1 Julius-Bernstein-Institut für Physiologie, Martin-Luther-Universität Halle-Wittenberg, D-06097 Halle/Saale; and 2 Abteilung für Molekulare Pharmakologie, Rheinisch-Westfälische Technische Hochschule Aachen, D-52074 Aachen, Germany
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ABSTRACT |
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A glutamate to alanine exchange at amino acid position 496 of the human P2X7 receptor was recently shown to be associated with a loss of function in human B lymphocytes in terms of ATP-induced ethidium+ uptake, Ba2+ influx, and induction of apoptosis (Gu BJ, Zhang WY, Worthington RA, Sluyter R, Dao-Ung P, Petrou S, Barden JA, and Wiley JS. J Biol Chem 276: 11135-11142, 2001). Here we analyzed the effect of the Glu496 to Ala exchange on the channel properties of the human P2X7 receptor expressed in Xenopus oocytes with the two-microelectrode voltage-clamp technique. The amplitudes of ATP-induced whole cell currents characteristic of functional expression, kinetic properties including ATP concentration dependence, and permeation behavior were not altered by this amino acid exchange. Also in HEK293 cells, the Ala496 mutant mediated typical P2X7 receptor-dependent currents like the parent Glu496 hP2X7 receptor. Because the function of the P2X7 receptor as an ATP-gated channel for small cations including Ba2+ remained unaffected by this mutation, we conclude that Glu496 plays a critical role in pore formation but does not determine the ion channel properties of the human P2X7 receptor.
Xenopus oocytes; whole cell current; permeation; kinetics; HEK293 cells
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INTRODUCTION |
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THE
P2Z/P2X7 receptor
belongs to the P2X family of ATP-gated ion channels, which are
nonselectively permeable to small cations (17, 21). ATP
has a rather low potency, whereas 2',3'-O-(4-benzoylbenzoyl) ATP (BzATP) is the most potent agonist at the P2X7 receptor
but not at other receptors of the P2X family (22, 24).
P2X7 receptors are mainly found in cells of the
hematopoietic system (4, 20) but also exist in epithelial
cells (25). In some of these cells (such as fibroblasts
and macrophages), prolonged application of high concentrations of
agonist leads to a slow pore dilation after channel opening, which
allows molecules of up to 900 Da to permeate the pore (4).
Other cells expressing P2X7 receptors (like salivary gland
cells, B lymphocytes, and Xenopus oocytes), however, are not
permeabilized by long-lasting exposure to ATP (16, 25). The P2Z receptor of human lymphocytes shows many of the typical features of the P2X7 receptor. The free acids
ATP4 and BzATP4
induce stable unitary
cationic currents with a conductance of 9 pS and mean open times of
~5 ms as measured by single-channel recordings. The currents do not
desensitize and can be activated and deactivated within <1 s
(18). P2X7 receptors in human B lymphocytes
display the same pharmacological profile as in other cells of the
immune system. Compared with other cells, such as fibroblast and mast
cells, a difference exists in the extent of ATP-induced pore dilation.
Although prolonged ATP exposure induces pores permeable to
ethidium+ (314 Da), permeation of molecules >400 Da was
not observed in human B lymphocytes (29). The recombinant
human P2X7 (hP2X7) receptors show a very
similar phenotype when expressed in Xenopus oocytes, i.e.,
they are activated and deactivated within a few seconds and are mainly
permeable to small inorganic cations. A detailed study of the
biophysical properties of hP2X7 receptors in
Xenopus oocytes revealed two distinct activation sites with apparent dissociation constants for ATP4
of ~4 and 200 µM (15).
Besides the opening of large pores, further
P2Z/P2X7-dependent effects like the activation of
phospholipase D (6), NF-B (8) and
interleukin-1
-converting enzyme (5) as well as the loss
of L-selectin from human lymphocytes (13) have been observed in some cell types of the immune system. The relationship between the initial opening of P2X7 receptor-dependent
small cationic channels and these agonist-induced metabotropic events
remains unexplained. The findings that
N,N-hexamethylene amiloride (HMA) (19) and propranolol (1) prevent the
formation of large pores without blocking the permeability for small
cations as well as the inverse effect of calmidazolium
(26) suggest that these functions may exist in part
independently of each other.
Recently, it was reported that some subjects express a nonfunctional P2X7 receptor in lymphocytes and monocytes, which results from a Glu to Ala mutation at position 496 of the 595-amino acid chain of the P2X7 receptor. In particular, ATP-induced uptake of ethidium+ (314 Da), shedding of L-selectin, and BzATP-evoked cytotoxicity of T lymphocytes were found to be abolished by this mutation (10, 11). On the other hand, in a P2X7 cDNA cloned from immortalized human B lymphocytes we found (14) that the amino acid exchange at two positions (the identical Glu496Ala and an additional Arg441Gly) of the published amino acid sequence of hP2X7 (22) did not obviously change the ionic current kinetics compared with the wild type. Therefore, we performed a more detailed study to investigate how these mutations influence the kinetics and agonist concentration dependence of hP2X7-dependent ionic currents. The results indicate that, in contrast to the other hP2X7-dependent effects, the biophysical properties of the small cationic channels are not influenced by these amino acid substitutions.
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MATERIALS AND METHODS |
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Chemicals. Chemicals were obtained from Sigma (Deisenhofen, Germany) unless otherwise stated.
P2X7 cDNA constructs. A P2X7 cDNA (designated Gly441Ala496-hP2X7) carrying two amino acid exchanges (Arg441Gly and Glu496Ala) compared with published sequences (GenBank accession nos. Y09561 and Y12851-5) was isolated from an immortalized human B lymphocyte cell line and cloned into the oocyte expression vector pNKS2 (9) as described previously (14). Gly441 and Ala496 were sequentially mutated to Arg and Glu to generate Arg441Ala496-hP2X7 (designated Ala496-hP2X7) and Arg441Glu496-hP2X7 (designated Glu496-hP2X7), respectively, with the QuikChange site-directed mutagenesis kit (Stratagene, Heidelberg, Germany). For expression in mammalian cells, the entire P2X7 coding regions were subcloned into the mammalian expression vector pcDNA3.1(+) (Invitrogen, Karlsruhe, Germany), yielding Ala496-hP2X7-pcDNA3.1 and Glu496-hP2X7-pcDNA3.1. Mutations and junction sequences were confirmed by sequencing.
Oocyte treatment and electrophysiology.
Preparation of Xenopus laevis oocytes, injection of
cRNA, and the protocols for the measurement of ATP-induced
hP2X7-dependent whole cell currents were performed as
described previously (15). Briefly, membrane currents were
measured by the two-microelectrode voltage-clamp method. Currents were
measured 1 or 2 days after injection of 20 nl of hP2X7 cRNA
(0.2 µg/µl). A fast and reproducible solution exchange was achieved
with a small tubelike chamber (0.1 ml) combined with fast superfusion
(~75 µl/s). Switching between different bathing solutions was
performed by a set of computer-controlled magnetic valves with a
modified U tube technique (3). Measurements of
hP2X7 receptor-dependent currents were carried out in a
bathing solution consisting of (in mM) 100 NaCl, 2.5 KCl, and 10 HEPES, pH 7.4, adjusted with NaOH. Ca2+ was omitted to avoid the
activation of endogenous currents by a Ca2+ influx through
the hP2X7 receptor channels. Mg2+ was omitted
to prevent the Mg2+ complexation of ATP4,
because otherwise the high ATP concentrations required for maximal activation of the hP2X7 receptor would have been insoluble
(4, 18). Accordingly, the concentrations of total ATP and
of free ATP4
were approximately equal in these solutions.
The removal of divalent cations from the extracellular solution evoked
a large conductance, which could be blocked by 0.1 mM flufenamic acid
(28, 32). Currents were recorded and filtered at 100 Hz
with an oocyte clamp amplifier (OC-725C; Warner Instrument, Hamden, CT)
and sampled at 85 Hz. Data were stored and analyzed on a personal
computer with software programmed at our department (Superpatch 2000, SP-Analyzer by T. Böhm). The holding potential was set to
40
mV. For characterization of permeation behavior, a bathing solution
with Na+ substituted by choline+ was used.
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Transfection of HEK293 cells. HEK293 cells were plated 1 day before transfection at a density of ~4 × 105 cells per 35-mm culture dish in 1 ml of DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) on small coverslips (diameter 5 mm) and maintained in a humidified atmosphere containing 5% CO2 at 37°C. A mixture of 0.8 µg of one of the hP2X7 receptor cDNAs, 0.8 µg of plasmid pEGFP-N1 encoding enhanced green fluorescent protein, 6 µl of LipofectAMINE 2000, and 0.1 ml of Opti-MEM was added to the culture dish according to the manufacturer's protocols (Invitrogen). After 12 h, the medium was replaced with DMEM, in which the cells were incubated for another 36 h.
Whole cell recordings from HEK293 cells.
Whole cell currents were measured similarly as previously described for
human B lymphocytes (2, 16). The coverslips with the
adhering HEK293 cells were transferred to a perfusion chamber and
rinsed with a bathing solution consisting of (in mM) 140 NaCl, 0.5 Ca,
10 HEPES, and 10 glucose, pH 7.4, adjusted with NaOH. Patch pipettes
were filled with a solution containing (in mM) 65 Cs aspartate, 65 CsCl, 1 EGTA, 10 HEPES, 5 NaATP, 5.5 MgCl2, and 10 glucose,
pH 7.2 adjusted with CsOH. For activation of
hP2X7-dependent currents, the bathing solution was quickly
replaced by the U tube technique (3) with a solution
containing 1 mM free ATP4 (same as bathing solution but
with 0.5 mM CaCl2 replaced by 3.9 mM CaCl2 and
6.3 mM total NaATP). All experiments were carried out at room
temperature (~22°C).
Statistical evaluation of data.
For quantitative analysis, the activating part of the hP2X7
receptor current [Iact(t)] was
fitted by Eq. 1 according to (15)
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(1) |
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(2) |
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(3) |
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RESULTS |
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Figure 1 demonstrates the time
courses of ion currents recorded from oocytes expressing the
Glu496-hP2X7 receptor (Fig. 1A;
corresponding to the sequence published in Ref. 22), the
Ala496-hP2X7 receptor (Fig. 1B;
corresponding to the mutated receptors described in Ref.
11), or the
Gly441Ala496-hP2X7 receptor (Fig.
1C; corresponding to the receptor investigated in Ref.
14) before, during (activation), and after (deactivation) extracellular application of ATP. The time course of activation and
deactivation was not obviously altered by these amino acid exchanges.
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The three versions of the hP2X7 receptor did not
significantly differ in their mean ATP-induced currents measured 1 and
2 days after cRNA injection (Fig.
2A). Likewise, the ATP
concentration dependence of the ATP-evoked currents remained unchanged
by the mutations (Fig. 2B).
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It is obvious from Fig. 1 that the activation and deactivation time
courses of the hP2X7-dependent currents displayed a complex kinetic behavior. This could be described by a kinetic model (see MATERIALS AND METHODS), which fitted the ATP-dependent
current traces similarly well for all three investigated
hP2X7 receptor versions as shown in Fig. 1. The
approximation of the ATP concentration dependence of the normalized
currents after 6 s of ATP application (Fig. 2B) as well
as of Iact,,rel(c) (Fig.
3A) did not reveal
statistically significant differences between the apparent dissociation
constants (KD,1, KD,2)
and the relative amplitudes of the current components
(Arel,
,1, Arel,
,2)
of the three hP2X7 receptor versions.
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A simpler model with two equal noncooperative activation sites (i.e.,
Arel,,2 = 0) could be used to assess the
ATP concentration dependence of the linearly activating current
component s (Fig. 3B) and of the deactivating
current components Ideact,1 (Fig. 3C)
and Ideact,2 (Fig. 3D). This
evaluation also provided no evidence for significantly different ATP
concentration dependencies of these two current components between the
three hP2X7 receptor versions (Fig. 3,
A-D). Furthermore, the time course, as characterized by
the time constants of activation (
act; Fig.
3E) and deactivation (
deact,1,
deact,2; Fig. 3F), was also not significantly
altered by the mutations.
It has been reported in several cell types that the prolonged
activation of P2X7 receptors induces a pore dilation such
that the receptor channel becomes permeable to large molecules of up to
900 Da (24, 27). The Glu496Ala amino acid
exchange has been reported to hinder the accumulation of
ethidium+ and hence the pore dilation in human B
lymphocytes challenged with 1 mM ATP (11). Therefore, we
investigated the effect of the Glu496Ala exchange on the
permeation behavior of the hP2X7 receptor by
electrophysiological techniques. As demonstrated in Fig.
4, the application of 1 mM ATP in a
choline+-based Na+-free medium evoked large
currents that, shortly after application of ATP, reversed at
approximately 60 mV. This behavior can best be reconciled with a much
higher permeability of the plasma membrane for the intracellular
K+ than for the much bigger extracellular
choline+ (104 Da). Prolonged application of 1 mM ATP for up
to 2 min shifted the mean reversal potential
(Vrev) equally (P > 0.05) to
about
40 mV in all P2X7 receptor versions, which
indicates a low choline+ permeability. The mean ATP-induced
conductance of the plasma membrane was not significantly different
among the three investigated P2X7 receptor versions (data
not shown).
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To test whether the permeation of divalent cations was affected by the
mutations, we performed similar experiments in extracellular solutions
containing different Ba2+ concentrations (Fig.
5). A significant shift of
Vrev was only induced by 1 mM Ba2+
in Na+-free (choline+-based) solution. In
identical bathing solutions, identical Vrev values were obtained for the three P2X7 mutants, indicating
that the Glu496 to Ala exchange has no effect on the
Ba2+ permeability of the hP2X7 receptor
channel.
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To exclude a possible contribution of the host cells to the behavior of
the expressed receptor, we also examined the electrophysiological function of the hP2X7 receptor versions in a mammalian
expression system. As shown in Fig. 6,
currents with similar amplitude and time courses of activation and
deactivation could be evoked by ATP in HEK293 cells expressing either
Glu496-hP2X7 or
Ala496-hP2X7 receptors. Accordingly, the lack
of effect of the Glu496 to Ala mutation on the
electrophysiological hP2X7 channel properties is not unique
to the Xenopus oocytes used as an expression system.
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DISCUSSION |
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A nucleotide polymorphism of the P2X7 receptor was recently shown to be present in the Caucasian population, coding for a Glu to Ala substitution at amino acid 496. Homozygous expression of the Ala496-hP2X7 receptor produced a nonfunctional receptor in B lymphocytes, as assessed by monitoring the ATP-induced uptake of lucifer yellow, ethidium+, and Ba2+ by fluorescence measurements. It has been inferred from this observation that Glu496 plays a critical role in the assembly of the P2X7 receptor (11). We observed (15) the same polymorphism to exist also in an immortalized human B lymphocyte cell line, which heterozygously expresses both the Glu496-hP2X7 and the Ala496-hP2X7 receptor. Here we demonstrate that the wild-type Glu496-hP2X7 receptor and the Ala496-hP2X7 receptor mutant exhibit the same electrophysiological phenotype on heterologous expression in Xenopus oocytes. This suggests that the operation of the hP2X7 receptor as an ATP-triggered cation-selective channel is not influenced by Glu496 and further suggests that it is in particular the induction of nonselective pores that is blocked by the Glu496 to Ala exchange.
The impaired function of the P2X7 receptor carrying the
Glu496Ala mutation was found to be reversed in terms of
ATP-gated ethidium+ uptake when the mutant receptor was
expressed in high density in HEK293 cells (11). Therefore,
it may be argued that expression of the hP2X7 receptor in
Xenopus oocytes leads to such a low channel density in our
experiments that the pore induction of the
Glu496-hP2X7 receptor is obscured and therefore
a loss of this function of the Ala496-hP2X7
receptor cannot be investigated in this expression system. With a
capacitance of 70 nF for stage VI oocytes (31), the
density of the current evoked by application of 1 mM ATP for 6 s
can be calculated to amount to 1.5 and 14 pA/pF on day 1 and
day 2 after cRNA injection, respectively (see Fig. 2).
Nontransfected human B lymphocytes show a very similar current density
of 15 pA/pF (300 pA/20 pF) when stimulated with 1 mM ATP4
(2). Even at a 10-fold lower current density of 1.5 pA/pF, as observed 1 day after cRNA injection, ATP-inducible currents could be
recorded from Xenopus oocytes expressing the
Glu496-hP2X7 receptor mutation. These
considerations exclude the possibility that the pore-forming function
of the Glu496-hP2X7 receptor was obscured in
our experiments by low-level expression.
Another cause for the apparent discrepancy of the effects of the mutations in Xenopus oocytes and B lymphocytes may be the different expression systems. However, as shown here, both Glu496 and Ala496 hP2X7 receptors mediated similar ionic currents when expressed in Xenopus oocytes as well as in HEK293 cells.
A further variation in the experimental protocol compared with the study of Gu et al. (11) is the temperature at which the experiments were conducted. The activation of the ionic channel permeable to small inorganic cations like Na+, K+, and Li+ seems to exhibit little temperature dependence (19) and can already be measured at room temperature according to our measurements. The pore-forming property leading to the permeation of larger organic cations like Tris+ (121 Da), N-methyl-D-glucamine (NMDG+) (195 Da), and ethidium+ (314 Da), however, occurs only (19) or is at least accelerated (30) at the higher temperature of 37°C. Effects similar to those of low temperature, i.e., minor blocking effects on the channel (14, 19) but strong inhibitory action on the pore (19, 29), are found for HMA. Therefore, it is conceivable that the effects of the mutations measured in B lymphocytes apply only to the temperature- and HMA-dependent pore formation and not to the opening of the ionic channel.
In our experiments in oocytes, we aimed to characterize a possible pore
formation by measuring the Vrev of ATP-induced
currents in extracellular solutions with Na+ replaced by
the larger organic cation choline+. A shift of
Vrev from the usual negative potential to a
value close to 0 mV would be indicative of a loss of permeability
selectivity of the receptor channel corresponding to pore formation.
Although we observed an initial shift of Vrev
from about 60 mV to
40 mV within 10-20 s,
Vrev remained constant thereafter in all three investigated hP2X7 receptor versions.
The underlying mechanism for the initial shift of
Vrev remains obscure. It is probably similar to
the permeability changes observed for P2X2 receptors in
Na+- and Ca2+-free extracellular solutions
(7) but dissimilar from the supposed pore dilation found
in HEK293 cells expressing the rat P2X7 receptor (27), because 1) in HEK293 cells
Vrev shifted within ~10 s after application of
the agonist BzATP (30 µM) to about 10 mV in extracellular solutions
containing Tris+ as the main extracellular cation compared
with a maximal shift to a Vrev of
40 mV
observed in our experiments using a choline+-based medium.
In our preparation, however, applications of 50 µM BzATP for 2 min in
choline+-containing bathing solution shifted
Vrev to values between
50 and
40 mV, similar
to the measurements using 1 mM ATP (data not shown). 2) As
shown in human B lymphocytes, 1 mM ATP applied at room temperature
induced a permeability state allowing ethidium+ to permeate
only after a delay of ~1-2 min (30), much slower than the Vrev shift observed here, which occurs
within seconds. 3) Ethidium+ uptake, considered
as a measure of pore dilation, is hindered by the Glu496Ala
mutation (11). Thus the different effects of the
Glu496 to Ala mutation in Xenopus oocytes and
human B lymphocytes can be explained by the assumption that it
selectively affects the pore formation and not the ion channel
properties of the hP2X7 receptor.
In hP2X7 receptor-expressing oocytes, a significant Ba2+ permeation seems to occur in Na+-free extracellular solution at a Ba2+ concentration of at least 1 mM, but this was similar for all three investigated mutants. To account for the apparent inability of the Ala496 mutant to mediate Ba2+ permeation in B lymphocytes as found by Gu et al. (11), we assume that the fura 2-based fluorescence measurements used there are less sensitive than the electrophysiological technique used by us to detect a small Ba2+ influx through hP2X7-dependent ion channels. It is possible that, in the experiments carried out by Gu et al. (11), the powerful depolarization induced by measuring Ba2+ uptake in 150 mM extracellular K+ reduced the driving force for the Ba2+ influx through the ion channel of hP2X7 to such an extent that the Ba2+ ions could permeate only through the pore and not through the ion channel. This would correlate to the finding that membrane depolarization exerts a strong inhibitory effect on the Ca2+ influx through P2Z/P2X7 receptors in human B lymphocytes (16).
The Glu496Ala mutation is located on the large cytoplasmic COOH-terminal tail of the hP2X7 receptor, confirming the suggestion that this part of the P2X7 receptor is necessary for pore formation but not for ion channel function (15, 24). Likewise, other hP2X7 functions like shedding of L-selectin and BzATP-evoked cytotoxicity of T lymphocytes seem to be dependent on Glu496 (10). The findings that pore formation depends on the expression density of P2X7 receptors (12) and that it can be blocked by mutation at a residue that is probably not part of the ion channel conducting pathway indicate that the pore formation is not a process of dilation of the ion channel to a pore within a single P2X7 molecule. The aggregation of several P2X7 molecules or association of other molecules is possibly involved in the process of pore formation.
Knowledge of the effect of the Glu496Ala mutation may be used for further investigations of the relations between the ion-conducting property and the other late effects of hP2X7 receptor activation.
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FOOTNOTES |
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Address for reprint requests and other correspondence: F. Markwardt, Julius-Bernstein-Institut für Physiologie, Martin-Luther-Universität Halle-Wittenberg, Magdeburger Straße 6, D-06097 Halle/Saale (E-mail: fritz.markwardt{at}medizin.uni-halle.de).
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.
First published November 13, 2002;10.1152/ajpcell.00042.2002
Received 27 January 2002; accepted in final form 7 November 2002.
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