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The Journal of Neuroscience, September 1, 1999, 19(17):7289-7299
Allosteric Control of Gating and Kinetics at P2X4
Receptor Channels
Baljit S.
Khakh1,
William R.
Proctor2,
Thomas
V.
Dunwiddie2,
Cesar
Labarca1, and
Henry A.
Lester1
1 Division of Biology, California Institute of
Technology, Pasadena, California 91125, and 2 University of
Colorado Health Sciences Center and Department of Veterans Affairs
Medical Center, Denver, Colorado 80262
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ABSTRACT |
The CNS abundantly expresses P2X receptor channels for ATP;
of these the most widespread in the brain is the P2X4
channel. We show that ivermectin (IVM) is a specific positive
allosteric effector of heterologously expressed P2X4 and
possibly of heteromeric P2X4/P2X6
channels, but not of P2X2, P2X3,
P2X2/P2X3, or P2X7 channels.
In the submicromolar range (EC50, ~250 nM)
the action of IVM was rapid and reversible, resulting in increased
amplitude and slowed deactivation of P2X4 channel currents
evoked by ATP. IVM also markedly increased the potency of ATP and that
of the normally low-potency agonist  , -methylene-ATP in a use- and
voltage-independent manner without changing the ion selectivity of
P2X4 channels. Therefore, IVM evokes a potent
pharmacological gain-of-function phenotype that is specific for
P2X4 channels. We also tested whether IVM could modulate
endogenously expressed P2X channels in the adult trigeminal
mesencephalic nucleus and hippocampal CA1 neurons. Surprisingly, IVM
produced no significant effect on the fast ATP-evoked inward currents
in either type of neuron, despite the fact that IVM modulated
P2X4 channels heterologously expressed in embryonic hippocampal neurons. These results suggest that homomeric
P2X4 channels are not the primary subtype of P2X receptor
in the adult trigeminal mesencephalic nucleus and in hippocampal CA1 neurons.
Key words:
ATP; ivermectin; ion channel; allosteric; modulation; P2X; purinoceptor
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INTRODUCTION |
ATP functions as a neurotransmitter
in the nervous system (Burnstock, 1972 ), in which it can mediate
(Edwards et al., 1992; Evans et al., 1992; Bardoni et al., 1997; Nieber
et al., 1997; Pankratov et al., 1998; Jo and Schlichter, 1999) and
possibly modulate fast synaptic transmission (Gu and MacDermott, 1997; Khakh and Henderson, 1998a). Furthermore, a class of ion channels exists that is a major target for synaptically released ATP; these are
the P2X receptor channels. P2X receptors are ATP-gated, nonselective cation channels (Surprenant et al., 1995), and they comprise a family
of seven distinct subunits that can form functional channels when
heterologously expressed (for review, see North, 1996; Ralevic and
Burnstock, 1998). Of these, P2X4 receptor
channels are notable because they are widely expressed in the brain
(Buell et al., 1996; Collo et al., 1996), they form functional channels
that can be both homomeric and heteromeric (Buell et al., 1996; Collo et al., 1996; Seguela et al., 1996; Soto et al., 1996; Le et al., 1998a), and they clearly can be localized to certain neurons (Le et
al., 1998b). P2X4 receptors have thus been
presumed to be an important target for endogenously released ATP.
However, previously there has been no way to specifically probe
P2X4 channels, and this represented a
considerable hindrance to investigating both their existence and
pathophysiological roles in native systems. As a consequence, the
prevalence and function of P2X4 channels in brain
neurons remained largely uninvestigated.
Many ligand-gated ion channels are allosterically modulated (Li et al.,
1997; Changeux and Edelstein, 1998), and such allosteric interactions
can be starting points for rational drug design. Moreover, some P2X
receptor channels are modulated by cations, e.g.,
P2X2 and P2X3 channels by
Zn2+, Ca2+,
and H+ (King et al., 1997; Stoop et al.,
1997; Wildman et al., 1997, 1998; Cook et al., 1998). There is also
evidence that an allosteric interaction at P2X4
channels regulates agonist binding (Michel et al., 1997), and recent
work demonstrates that ATP receptor antagonists can also potentiate
ATP-evoked currents at P2X4 channels (Bo et al.,
1995; Miller et al., 1998); however, the mechanism(s) and consequences
for P2X4 channel properties remain to be fully explored. Furthermore, such compounds are of limited value to probe
P2X4 channels, because they are antagonists of
most other ATP receptors, they potently inhibit ATP-hydrolyzing
enzymes, and some are chemically impure (see Humphrey et al.,
1995).
Ivermectin (IVM) is an agonist of glutamate-gated chloride channels of
invertebrates from several phyla (Cully et al., 1994), and this is
thought to be its mechanism of action when used clinically as a
treatment for river blindness caused by the nematode Onchocerca volvulus (Fisher and Mrozik, 1992; Van Laethem and Lopez, 1996). Recent evidence shows that in addition to its action on glutamate-gated chloride channels, IVM can also allosterically modulate mammalian GABAA (Krusek and Zemkova, 1994) and
7 nicotinic channels (Krause et al., 1998),
and this prompted us to test its actions on P2X channels.
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MATERIALS AND METHODS |
Molecular biology. Wild-type
P2X2, P2X3,
P2X4, and P2X7 cDNAs cloned
into pcDNA3 or pcDNA1 were obtained from Glaxo Wellcome (Greenford,
Middlesex, UK). cDNAs were linearized at unique restriction sites
downstream of the poly(A) tail. The cDNAs were transcribed in
vitro using the mMESSAGE mMACHINE kit (Ambion Inc., Austin, TX). The capped cRNA was dissolved in DEPC-treated water at a concentration of ~1 µg/µl and stored at  80°C until required
in 2 µl aliquots. Five to 10 ng of cRNA were injected into individual Xenopus laevis oocytes in a volume of 50 nl/oocyte using a Drummond micropipettor (Quick and Lester, 1994); the
preparation and maintenance of the oocytes was performed as described
previously (Quick and Lester, 1994), and all electrophysiological
recordings were made 1-4 d after injection.
Oocyte studies. Two-electrode voltage-clamp recording of
oocytes was performed using the Geneclamp 500 amplifier (Axon
Instruments, Foster City, CA). Electrodes were pulled (Flaming-Brown
type horizontal puller; Sutter Instruments, Novato, CA) from
borosilicate glass (Sutter Instruments) and back-filled with 3 M KCl to yield resistances of 1-2 M . Recordings were
made in solution consisting of (in mM): 98 NaCl, 5 HEPES,
and 1 MgCl2, pH 7.45, which was superfused over the oocytes by gravity flow at a rate of ~3 ml/min (chamber volume was ~300 µl). Solutions containing ATP were applied to the
oocyte using a solenoid-operated solution switcher (General Valve Co.,
Fairfield, NJ); complete solution exchange around the oocyte occurred
within 0.5-1.0 sec. We used IVM at a concentration of  10
µM, because at higher concentrations we occasionally
observed some precipitation in physiological solutions. The vehicle for IVM is DMSO. In vehicle controls 1% DMSO did not affect ATP-evoked currents (control 100 µM ATP-evoked current was
2611 ± 186 nA, whereas in the presence of 1% DMSO it was
2789 ± 69 nA; p > 0.05; n = 4;
also see Results). Voltage control of oocytes was maintained using a
Digidata 1200 interface and a personal computer running pCLAMP 6 or
pCLAMP 7 software (Axon Instruments). Data were filtered at 200-500 Hz
and digitized at three to five times this rate. Current-voltage
relation data were filtered at 1 kHz and digitized at 3 kHz. All
experiments were performed at 20-23°C.
Human embryonic kidney cells. Human embryonic kidney (HEK)
293 cells were transfected using Superfect (Qiagen, Hilden, Germany), and all recordings were made 24-48 hr later using an Axoclamp 2A
amplifier (Axon Instruments). The extracellular recording solution used
for mammalian cells comprised (in mM): 147 NaCl, 2 KCl, 1 MgCl2, 2.5 CaCl2, 10 HEPES,
and 10 dextrose, and the pipette solution comprised (in
mM): 154 NaCl (or KCl), 11 EGTA, and 10 HEPES. Whole-cell voltage-clamp recordings were made with 5 M borosilicate glass electrodes (Sutter Instruments) using methods that are essentially identical to those already described (Evans et al., 1996).
Adult rat brain slices. All methods used were similar to
those already described previously for brainstem neurons (Khakh et al.,
1997; Khakh and Henderson, 1998a,b) and hippocampal neurons (Dunwiddie
et al., 1997; Frazier et al., 1998a,b). Briefly, young (18- to
27-d-old) male Sprague Dawley rats were killed by decapitation, and a
vibratome (model 1000; Pelco, Clovis, CA) was used to prepare 300-µm-thick coronal slices of hippocampus and brainstem. During incubation the slices were submerged at 32-34°C in artificial CSF
comprising (in mM): 124 NaCl, 3.3 KCl, 2.4 MgSO4, 10 D-glucose, 2.5 CaCl2, 1.2 KH2PO4, and 25.9 NaHCO3 saturated with 95%
O2 and 5% CO2 gas. All
experiments were performed at 32-34°C while the tissue was
superfused with buffer at a rate of 2 ml/min. Whole-cell patch-clamp
recording was accomplished by using glass pipettes pulled using a
Flaming-Brown electrode puller. The resistance of the pipettes was
6-10 M when filled with a potassium gluconate-based internal
solution, which comprised (in mM): 130 K gluconate, 1 EGTA,
2 MgCl2, 0.5 CaCl2, 2.5 ATP, 0.3 GTP, and 10 HEPES, pH 7.25, adjusted to 290 mOsm. Cells were
visualized with an upright microscope (AxioSkop; Zeiss, Thornwood, NY)
equipped with differential interference contrast optics. Puffs (5-20
msec) of ATP (50 µM) were applied directly to the cell
body via pressure microejection (5-20 psi) from pipettes identical to
the recording pipettes, using a Picospritzer II (General Valve,
Fairfield, NJ). Inward currents were recorded in neurons
voltage-clamped at 50 to 60 mV using HEKA (Aalen, Germany) software
and hardware. Calibrated syringe pumps (Razel, Stamford, CT) were used
to add IVM from a concentrated stock solution directly to the
superfusion system. Trigeminal mesencephalic nucleus (MNV) neurons were
identified by their location (lateral to the locus coeruleus and the
fourth ventricle), large pseudomonopolar appearance, and prominent
IH current (Khakh et al., 1997; Khakh and Henderson, 1998b). CA1 pyramidal cells were identified on the basis
of their anatomical location, long dendrite projecting into the stratum
radiatum, and distinctive electrophysiological characteristics
(Dunwiddie et al., 1997; Frazier et al., 1998a,b).
Embryonic hippocampal neurons. Neurons were prepared as
described (Li et al., 1998). Hippocampal neuron cultures were
transfected with plasmids for P2X4 and for
enhanced green fluorescent protein (EGFP) using the Bio-Rad (Hercules,
CA) Helios gene gun. Gold particles coated with plasmids were prepared
as recommended by the manufacturer, and the gene gun was pressurized
between 60 and 100 psi. Neurons were transfected after 14 d in
culture, and recordings were made 24-48 hr later. Recordings were made
using borosilicate glass electrodes of 4-7 M resistance. The bath
solution was (in mM): 110 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgCl2, 10 HEPES, and 10 D-glucose, pH 7.4, osmolarity 230 mOsm. Patch
pipettes were filled with a solution containing (in mM):
100 potassium gluconate, 0.1 CaCl2, 1.1 EGTA, 5 MgCl2, 10 HEPES, 3 ATP, 3 phosphocreatine, and
0.3 GTP, pH 7.2, 215 mOsm. Whole-cell voltage clamp was maintained using an Axopatch-1D amplifier controlled by a personal computer running pCLAMP 6 software via a Digidata 1200 interface (Axon Instruments). Data were filtered at 2 kHz, digitized at 5 kHz, and
recorded on computer for later analysis. Drugs were applied to single
voltage-clamped neurons using a U-tube concentration clamp system
(Khakh et al., 1995a,b).
Data analysis. Data were analyzed using Clampfit (Axon
Instruments) or Origin 5.0 (Microcal Software Inc., Northampton, MA). Data in the text and graphs are shown as mean ± SEM from
n determinations as indicated (always more than four
experiments). All current-voltage relations shown in the figures are
leak-subtracted. The ratio of NMDG+ to
Na+ permeability was calculated for
control ATP-evoked currents and those in the presence of IVM using the
relation,
where pNMDG+ is the
permeability to NMDG+,
pNa+ is the permeability to
Na+,
 Erev is the shift in reversal
potential, and F, R, and T have their
usual meaning (Khakh et al., 1999). Concentration-effect curves were
fitted where appropriate, as indicated, to the Hill equation of the
form,
where y is current evoked by ATP,
EC50 is the response producing 50% of the
maximal response, nH is the Hill
coefficient, and [A] is the concentration of ATP. Membrane
capacitance was determined from the capacitive transients produced by
5-10 mV steps of >200 msec duration using the membrane test
capability of pCLAMP 7 (Axon Instruments). Mean data in Figure
3A were fitted to exponential functions (Origin 5.0) to
determine time constants for association ( on)
and dissociation (off). The equilibrium dissociation constant (kd) for the
receptor-IVM interaction was determined by considering the interaction
of IVM with P2X4 channels by the following
reaction scheme:
where
and
where the forward binding rate constant is
k+1, [A] is the
concentration of IVM used, and k 1 is
the rate constant for dissociation. Total charge transfer during
ATP-evoked currents was determined by integration of the voltage-clamp
records. In some of the figures background noise has been minimized in
the original traces by use of a Savitsky-Golay digital smoothing
paradigm (Origin 5.0). Statistical tests were performed using the
paired or unpaired Student's t test, as appropriate, and
p < 0.05 was taken to indicate significance.
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RESULTS |
IVM potentiates P2X4 but not P2X2,
P2X3,
P2X2/P2X3, or P2X7
channel currents
We expressed homomeric P2X4 channels in
Xenopus oocytes and tested for responses to ATP applications
(5-20 sec in duration). Suramin (30 µM), which
is an antagonist of some types of P2X ATP receptors, did not inhibit
peak currents evoked by 100 µM ATP at
P2X4 channels (n = 4). However,
suramin blocked by >80% the current evoked by 100 µM ATP at both P2X2 and
P2X3 channels (Fig. 1; also see Buell et al., 1996; Seguela
et al., 1996). We next tested the action of IVM (10 µM) on currents evoked by ATP (100 µM) at P2X2,
P2X3,
P2X2/P2X3,
P2X4, and P2X7 channels. In
cells expressing P2X2,
P2X3, or
P2X2/P2X3 channels, IVM
produced no significant effect on ATP-evoked currents (Fig. 1);
however, in P2X4-expressing cells, IVM increased
the peak ATP-evoked current significantly in all cells tested (by
50-300% depending on the concentration and incubation time; see
below). ATP-evoked currents at P2X7 channels
expressed in oocytes have two phases (Nuttle and Dubyak, 1994; Khakh et
al., 1999), and we tested whether IVM could affect either phase of
these currents evoked by ATP at P2X7 channels.
Neither phase of the 100 µM ATP-evoked current
at P2X7 channels was potentiated by IVM (10 µM); the initial current and secondary current
peaks were 512 ± 76 and 7490 ± 538 nA for ATP alone and
371 ± 108 and 5257 ± 945 nA for ATP plus IVM, respectively (n = 3; unpaired Student's t
test, p > 0.05). Homomeric P2X1
and P2X5 channel assemblies were not tested for
IVM sensitivity.
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Figure 1.
IVM potentiates P2X4 but not
P2X2 or P2X3 channel currents.
A, Representative recordings of currents mediated by
P2X4 channels expressed in oocytes. Left
trace, 100 µM ATP-evoked current before and after
(right trace) addition of IVM (10 µM); IVM
potentiates the amplitude as well as duration of the ATP-evoked
current. B, Representative recordings for
P2X2 channels expressed in oocytes. Left
trace, 100 µM ATP-evoked current before and after
(right trace) addition of IVM (10 µM); IVM
causes no change in either the holding current or ATP-evoked current in
P2X2-expressing cells. C, Summary of data
from a number of cells (n > 5 for each) showing
that IVM (10 µM) potentiates ATP-evoked currents at
P2X4 channels but not at P2X2,
P2X3, and P2X2/P2X3
channels. D, Summary of data from a number of cells
(n > 5 for each) showing that suramin (30 µM) can block ATP-evoked currents at
P2X2, P2X3, and
P2X2/P2X3 channels but not at
P2X4 channels. Subsequent figures show a more pronounced
potentiation of P2X4 channels at lower IVM and/or ATP
concentrations. The data are derived from measurements of peak
ATP-evoked currents.
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Functional consequences of P2X4 channel
allosteric modulation
IVM (3 µM) produced a maximal potentiation of
ATP-evoked currents at P2X4 channels within 30 sec of application with a time constant
(on) of 14 sec (Fig.
2A; the apparent
bimolecular rate constant for association was 2.2 × 104 M/sec). The
potentiation by IVM was fully reversible within 10 min of washing with
a time constant (off) of 165 sec. The data presented in Figure 2 show that the action (and reversal) of IVM is
use-independent, suggesting that permeation through open P2X channels
is not required. The action of IVM depends on concentration, with an
apparent half-maximal effect (EC50) at ~257
nM (for currents evoked by 3 µM ATP), which is in good agreement with a
kd of 278 nM
determined from kinetic experiments shown in Figure 2, A and B (see Data analysis). However, the IVM
concentration-effect relationship did not reach a well defined plateau
at high concentration (Fig. 2C), vitiating a systematic
description in terms of the Hill function or other dose-response
formulations. With IVM at concentrations  10
µM we observed a downward phase of the
concentration-effect relationship, and this may represent increased
desensitization of P2X4 channels during ATP
application. Importantly, the augmentation by IVM is not attributable
to DMSO, because DMSO caused no marked change in the ATP-evoked current
when it was added to the buffer at 0.1% (control current evoked by ATP
was 181 ± 54 nA, whereas in the presence of DMSO it was
167 ± 91 nA), but 1 µM IVM solutions also contained 0.1% DMSO and produced a large increase in ATP-evoked current (Fig. 2C).
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Figure 2.
Properties of the IVM potentiation of
P2X4 channel currents. A, Time course of
potentiation by IVM. IVM was added at t = 0, just
after the first puff of ATP (3 µM). Potentiation by IVM
occurs within 15 sec (time of the second ATP pulse) and is maximal by
30 sec. B, The oocyte was exposed once to ATP (3 µM) in the absence of IVM (t = 5
min). Fifteen seconds later, IVM was applied for 5 min. At
t = 0, a second ATP application evoked a greater
than fourfold larger ATP response. Thus the ATP-evoked current is
potentiated in the absence of ATP, showing that IVM action is
use-independent. After washout of IVM, the ATP-evoked current returns
to baseline levels within 10-20 min. For A and
B the period of IVM application is shown by the
horizontal bar, and the bold line is an
exponential fit to the data. C, Concentration dependence
of IVM action, tested with pulses of 1 µM ATP. IVM causes
significant potentiation at concentrations 0.1 µM. The
superimposed heavy line represents a fitted curve (Hill
equation) with an EC50 of 257 nM and a Hill
coefficient of 1; note, however, that the IVM potentiation decreases at
10 µM. D. Concentration-effect
relationship for ATP in the absence ( ) and presence ( ) of 3 µM IVM. IVM both increases the maximal current and
decreases the EC50 for ATP. E,
Leak-subtracted current-voltage relationships for the ATP-evoked
current in the absence () and presence () of 3 µM
IVM; note that there is no shift in the reversal potential.
F, Data from experiments like that shown in
D. The ratio of the IVM potentiated current to that of
the ATP-evoked current alone is plotted versus the membrane voltage.
IVM can potentiate the ATP-evoked current at all membrane voltages
tested; in addition, the potentiation is slightly greater at positive
voltages. For this figure the data are plotted as mean ± SEM.
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In control cells P2X4 channels responded to ATP
with an EC50 of 3 µM (Khakh et al.,
1999), with a maximal current of ~2 µA. However, in oocytes
pretreated with 3 µM IVM for 2 min before each
application of ATP, the EC50 was 0.3 µM, and the maximal current increased to ~4 µA (Fig.
2D). This profile of action is similar to the effect
of IVM on 7 nicotinic channels (Krause et al., 1998) and indicates
an allosteric action on P2X4 channels.
We tested for voltage dependence for the action of IVM, as would be
expected if its site of action were inside the pore. The data show that
IVM does not cause any shift in the reversal potential of the
ATP-evoked current-voltage relationship and that the IVM potentiation
occurs at all membrane voltages (120 to + 60 mV with 3 µM IVM and 10 or 100 µM ATP; Fig.
2E). Figure 2F shows these data
plotted as a ratio of the IVM-potentiated current to the ATP-evoked
current alone versus membrane potential. Although the potentiating
effect of IVM clearly occurs at all membrane potentials, it does in
addition display weak voltage dependence; the effect is smaller at
voltages negative to 60 mV than at voltages more positive than this.
In addition to increasing the peak current mediated by
P2X4 channels, IVM also increased the decay time
of the ATP-evoked currents at P2X4 channels by
~30-fold (Fig.
3A,C).
This suggests that IVM increases the affinity of
P2X4 channels for ATP, and that once bound in the
presence of IVM, ATP dissociates slowly from the channel. Alternatively
P2X4 channels may be less likely to enter a
desensitized state after IVM application. We also determined the effect
of IVM on total charge transfer during ATP-evoked currents; this was
increased markedly (36 ± 8-fold) by IVM from 4.1 ± 0.8 µC
in control to 149 ± 37 µC with IVM. There was no change in membrane capacitance (Fig. 3D) during IVM application,
suggesting that the effect of IVM on the peak ATP-evoked currents is
probably not attributable to the insertion of vesicles containing a new population of P2X4 channels. Instead the increase
in peak ATP-evoked current, slowing of deactivation, and decrease in
EC50 for ATP are most readily explained by
modulation of the existing population of P2X4
channels.
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Figure 3.
IVM affects P2X4 deactivation kinetics
but neither basal current nor membrane capacitance. A,
Representative recording showing that IVM produces a large increase in
P2X4 channel current evoked by 3 µM ATP,
although IVM produces no change in the holding current.
B-D, Bar graphs showing summary of ATP-evoked current
properties from cells recorded in the experiment shown in A. B, peak response; C, deactivation half-time;
D, cell capacitance.
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The ATP analog ,-methylene-ATP is a weak agonist at
P2X4 channels, producing <10% of the current
evoked by ATP (Fig. 4A) even at high concentrations (Buell et al., 1996; Seguela et al., 1996;
Michel et al., 1997; Le et al., 1998a), indicating that it is a
low-efficacy agonist (Colquhoun, 1998). An allosteric effector acts
independently of the agonist binding site and would therefore be
expected to affect both ATP and ,-methylene-ATP responses.
Indeed, IVM (3 µM) increased the inward current
evoked by 30 µM ,-methylene-ATP
significantly from 15 ± 6 nA with ,-methylene-ATP alone
to 357 ± 147 nA for ,-methylene-ATP plus IVM
(n = 5); on the same occasions as these experiments
were performed, IVM (3 µM) increased the
current evoked by ATP (30 µM) from 273 ± 75 to 857 ± 284 nA (n = 5). These data
prompted us to investigate the action of IVM (3 µM) on the concentration-effect relationship
of ,-methylene-ATP. IVM converted ,-methylene-ATP from a
weak agonist with an EC50 of >300
µM to a more potent agonist, one with an
EC50 of 19 µM (Fig.
4A), and thus provides direct evidence for allosteric
modulation of P2X4 channels by IVM.
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Figure 4.
Effect of ,-methylene-ATP on homomeric
P2X4 and heteromeric
P2X4/P2X6 channels. A,
P2X4 channels: concentration-effect curves for
,-methylene-ATP in the absence and presence of 3 µM
IVM. Right graphs, Same data on an expanded current
scale. B, Co-expression of
P2X4/P2X6 channels:
concentration-effect curves for ,-methylene-ATP in the
absence and presence of 3 µM IVM. Right
graphs, Same data on an expanded current scale. The
asterisks indicate the threshold concentration for
,-methylene-ATP (see Table 1).
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Channels formed by co-expression of P2X4 and
P2X6 channels
We found no evidence for ATP-evoked or
,-methylene-ATP-evoked currents at homomeric
P2X6 channels expressed in oocytes (with or
without 3 µM IVM), and this indicates that
P2X6 channels are nonfunctional in oocytes (also
see Le et al., 1998a). However, in agreement with previous data when
P2X4 and P2X6 channels were co-expressed, we observed ,-methylene-ATP-evoked currents with a
threshold concentration of 10 µM, and
EC50 of 25 µM (Table
1). The heteromeric
P2X4/P2X6 channel displays
a phenotype distinct from either P2X4 or
P2X6 channels alone (Le et al., 1998a); in particular, the threshold concentration for ,-methylene-ATP at
P2X4 channels was 300 µM (Fig.
4A, Table 1), indicating that the heteromer formed by
co-expression of P2X4 and
P2X6 channels has a higher sensitivity to
,-methylene-ATP than does homomultimeric P2X4 alone (Le et al., 1998a). We next tested the
action of 3 µM IVM in cells expressing
P2X4/P2X6 channels. In the
most important observation, IVM decreased the ,-methylene-ATP
threshold concentration from 10 to 3 µM (Fig.
4B, asterisk). Because homomeric
P2X4 channels do not respond detectably to 3 µM ,-methylene-ATP, this observation suggests but does not provethat IVM also potentiates a distinct receptor species, presumably the
P2X4/P2X6 receptor.
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Table 1.
Properties of ,-methylene-ATP concentration-effect
curves with and without IVM on P2X4 receptors alone or when
expressed in combination with P2X6 receptors
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In oocytes co-injected with P2X4 and
P2X6 mRNA, IVM also potentiated agonist-evoked
currents at ,-methylene-ATP concentrations >10 µM
and decreased the EC50 from 25 to 16 µM (Table 1). These IVM actions could arise via effects
at homomeric P2X4 channels that may also be
present in the oocytes (Fig. 4, Table 1) and are therefore less
convincing than the differences in threshold ,-methylene-ATP concentration.
IVM modulates only the I1 states of
P2X4 channels
Recent data show that P2X4 channels display
biphasic currents during prolonged ATP applications in
Na+ solutions (Fig.
5, I1,
I2). The second phase is attributable to a
history-dependent increase in permeability (Khakh et al., 1999;
Virginio et al., 1999). We tested whether IVM modulates both
I1 and
I2, and found that only the initial ATP-evoked current (I1, 10-20 sec ATP
applications), is potentiated (Fig. 5). This provides further evidence
that I2 occurs independently of the
magnitude of inward current during I1
(Khakh et al., 1999). However, it remained possible that the
potentiated I1 current was in fact
caused by a recruitment of some channels from the I2 state, such that they now opened
with brief ATP applications. To address this directly, we determined
NMDG+ permeability ratios for the control
and IVM-potentiated currents, because
NMDG+ is substantially permeable during
the I2 current, and is only weakly
permeable during I1. The reversal
potential for ATP-evoked current alone
(I1) was 50.5 mV, whereas that for
the IVM-potentiated current was 47.9 mV (see Fig. 5;
n = 5). In comparison, the reversal potential for
I2 is typically 30 mV (Khakh et al.,
1999). The pNMDG+/pNa+
ratios for control ATP-evoked currents and the IVM-potentiated currents
were identical (Fig. 5D). This indicates that control currents and IVM-enhanced currents have equally low permeability to
NMDG+. Therefore, the previously reported
low-selectivity I2 state (Khakh et
al., 1999) does not contribute detectably to the IVM-potentiated current; IVM potentiates only I1. The
mechanism(s) of ion selectivity changes are not fully understood but
likely involve structural rearrangements in the permeation pathway
(Khakh et al., 1999; Virginio et al., 1999). We presume that IVM binds
less strongly to the I2 state than to
the I1 state or does not enhance the
conformational change that leads to opening of this state. This extends
previous findings demonstrating that the distinct selectivity states of P2X7 channels can be probed differentially by
blockers (for review, see Ralevic and Burnstock, 1998).
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Figure 5.
IVM potentiates the high-selectivity
(I1) but not the low-selectivity
(I2) state of the P2X4 channel.
A, Representative 3 µM ATP-evoked current
from an oocyte expressing P2X4 channels; the solid
bar indicates the period of ATP application. B,
Representative recording of a 3 µM ATP-evoked current
during exposure to IVM (3 µM). C, Bar
graph summarizing data from experiments such as those illustrated in
A and B from seven cells; only
I1 is potentiated. D, Table
showing reversal potentials for control and IVM-potentiated
I1 in both Na+ and
NMDG+ solutions, as well as the ratio of
NMDG+ to Na+ permeability
(n = 5). The NMDG+ reversal
potential for I2 is typically 30 mV (Khakh
et al., 1999).
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Allosteric modulation in a clonal mammalian cell line
To circumvent desensitization we applied ATP every 2 min to HEK
293 cells transfected with P2X4 channels. After
three doses of ATP we applied IVM (3 µM) for 4 min and
repeated the ATP applications. These experiments show that IVM can
augment ATP-evoked currents in P2X4-transfected
mammalian cells. The peak effect and kinetics resemble those observed
for IVM action in oocytes, demonstrating that the effect of IVM on
P2X4 channels is independent of the host cell
(Fig. 6; top graph is from a
single HEK 293 cell; bottom graph is an average from four
such cells).
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Figure 6.
IVM potentiates P2X4 channel currents
in a clonal mammalian cell line. Top panel, Amplitudes
of ATP (10 µM)-evoked currents from a single HEK 293 cell; ATP was applied every 2 min and IVM (3 µM) was
added for the period indicated. The sample traces at the
right were elicited from this cell at the time points
indicated. Bottom panel, Averaged data from four such
cells tested with the same protocol.
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Brainstem and hippocampal neurons expressing P2X channels
The brainstem MNV contains proprioceptive neurons that express
transcripts for P2X4, P2X5,
and P2X6 (Collo et al., 1996); P2X responses are
recorded on MNV neuron somata and central terminals (Cook et al., 1997;
Khakh et al., 1997; Khakh and Henderson, 1998a). In agreement with
these studies, ATP (50 µM) puffed directly onto the
somata evoked inward currents in these neurons, but IVM (5 µM) did not affect these ATP-evoked currents (<5%
change; n = 3; Fig. 7).
These data provide further evidence that the P2X channels in MNV
neurons do not comprise homomeric P2X4 channels
and thus extend previous findings using electrophysiological approaches (Cook et al., 1997; Khakh et al., 1997).
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Figure 7.
Endogenous P2X channels in MNV and hippocampal CA1
neurons. A, Bright-field image of a brainstem slice
containing the MNV. MNV neurons can be seen as round neurons. Scale
bar, 10 µm. B, Image of the CA1 region in a
hippocampal slice. CA1 pyramidal neurons can be seen extending a
dendrite into the stratum radiatum. Scale bar, 10 µm.
C, Representative ATP-evoked currents from an MNV
neuron. D, Representative traces of ATP-evoked currents
from a CA1 neuron. In both cases recordings in the presence of 2-5
µM IVM are also shown. The bar graph
represents average values for MNV neurons (n = 3),
whereas the scatter graph shows data for nonresponding
CA1 neurons (), the three responding neurons ( ;
arrows), and the average for all 13 neurons ().
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Recent studies in CA1 pyramidal neurons have demonstrated rapid
ATP-evoked currents and fast ATP synaptic transmission (Pankratov et
al., 1998; Proctor and Dunwiddie, 1998), but the identity of the P2X
channels in CA1 neurons is uncertain, in part because they express many
mRNA species, including P2X4 (Collo et al., 1996). If IVM could modulate the ATP-evoked currents in these neurons,
this might indicate the presence of homomeric
P2X4 or possibly
P2X4-containing channels. However, when responses
to locally applied ATP were recorded from identified hippocampal CA1
pyramidal neurons, ATP-evoked currents in the majority of neurons were
unaffected by IVM (13 of 13 neurons responded to ATP, but 10 of these
13 did not respond to IVM; Fig. 7). A potential concern in these
experiments was the possibility that IVM does not penetrate brain
slices effectively. However, in parallel experiments we found that 2 µM IVM markedly potentiated fast
GABAA channel currents evoked by puffing GABA on
to CA1 neurons; the effect of IVM was both rapid and reversible
(n = 3), suggesting that IVM does in fact penetrate
brain slices. In three neurons we observed larger ATP-evoked currents
in the presence of IVM than in control. However when the data from all
13 cells were averaged, the increase was not significant (174 ± 85%
of control). To gain further insight as to the nature of the P2X
channels expressed on CA1 neurons and to allow comparison with other
neurons, we next tested the action of ,-methylene-ATP.
,-Methylene-ATP is a weak agonist at homomeric
P2X4 channels (Fig. 4A) but is
a potent agonist at known CNS heteromeric channels (Le et al.,
1998a; Torres et al., 1998); it is known to activate P2X
channels that are endogenously expressed in neurons, some of which are
most likely heteromeric assemblies [e.g., sensory neurons (Chen et
al., 1995; Khakh et al., 1995a; Lewis et al., 1995; Cook et al., 1997),
medial habenula neurons (Edwards et al., 1992), and locus coeruleus
neurons (Nieber et al., 1997)]. When we puffed ,-methylene-ATP
(50 µM) onto CA1 neurons we observed large fast
inward currents (98 ± 19 pA; six of six neurons). This
observation, in combination with the lack of effect of IVM on most
cells, provides the first functional evidence to indicate that
homomeric P2X4 channels are not expressed in the
majority of adult hippocampal CA1 region neurons, despite the fact that
in situ hybridization studies show that their transcripts are abundant here (Buell et al., 1996; Collo et al., 1996); overall this supports and extends previous findings using immunocytochemistry (Le et al., 1998a).
Conclusions about the lack of effect of IVM against ATP-evoked currents
in brain slice neurons would be invalid if for unknown reasons IVM does
not modulate P2X4 channels when expressed in neurons. To address this directly we transfected embryonic hippocampal neurons with P2X4 and used EGFP as a transfection
reporter. Untransfected neurons show no ATP-evoked responses (Fig.
8C,D, top; 0.1-1
mM ATP), consistent with in situ
hybridization studies showing minimal levels of
P2X4 transcripts in the embryonic hippocampus
(Buell et al., 1996). We used the presence of EGFP fluorescence (24-48 hr after transfection) to identify transfected neurons; such neurons responded with large ATP-evoked inward currents or membrane
depolarizations (Fig. 8C,D). We next tested the action of
IVM (3 µM) on ATP-evoked currents in
transfected hippocampal neurons, and IVM markedly augmented the
ATP-evoked currents in all neurons tested (Fig. 8E,F). These experiments provide direct
evidence that IVM can modulate P2X4 channels when
expressed in neurons and allow unambiguous interpretation of the data
showing little effect of IVM on neurons in hippocampal slices (see
above and Fig. 7).
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Figure 8.
P2X4 channels expressed in embryonic
hippocampal neurons. A, B, Bright-field
(A) and fluorescence (B)
image of embryonic hippocampal neurons transfected with plasmids for
P2X4 and EGFP. Note that the morphology of transfected and
nontransected neurons is very similar, and this appearance is typical
of healthy cells. The extended fluorescent cells in B
are glial cells. C, Top trace,
Representative voltage-clamp currents recorded from an untransfected
neuron showing no ATP-evoked currents (holding potential, 60 mV);
bottom trace, large ATP-evoked currents in a transfected
hippocampal neuron. D, Top trace,
Representative current-clamp recording (resting potential, 52 mV)
from an untransfected neuron showing no ATP-evoked change in membrane
potential; bottom trace, large ATP-evoked depolarization
in a transfected hippocampal neuron. Action potentials have been
clipped. E, Representative traces from a transfected
hippocampal neuron showing 10 µM ATP-evoked currents
before and after 3 µM IVM application. F,
Summary of experiments such as those illustrated in E
from six neurons. Note that the size of the ATP-evoked current varies
markedly between neurons, but in all neurons IVM potentiates the
current.
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DISCUSSION |
The main finding of the present study is that IVM can augment
ATP-evoked currents at homomeric P2X4 channels,
and possibly P2X4/P2X6
channels, by an allosteric mechanism.
Functional sequelae of P2X4 channel allosterism
IVM is an agonist at glutamate-gated chloride channels of
invertebrates (Cully et al., 1994) and is a positive allosteric effector of GABAA and 7 nicotinic channels
(Krusek and Zemkova, 1994; Krause et al., 1998). These ligand-gated
channels all belong to the four transmembrane domains per subunit
superfamily (Krusek and Zemkova, 1994; Krause et al., 1998). In
contrast, the P2X channels are unrelated to these channels both at the
level of amino acid sequence and protein topology (North, 1996). At
present the binding site for IVM on any ion channel is unknown;
sequence comparisons among P2X4 channels,
glutamate-gated chloride channels, or 7
nicotinic channels reveal no real clues. Nevertheless, IVM is most
likely acting on P2X4 channels themselves and not
on components that are endogenous to oocytes, such as calcium-activated
chloride channels, for two reasons. First, the effect of IVM was
specific to P2X4 and possibly
P2X4/P2X6 channels;
P2X2, P2X3,
P2X2/P2X3, or
P2X7 channels were unaffected, although these
channels are also calcium-permeable. The evidence for potentiation of
P2X4/P2X6 channels is not
decisive. Second, the kinetics and degree of potentiation for IVM were
similar in oocytes, HEK-293 cells, and embryonic hippocampal neurons,
clearly demonstrating that the effect of IVM is independent of the host
cell in which P2X4 channels are expressed. The
present results demonstrate that IVM specifically increases ATP-evoked
currents mediated by P2X4 channels and suggest that P2X4 channels contain an allosteric binding
site for IVM. IVM does not markedly affect ATP-evoked currents mediated
by other P2X subunits expressed in oocytes, such as the
P2X2 channels, which are found in the central and
peripheral nervous systems (for review, see Ralevic and Burnstock,
1998), P2X3 channels, which are abundant in pain
sensors (Chen et al., 1995; Lewis et al., 1995; Cook et al., 1997), or
P2X7 channels, which are found in immune cells
(Nuttle and Dubyak, 1994; Surprenant et al., 1996; Collo et al., 1997).
Thus, IVM may be a useful pharmacological tool that can discriminate
central P2X4 from other P2X channels.
Previously, radioligand-binding studies have suggested that ATP binding
to P2X4 channels can be modulated allosterically
(Michel et al., 1997). By using IVM we (1) identify a novel chemical
structure that specifically probes P2X4 channels,
(2) characterize the effects of this modulation on the properties of
P2X4 channels, and (3) show that
P2X4 channel modulation by IVM is by an
allosteric mechanism. In agreement with published functional studies
that show suramin to be ineffective at P2X4
channels (Buell et al., 1996; Seguela et al., 1996; Soto et al., 1996),
we find no evidence to show that suramin can modulate
P2X4 channel currents.
An allosteric action of IVM on P2X4 is supported
by the observations that IVM rarely produced a change in the holding
current by itself but potentiated the current evoked by ATP and slowed its deactivation. The ATP analog ,-methylene-ATP is a weak
agonist at P2X4 channels (Buell et al., 1996) and
evokes ~10% of the current evoked by ATP at similar concentrations.
Interestingly, however, ,-methylene-ATP and ATP both bind with
relatively high affinity to P2X4 channels (Michel
et al., 1997), suggesting that the low-potency ,-methylene-ATP in
functional experiments may be attributable to a lower intrinsic
efficacy or a lower ability to gate the channel once bound. In the
presence of IVM, ,-methylene-ATP becomes a potent agonist,
another familiar property of allosteric effectors. If further
experiments lead to knowledge about the binding site and mechanisms of
allosterism by IVM, this will be a useful approach to study the events
leading to P2X channel gating after ATP binding.
Responses in neurons
Having established the specificity of IVM as an allosteric
effector of P2X4 channels in oocytes, HEK cells,
and embryonic hippocampal neurons, we next tested its actions in adult
brain neurons that express mRNA transcripts for
P2X4. Our initial goal was to determine whether
homomeric P2X4 channels are expressed in these
neurons. In the brainstem MNV we found that IVM did not affect
ATP-evoked currents, and this is consistent with previous conclusions
that the P2X channels in MNV neurons are most like P2X5 (Cook et al., 1997) or a combination of P2X
channels (Khakh et al., 1997). The CA1 field of the hippocampus is
abundant in P2X4 mRNA, and previous investigators
therefore suggested that homomeric P2X4 channels
might be expressed in these cells (Buell et al., 1996; Collo et al.,
1996). To our surprise, IVM did not affect ATP-evoked currents in these
neurons, suggesting that homomeric P2X4 channels
are not located on the soma of pyramidal cells, but functional activity
of these channels on the dendrites was not tested. To the extent that
our data suggest that heteromeric P2X4/P2X6 channels are also
potentiated by IVM, we may conclude that such channels are also not
expressed in these cells. It is possible that the endogenous P2X
channels in CA1 comprise (1) other heteromeric P2X channels, (2) a
mixed population of P2X channels, or (3) a new P2X channel and/or (4)
that homomeric P2X4 channels are found in only a
subset of CA1 neurons. The marked enhancement of responses in a small
fraction of hippocampal neurons (Fig. 7F) is
consistent with this last conclusion, but further studies are needed to
discriminate between these four possibilities. Known heteromeric P2X
channel assemblies are activated by ,-methylene-ATP, and it has
been proposed that they may constitute some of the P2X channels found
in the CNS, possibly including those in the hippocampus (Le et al.,
1998a; Torres et al., 1998, 1999). This proposal is supported by
data showing overlapping distribution of many P2X subunit mRNAs
throughout the nervous system (Collo et al., 1996). Interestingly,
recent data from the CA1 region of the hippocampus also show that the
P2X channels can be activated by ,-methylene-ATP (this study) and
partially blocked by antagonists (Pankratov et al., 1998; Proctor and
Dunwiddie, 1998). These properties, along with the insensitivity to
IVM, suggest that the predominant ATP-evoked current (Pankratov et al.,
1998) in the CA1 of the hippocampus is mediated by neither homomeric
P2X4 nor heteromeric P2X4/P2X6 channels but
rather by channels with properties that may be most similar to those
described for other heteromeric channels (Lewis et al., 1995; Le et
al., 1998a; Torres et al., 1998, 1999). The P2X4
message is not restricted to the hippocampus and is found in many brain
areas (Collo et al., 1996); it remains to be determined whether some of
these neuron subsets do indeed express homomeric P2X4 channels.
Allosteric modulators of other ligand-gated ion channels have become
important experimental tools to determine the contribution of distinct
channel types to the function of brain synapses.
P2X2 and P2X3 receptors are
modulated by cations such as Zn2+,
H+, and Ca2+,
and it is possible that such modulation has a physiological role as
well (King et al., 1997; Stoop et al., 1997; Wildman et al., 1997,
1998; Cook et al., 1998). The lack of pharmacological probes for
P2X4 channels has hindered understanding of their
function, but in the present study we have identified IVM as the first
agent that discriminates P2X4 from other P2X
channels. Remarkably, IVM can increase the gating efficiency and slow
the deactivation kinetics of ATP at P2X4
channels, suggesting that the open probabilities of
P2X4 channels increase in the presence of IVM.
Overall IVM evokes a profound gain-of-function phenotype in
P2X4 channels. It remains to be determined
whether the site on the P2X4 protein that IVM
probes is also the target for an endogenous allosteric effector.
| |
FOOTNOTES |
Received April 5, 1999; revised June 1, 1999; accepted June 14, 1999.
This work was supported by a Wellcome Trust (UK) International Prize
Fellowship (to B.S.K.), National Institutes of Health Grant NS-11756
and NS-29173, and grants from the Department of Veterans Affairs.
Thanks to Dr. I Chessell (Glaxo Wellcome) for providing P2X cDNA clones
and Dr. Phillipe Seguela for the P2X6 cDNA. We thank S. McKinney and H. Li for help with preparation of hippocampal
cultures and Xenopus oocytes and W. B. Smith for guidance on use of the Biolistics gene gun.
Correspondence should be addressed to Baljit S. Khakh, Division of
Biology, 156-29, California Institute of Technology, Pasadena, CA 91125.
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TOP
ABSTRACT
INTRODUCTION
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