HEPES, 0.1% BSA, 5 mM D-glucose, 145 mM NaCl, and 5 mM
KCl). Mononuclear cells were separated by density gradient
centrifugation over Ficoll-Paque and washed twice, and monocytes were
depleted by adhesion to plastic. Cytocentrifuge preparations showed
that >98% of cells were small mature lymphocytes. Immunophenotype
analysis of these lymphocytes showed 94 ± 4% B cells
(CD5+,
CD19+) and 3 ± 2% T cells
(CD3+).
Monocyte/macrophage preparation.
Mononuclear cells from normal subjects were prepared as above, and
monocytes were allowed to adhere to plastic flasks for 1 h at 37°C.
Nonadherent cells were then washed away, and adherent cells were
cultured for 7 days in RPMI 1640 medium with 20% FCS plus
interferon-
(100 U/ml).
RT-PCR analysis.
Total RNA was isolated from cells using RNA isolation reagent (Advanced
Biotechnologies) according to the manufacturer's recommendations. For
cDNA synthesis, 1 µg of total RNA was used in the reverse transcription reaction with 0.5 µg of primer oligo(dT) with 200 units
SUPERSCRIP II RT (Life Technologies), 25 units RNase inhibitor, 2 mM
dNTPs, 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM
MgCl2, and 10 mM dithiothreiotol
in a total volume of 29.5 µl. Primers for RT-PCR were designed from
the human P2X7 mRNA sequence in the area of the COOH-terminal tail for bp 1425-1780 (GenBank
accession no. Y09561), using the computer program PRIMER (version 0.5; S. E. Lincoln, M. J. Daly, and E. S. Lander, unpublished). The forward
primer was bp 1425-1446
(5'-ACTCCTAGATCCAGGGATAGCC-3'), and the reverse primer was
bp 1780-1759 (5'-TCACTCTTCGGAAACTCTTTCC-3'). For PCR
amplification, 30-120 ng of cDNA were used as template DNA in a
final volume of 25 µl, containing 0.75 units
Taq DNA polymerase, 2 mM
MgCl2, 100 µM dNTPs (dATP, dGTP,
dCTP, and dTTP), 1× Taq DNA
polymerase buffer, and 10 pmol of each human
P2X7
(hP2X7) primer. The reaction
solution above was overlaid with one drop of mineral oil, and the PCR
was carried out with a thermocycling program as follows: initial
denaturation at 95°C for 5 min, and then 35 cycles of 95°C for
30 s, 55°C for 30 s, and 72°C for 1 min; the final extension
step was carried out at 72°C for 10 min. The RT-PCR products were
electrophoresed in a 2% agarose gel at 100 V for 1 h. The size of the
fragment was determined with a 100-bp DNA molecular weight ladder
track.
Uptake of
133Ba2+.
Ba2+ is a good surrogate for
Ca2+ and once inside the cell is
neither pumped nor sequestered by transport ATPases (39).
Ba2+ uptake was measured over 60 s
using
133BaCl2
(final concentration, 0.2 mM). At time
0, a prewarmed stock solution of
133Ba2+
(0.4 mM and 1 µCi/ml) was added in equal volumes to prewarmed lymphocytes (2.5 × 107 cells/ml, in 150 mM KCl with
HEPES, pH 7.4) at 37°C or 24°C. ATP (1 mM) was added either 10 min before or simultaneously with the
133Ba2+
isotope. Aliquots of 0.8 ml taken at time points between 0 and 60 s
were immediately mixed with 0.2 ml of ice-cold 50 mM
MgCl2 (in KCl-HEPES medium) that
had been previously layered over 250 µl of oil mixture
(di-n-butyl phthalate and
di-iso-octyl phthalate, 7:3 vol/vol)
and then centrifuged at 8,000 g for 30 s. The supernatants and the oil were aspirated, and the cell pellets
were counted in a Wallac Wizard 3 automatic gamma-counter. The uptake
was converted to picomoles per 107
cells using the relative specific activity of the
133Ba2+
stock.
Ethidium and YO-PRO-1 measurements by flow cytometry.
Flow cytometry was used to quantitate the uptake of ethidium bromide
(39). Cells were suspended at 106
cells/ml in medium containing 150 mM KCl buffered with 10 mM HEPES (pH
7.4), and either 1) the cells were
incubated for 10 min at 37 or 24°C with or without ATP (0.5 mM)
before addition of 25 µM ethidium or
2) ATP was added 30 s after
ethidium. Samples in which ethidium uptake was measured were kept
stirred and with the temperature controlled at 37°C (unless
otherwise stated) using a Time Zero module (Cytek, Fremont, CA).
Histograms (256 channels) of lymphocyte-associated fluorescence signals
were collected over consecutive 6-s intervals for 5 min using a Coulter
Elite flow cytometer (Coulter, Hialeah, FL) with argon laser excitation
at 488 nm. Fluorescent emission was collected using a 590-nm long-pass filter. The mean channel of fluorescence intensity was then calculated for each of the histograms collected for each of the 6-s intervals and
plotted against time. Uptake of YO-PRO-1 (5 µM) was
measured by an identical procedure.
Data presentation.
Data are expressed as means ± SE.
 |
RESULTS |
Lymphocyte P2Z is the hP2X7 receptor.
Total RNA from human lymphocytes from six different patients was
subjected to RT-PCR using primers specific for the long COOH-terminal tail of the hP2X7. Agarose gel
electrophoresis showed a single oligonucleotide product, which migrated
at the predicted 356-bp position (Fig.
1). Single bands at the
same position were also observed for RT-PCR products from RNA of
macrophages and liver, which also express
P2X7 (27, 32). One feature of
hP2X7 heterologously expressed in
HEK-293 cells is that it allows ATP-induced permeation of the
fluorescent dye YO-PRO-1 (375.5 Da) (27, 32). Human lymphocytes
incubated with ATP (500 µM) took up the divalent YO-PRO-1 cation at a
rate comparable to that for the smaller, monovalent ethidium cation
(data not shown).

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Fig. 1.
Agarose gel electrophoresis of RT-PCR products from liver, lymphocyte,
and macrophage cDNA using human
P2X7
(hP2X7) purinoceptor-specific
primers; 2% agarose gel electrophoresis reveals a single 356-bp
product following amplification by PCR using
hP2X7-specific primers and cDNA
from lymphocytes of 6 different patients
(lanes 1-4,
9, and
10), macrophages
(lanes 5 and
6), liver
(lanes 7 and
8), and
H2O
(lane 11). M, molecular weight markers
(100-bp ladder).
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ATP-induced influx of
133Ba2+.
The Ba2+ permeability of
lymphocytes at 37°C was compared immediately after ATP addition and
after a 10-min preincubation with ATP. Figure
2A shows
that the
133Ba2+
uptake into lymphocytes incubated with ATP commenced within 10 s and
continued throughout 60 s for each condition. The influx of
133Ba2+
was greater for cells preincubated with ATP than for cells to which
coaddition of ATP and
133Ba2+
was made at time 0. Under both
conditions, the basal
133Ba2+
influx was negligible. The
133Ba2+
influx under both conditions was also measured at 24°C (Fig. 2B), and again the ATP-induced
133Ba2+
influx was greater for cells preincubated with ATP than for cells to
which ATP and
133Ba2+
were added simultaneously. The relative difference between the two
conditions was greater at 24°C than at 37°C.

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Fig. 2.
Extracellular ATP-induced isotopic
Ba2+ influx into fresh leukemic
lymphocytes measured either at 37°C
(A) or at 24°C
(B). Cells were suspended at 1.2 × 107 cells/ml in
HEPES-buffered isotonic KCl medium with no added
Ca2+. Either ATP (1.0 mM) was
added to suspension 10 min before
133Ba2+
(0.2 mM) or ATP and
133Ba2+
were added simultaneously at time 0.
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ATP-induced uptake of ethidium measured by flow cytometry.
We have previously shown that ethidium is a large organic permeant ion
for the lymphocyte
P2Z/P2X7-operated ion channel and that uptake of this cation can be readily followed by time-resolved flow cytometry (10, 39). Lymphocytes were incubated at 37 and 24°C
for 10 min, either with or without ATP (0.5 mM). In cells preincubated
with ATP, influx was initiated by addition of 25 µM ethidium bromide,
and uptake of this dye was compared with that into cells to which
ethidium cation was added just before ATP (Fig.
3). Cells preincubated with ATP showed a
greater influx of ethidium than cells for which ATP was added at
time 0. However, the uptake of
ethidium was linear with time only for those cells that had been
preincubated with ATP. In contrast, a delay phase was observed before
ethidium uptake commenced in cells exposed to ATP at
time 0, and the delay was more
prominent and prolonged at 24°C than at 37°C (Fig. 3,
A and
B). At 12°C, the ATP-induced uptake of ethidium was insignificant whether or not cells were preincubated for 30 min with ATP at this temperature (data not shown).

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Fig. 3.
Extracellular ATP-induced uptake of ethidium cation into fresh
lymphocytes measured either at 37°C
(A) or at 24°C
(B). Cells were suspended at
106 cells/ml in HEPES-buffered KCl
medium with no added Ca2+ and
incubated with or without ATP (0.5 mM) at designated temperature. In
cells with ATP preexposure, an addition of ethidium bromide (Et; 25 µM) was made as indicated. In cells without ATP preexposure,
additions of ethidium cation
(Et+) and ATP were made in quick
succession as indicated. Mean channel cell-associated fluorescence
intensity was measured at 6-s intervals.
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The delay in ethidium uptake was not due to the slow rate of diffusion
or reaction of the dye with cellular nucleic acids, since streptolysin
O (2 U/ml), which produces membrane lesions 15 nm in diameter in mast
cells and HL-60 leukocytes (12, 16), produced immediate and maximal
cell-associated fluorescence when added to chronic lymphocytic leukemia
lymphocytes simultaneously with ethidium (data not shown).
Partial agonists produce ethidium uptake only after a delay.
The time course of ethidium uptake into lymphocytes was compared for
ATP and its three analogs BzATP, 2-MeS-ATP, and ATP
S added at
maximal effective concentrations immediately after 25 µM ethidium
addition. Ethidium influx commenced immediately after addition of the
most efficacious agonist, BzATP (50 µM), at a rate that was linear
with time (Fig.
4A). In
contrast, a delay was observed before uptake of ethidium commenced
after the addition of ATP (500 µM), 2-MeS-ATP (500 µM), and ATP
S
(2.0 mM). The delay before maximal ethidium influx was reached was
estimated for each agonist by extrapolation of the line of maximal
slope back to its intersection with the line of basal ethidium influx
and measurement of the time interval between agonist addition and this
intercept. Figure 4B shows the inverse
relationship between this estimate of delay time and the maximal
ethidium influx achieved for each agonist
(J) relative to that of BzATP
(Jmax, taken as
100%). All three partial agonists for the
P2Z/P2X7 receptor allowed ethidium cation entry, but only after a delay, which became longer as the efficacy of the partial agonist became less.

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Fig. 4.
Agonist-induced ethidium cation uptake at maximal concentrations.
A: cells
(106 cells/ml) suspended in
HEPES-buffered KCl medium at 37°C were incubated with 50 µM
3'-O-(4-benzoyl)benzoyl-adenosine
5'-triphosphate (BzATP), 500 µM ATP, 500 µM
2-methylthioadenosine 5'-triphosphate (2-MeS-ATP), or 2 mM
adenosine
5'-O-(3-thiotriphosphate)
(ATP S) added 30 s after 25 µM ethidium. Control cells were
incubated with 25 µM ethidium alone. Mean channel cell-associated
fluorescence was measured at 6-s intervals by flow cytometry. Results
are from 1 experiment representative of 3 or 4. B: maximal rates of ethidium uptake
for each agonist (J) calculated as a
percentage of response to 50 µM BzATP
(Jmax) are
plotted against length of delay phase of uptake. Line of best fit
through data points from 4 experiments was calculated by least squares
analysis. Correlation between
J/Jmax
and length of delay phase was 0.96 (Pearson's
r; P < 0.001).
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Delay phase with low BzATP concentrations.
Although no delay phase was observed for BzATP concentrations (
50
µM) producing maximal responses, a delay in ethidium uptake became
apparent when submaximal concentrations of BzATP were added 30 s after
ethidium (Fig. 5). Furthermore, no delay
was observed when cells were incubated with BzATP (10-20 µM) for
10 min before the addition of ethidium (data not shown).

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Fig. 5.
BzATP-stimulated ethidium cation uptake. Cells
(106 cells/ml) suspended in
HEPES-buffered KCl medium at 37°C were incubated with BzATP added
30 s after ethidium, and mean channel cell-associated fluorescence was
measured at 6-s intervals by flow cytometry. Control cells were
incubated with 25 µM ethidium alone. Results are from 1 experiment
representative of 3.
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P2Z/P2X7 inhibitors lengthen the delay
phase.
We have previously shown that the isoquinoline derivative KN-62 is a
potent antagonist of the P2Z/P2X7
receptor and inhibits ATP-stimulated ethidium uptake with an
IC50 of 13.1 ± 2.6 nM and with
complete inhibition at 500 nM (11). However, KN-62 is a less potent
inhibitor of BzATP, and even 2,000 nM KN-62 blocked only 70% of the
ethidium influx (Fig. 6). In addition, Fig.
6 shows that KN-62 added 10 min before ethidium (25 µM) and BzATP (50 µM) not only inhibited ethidium influx through the P2Z channel but
lengthened the delay before significant ethidium uptake was detected.

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Fig. 6.
KN-62 delays BzATP-stimulated ethidium cation uptake. Fresh lymphocytes
were suspended at 106 cells/ml in
buffered KCl medium and preincubated with or without KN-62 for 10 min
at 37°C, followed by additions of 25 µM ethidium and 50 µM
BzATP as indicated. Mean channel cell-associated fluorescence intensity
was measured at 6-s intervals by flow cytometry. Results are from 1 experiment representative of 4.
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The amiloride analog HMA is another inhibitor of the
P2Z/P2X7-agonist-operated ion
channel (25, 40). This analog was added (3-30 µM) to fresh
lymphocytes suspended in KCl medium at 37°C, followed immediately
by addition of ethidium (25 µM) and ATP (0.5 mM). In the absence of
HMA, there was no uptake of ethidium for ~20 s, but the uptake of dye
then increased and finally became linear with time after 60 s (Fig.
7). At HMA concentrations between 3 and 30 µM, the linear phase of ethidium uptake was inhibited, but even 30 µM (Fig. 7) or 100 µM (data not shown) inhibitor failed to
completely abolish dye uptake. A second effect of HMA was to lengthen
the delay phase, since in the presence of 30 µM HMA there was no
significant uptake of ethidium until ~90 s after ATP addition.

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Fig. 7.
Inhibition of ATP-induced uptake of ethidium cation by the amiloride
analog
5-(N, N-hexamethylene)-amiloride
(HMA). Fresh lymphocytes were suspended at
106 cells/ml in buffered KCl
medium without Ca2+ at 37°C.
Additions of HMA (0-30 µM) were made, followed immediately by
ethidium bromide (25 µM) and ATP (0.5 mM) as indicated. Mean channel
cell-associated fluorescence intensity was measured at 6-s intervals.
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 |
DISCUSSION |
Our study has shown that the P2Z responses of human lymphocytes (3, 10,
11, 22, 38-40) are mediated by the
hP2X7 receptor that was recently
cloned from a human monocyte library (27) and is the latest member of
the P2X family of receptors. RT-PCR analysis shows an identically sized
product for human lymphocytes and monocytes, and the rank order of
agonist efficacy and the upper limit of permeant ion size are also the
same for the P2Z/P2X7 channel in
its native lymphocyte and for HEK-293 cells heterologously expressing
the cDNA for hP2X7.
The major finding of this study is the slow further increase in
permeability that develops after an initial opening of the P2Z/P2X7 receptor channel induced
by ATP and other partial agonists. The data in Fig. 2 show that ATP
induces an immediate
133Ba2+
influx into cells at both 37 and 24°C, which is in line with electrophysiological data showing rapid (<1 s) channel opening following ATP addition (3, 25, 31, 34). This influx occurs only via the
P2Z/P2X7 ion channel, since
inhibitors of L-type voltage-gated
Ca2+ channels are without effect
on ATP-induced Ba2+ influx (39).
133Ba2+
influx into lymphocytes was always greater for cells preincubated with
ATP than for cells to which simultaneous addition of ATP and
133Ba2+
were made (Fig. 2), suggesting a second increase in permeability to
Ba2+ had developed within 2 min at
37°C. This second permeability increase could be conveniently
studied with the larger ethidium cation, since the large size (314 Da)
of this cation used at low concentrations (25 µM) revealed the slow
kinetics of channel enlargement. Previous studies by Gomperts, Tatham,
and colleagues (13, 33, 34) measured uptake of ethidium
cation by the P2Z/P2X7 ion channel of mast cells using a fluorometric technique that relies on the enhancement of fluorescence after ethidium cation enters the cell and
binds to nucleic acids, a step that eliminates any backflux of the
permeant ion. We have adapted this method to flow cytometry, which
offers unique advantages for studying permeability states of the
P2Z/P2X7 ion channel.
Time-resolved flow cytometry generates the mean fluorescence intensity
of 3,000 cells per 6-s interval while mainly cell-associated
fluorescence is measured, since very little of the bulk medium is
illuminated in the laser light path. Thus this technique allows a
sensitive measurement of the initial rates of ethidium cation uptake,
which are essentially unidirectional because of binding of permeant
ions to nucleic acids. Figure 3, A and
B, shows that the rapidly activated
P2Z/P2X7 channel is initially
impermeable to ethidium cation, but within the first 1 min after
addition of ATP a steady increase in ethidium influx was observed. This
delay and slow increase in the rate of ethidium cation uptake contrast
with the immediate uptake of
133Ba2+
observed following ATP addition and suggest that the initial P2Z/P2X7 channel, once opened, is
slowly modified to accept larger permeant ions. Uptake of ethidium
after ATP addition was strongly temperature dependent (Fig. 3), and no
uptake occurred at 12°C. A strong temperature dependence has also
been noted previously for uptake of Lucifer yellow through the
P2Z/P2X7 channel of
murine macrophages, with no dye entering at 18°C (30). A recent
patch-clamp study of human B lymphocytes showed that
P2Z/P2X7 unitary channel currents
could be carried by Na+ or
Cs+ but not by choline ions (22).
However, the reduced temperature (22°C) and short time frame (~2
min) of these measurements may not have allowed sufficient time to
elapse for channel enlargement.
We and others have previously shown that the influx of ethidium
stimulated by maximally effective agonist concentrations follows a rank
order BzATP > ATP > 2-MeS-ATP > ATP
S (10, 32). These data
together with the results of competitive experiments show that the
natural ligand ATP is a partial agonist for the
P2Z/P2X7 receptor, whereas BzATP
is a full agonist (10). Two other less potent agonists for the
P2Z/P2X7 receptor, 2-MeS-ATP and
ATP
S, are also partial agonists, and all three partial agonists
stimulate the uptake of ethidium only after a delay phase of 30-90
s (Fig. 4A). Partial agonism is a
well-recognized feature of many receptor-operated channels at which the
partial agonist, added at maximally effective concentrations, produces
less permeant ion flux (J) than does the full agonist
(Jmax). This
lower maximal response
(J/Jmax) is a measure of the intrinsic efficacy of the partial agonist, and it
is striking that
J/Jmax
shows an inverse relationship to an estimate of the duration of the
delay phase (Fig. 4B). It is generally accepted that partial agonists are unable to induce the same
change in receptor conformation as full agonists and thus maintain a
lower proportion of the channels in the open conformation (21).
Although the full agonist BzATP (50 µM) produced an immediate influx
of ethidium with no sign of delay, lower BzATP concentrations (10-20 µM) stimulated ethidium uptake only after significant
delays of up to 30 s (Fig. 5). Such low concentrations of the full
agonist also maintain a lower proportion of the channels in the open
state. A similar effect was observed with the inhibitors KN-62 and HMA, both of which slowed agonist-induced ethidium influx with an associated increase in the delay before significant ethidium entry could be
detected (Figs. 6 and 7). Taken together, these data
support a model proposed by Tatham and Lindau (34) and by Nuttle and Dubyak (25) in which agonists for the
P2Z/P2X7 receptor activate immediate opening of an ion channel
(P2Zopen), followed by a slower transition to a permeability pathway that passes larger cations up to
the size of ethidium and YO-PRO-1 cations
(P2Zpore). Thus
in
which the proportion of channels in the open conformation is determined
by the efficacy of agonist as well as agonist concentration. Our data
show that the rate of formation of the larger "pore" is slow
(k2
k1), so that
the model predicts the second step (or series of steps) occurs at a
rate (k2 × P2Zopen) that is largely
determined by the abundance of the open channels in the membrane.
Amiloride analogs, even at high concentrations (30-100 µM),
block only 70-85% of the flux of monovalent or divalent cations
through the P2Z/P2X7 channel,
despite having inhibitor constants of ~2-10 µM (25, 40). A
similar and incomplete inhibition of ethidium influx by the amiloride
analog HMA was observed in this study (Fig. 7). Even more striking was
the finding that the most potent inhibitor described to date, KN-62,
could only inhibit 75% of the BzATP-induced ethidium influx (Fig. 6)
although KN-62 could completely abolish the ATP-induced ethidium influx
(11). One explanation is that the two permeability states of the
channel have different sensitivity to inhibitor (25). However, it is equally possible that a fraction of the
P2Z/P2X7 pores, once formed, develop firm interactions with other components of the plasma membrane
(k
2
becomes smaller) and that these complexes resist the action of
inhibitors. Recently it has been reported that calmidazolium inhibits
agonist-induced ionic currents through the
hP2X7 channel by up to 90% with
little effect of inhibitor on YO-PRO-1 cation uptake (37). Although
this finding supports the suggestion that conformational changes
accompany channel-to-pore transition (32), confirmatory findings are
needed in a cell type expressing native
hP2X7.
There is electrophysiological evidence that the macrophage
P2Z/P2X7 receptor ion channel has
variable conductance states. Thus single channel recordings of
patch-clamped membranes showed a number of conductance sublevels
ranging from 3.5 to 15 pS with Ba2+ as the permeant cation (22,
23). Multiple conductance states for receptor-operated single channels
have also been documented for the nicotinic ACh receptor (15) and for
-aminobutyric acid-operated Cl
channels (7, 9).
However, slow changes in channel conductance have not been reported at
37°C over the time frame (1-5 min) of the present ethidium
uptake studies. There is good evidence for phosphorylation of ion
channel subunits (20), and it is possible that the phosphorylation
state may influence the channel flux. Thus the permeability of the
P2Z/P2X7 receptor channel of mouse lacrimal acinar cells or mast cells is potentiated by stimulation of
cAMP-dependent protein kinase activity (24, 29), although there is
conflicting evidence (35). Figure 6 shows that the inhibitor KN-62 at
10 nM induces a delay before BzATP-induced uptake of ethidium is
observed. Although KN-62 inhibits
Ca2+/calmodulin-activated kinase
II with an IC50 of 0.9 µM (36), phosphorylation of the channel by this enzyme is unlikely to be involved, since KN-62 blocks the receptor at a concentration too low to
inhibit this kinase. Another possibility is that oligomerization of
open P2Z/P2X7 monomeric channels
might occur on ATP binding and modulate the permeability pathway to
allow larger cations to permeate. Aggregation of channel monomers to
form a larger oligomer pore has been suggested previously for the
P2Z/P2X7 channel of mast cells
(34) and by Nuttle and Dubyak (25) for the
P2Z/P2X7 channel of macrophages.
There is evidence that the long COOH-terminal tail of the
P2Z/P2X7 receptor is required for
permeation through the channel by large fluorescent dyes (32), although
the molecular mechanisms involved are unclear.
There is increasing evidence for protein-protein interactions in the
plasma membrane between receptor/channel monomers that may allow their
recruitment and assembly into larger complexes. Thus the nicotinic ACh
receptor of Torpedo membranes
undergoes ligand-induced dimerization (19), whereas the T-cell receptor for antigen undergoes spontaneous oligomerization on engagement of
peptide-major histocompatibility complex molecules (28). Recently the postsynaptic density protein PSD-95 has been shown to
mediate the clustering of both
N-methyl-D-aspartate
receptors (18) and K+ channels
(17) via specialized domain interactions. However, it is
not known whether oligomerization of these channels allows the
permeation of larger molecules. Whatever the mechanism, the use of
time-resolved flow cytometry allows the slow second-phase permeability
changes of the P2Z/P2X7 channel to
be studied, with results that suggest that the formation of the
P2Z/P2X7 "pore pathway"
occurs at a rate that is proportional to the fraction of
P2Z/P2X7 receptors that are held
in the open channel state.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Arthur Conigrave, Bruce Livett, and Bill Sawyer for
helpful comments and Tina Murphy for typing the manuscript.
 |
FOOTNOTES |
This work was supported by grants from the National Health and Medical
Research Council of Australia.
Address for reprint requests: J. S. Wiley, Sydney University Dept. of
Medicine, Nepean Hospital, Penrith, NSW 2750, Australia.
Received 22 September 1997; accepted in final form 25 June 1998.
 |
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