Tightening of the ATP-binding sites induces the opening of P2X receptor channels.

Abstract

The opening of ligand-gated ion channels in response to agonist binding is a fundamental process in biology. In ATP-gated P2X receptors, little is known about the molecular events that couple ATP binding to channel opening. In this paper, we identify structural changes of the ATP site accompanying the P2X2 receptor activation by engineering extracellular zinc bridges at putative mobile regions as revealed by normal mode analysis. We provide evidence that tightening of the ATP sites shaped like open 'jaws' induces opening of the P2X ion channel. We show that ATP binding favours jaw tightening, whereas binding of a competitive antagonist prevents gating induced by this movement. Our data reveal the inherent dynamic of the binding jaw, and provide new structural insights into the mechanism of P2X receptor activation.

Full text

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Tightening of the ATP-binding sites induces the opening of P2X receptor
channels

By Ruotian Jiang1, Antoine Taly1, Damien Lemoine, Adeline Martz, Olivier Cunrath &
Thomas Grutter*


Laboratoire de Biophysicochimie des Récepteurs Canaux, UMR 7199 CNRS, Conception et
Application de Molécules Bioactives, Faculté de Pharmacie, Université de Strasbourg, 67400
Illkirch, France. 1 These authors contributed equally to this work.

* To whom correspondence and request for materials should be addressed
(grutter@unistra.fr).

Running title: Inherent dynamics of the ATP sites in P2X receptor

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Abstract
The opening of ligand-gated ion channels in response to agonist binding is a fundamental
process in biology. In ATP-gated P2X receptors, little is known about the molecular events
that couple ATP binding to channel opening. In this paper, we identify structural changes of
the ATP site accompanying the P2X2 receptor activation by engineering extracellular zinc
bridges at putative mobile regions as revealed by normal mode analysis. We provide evidence
that tightening of the ATP sites shaped like open ‘jaws’ induces opening of the P2X ion
channel. We show that ATP binding favors jaw tightening, whereas binding of a competitive
antagonist prevents gating induced by this movement. Our data reveal the inherent dynamic of
the binding jaw, and provide new structural insights into the mechanism of P2X receptor
activation.

Keywords: purinergic receptors/ ligand-gated ion channels/ normal mode analysis/ zinc-
binding sites/ allosteric mechanism

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P2X receptors are membrane cation channels gated by extracellular ATP. They are
involved in many physiological functions as diverse as synaptic transmission, presynaptic
modulation, taste sensation, pain signaling, inflammation and intestinal motility (Burnstock,
2008; Khakh & North, 2006). Upon ATP binding, a large conformational change opens the
transmembrane pore. This process, referred to as gating, allows Na+, K+ and Ca2+ ions to
rapidly flow down their electrochemical gradients, leading to the depolarization of the cell
and downstream signaling.
The crystal structure of the zebrafish P2X4 (zfP2X4) receptor has recently been solved
in the absence of ATP by X-ray crystallography at 3.1 Å resolution (Kawate et al, 2009). It
confirms previous biochemical evidence that the association of three subunits arranged
around the three-fold axis of symmetry forms the ion channel (Jiang et al, 2003; Nicke et al,
1998). Each subunit (there are seven identified so far in mammals, termed P2X1 through
P2X7) has intracellular amino and carboxyl termini, and two transmembrane α-helices,
termed TM1 and TM2, joined by an ectodomain, which contains highly conserved amino acid
residues necessary for ATP function (Browne et al, 2010; Evans, 2010; Young, 2009). The
ion-conducting pathway is defined by three TM2, each coming from a distinct subunit,
surrounded by three peripheral TM1 (Kawate et al, 2009).
Very recently, we have localized ATP-binding sites in the rat P2X2 (rP2X2) receptor
ectodomain through an engineered affinity labeling approach (Jiang et al, 2011). This consists
in creating a covalent bond between a single cysteine mutation introduced in the putative
binding site and a sulfhydryl-reactive 8-thiocyano-ATP derivative (NCS-ATP). This reagent
is a rP2X2 agonist that selectively cross-linked ATP sites at the level of positions Asn140 and
Leu186. In the light of the crystal structure, we showed that these sites are located in large
and deep intersubunit cavities, separated by ~45 Å from the ion pore region (Jiang et al,
2011). Interestingly, the binding cavity is shaped like an open jaw, which is framed in its

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upper side by the cysteine-rich ‘head’ domain, which contains the identified residue Asn140,
and in its bottom side by the ‘dorsal fin’ domain (Figure 1A). Whether or not the open jaw
deforms upon ATP binding remains undetermined.
Extracellular zinc modulates many different synaptic targets in the forebrain, including
ligand-gated ion channels (LGICs) (Paoletti et al, 2009), among which the P2X receptor is an
emerging candidate (Huidobro-Toro et al, 2008; Lorca et al, 2011). Depending on P2X
subunit subtypes (Jarvis & Khakh, 2009) and species (Tittle & Hume, 2008), zinc displays
opposite effects, ranging from inhibition to potentiation of the ATP response. For instance, it
is known that the extracellular divalent cation inhibits the human P2X2 receptor activity,
while it strongly potentiates the ATP response in the rP2X2 receptor (Tittle & Hume, 2008).
More detailed studies identified His120 and His213 from two adjacent subunits as
contributing residues to potentiation in the rat receptor (Nagaya et al, 2005; Tittle et al, 2007).
Interestingly, these two histidines are located respectively in the head and dorsal fin domains,
and therefore are positioned on the two ‘lips’ of the open jaw. In the rP2X2 homology model
based on the X-ray structure of zfP2X4, these two residues are too far apart to coordinate Zn2+
(Figure 1B). Because the structure was solved in the absence of ATP, and thus likely
represents a closed channel state, this suggests that during ATP activation the head and dorsal
fin domains must move closer to each other, allowing Zn2+ coordination. This hypothesis,
although attractive, has no direct support.
In this paper, we studied protein conformational changes in the rP2X2 receptor
accompanying ion channel activation by combining normal mode analysis (NMA) and
experimental data. We successfully engineered receptors with histidines, in which
extracellular Zn2+ was able to bridge specifically distant regions of the jaw that were predicted
to come closer to each other during gating. By using a pore mutation background that
displayed spontaneous openings (Cao et al, 2007), we showed that Zn2+ not only gated

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channel receptors in which the native Zn2+-potentiating site was present, but also in receptors
in which the native site was transferred to another part of the jaw. Finally, we provided
evidence that binding of ATP favored zinc activation through an allosteric conformational
change, whereas binding of the competitive P2X antagonist 2',3'-O-(2,4,6-trinitrophenyl)-
ATP (TNP-ATP) prevented Zn2+ activation. Based on these results, we propose a model in
which ATP gating requires tightening of the two lips of the binding jaw, whereas binding of
TNP-ATP prevents jaw closure-induced gating. This original mechanism is reminiscent of
those found in other LGICs.

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RESULTS
NMA suggests motions of the head domain coupled to ATP binding. NMA is a
computational approach that can efficiently predict the collective motions and conformational
flexibilities of biological macromolecules (Bahar, 1999). This method approximates the
surface of the conformational landscape of the macromolecule and gives a decomposition of
the movements into discrete modes. The elastic-network model, which is a simplified, but
physically meaningful, representation of the interaction between atoms, is based on simple
springs that connect close pairs of atoms in the structure (Tirion, 1996). At variance to
molecular dynamics, this method leads to a time-independent equation that can be solved in
closed form analytically, and therefore allows studying slow and collective conformational
transitions that are biologically relevant (Krebs et al, 2002). More recently, NMA has been
useful in studying gating transitions in other ion channels (Amiri et al, 2005; Cheng et al,
2006; Sukumaran et al, 2011; Taly et al, 2005; Yang et al, 2009; Zhu & Hummer, 2009). To
speed up simulation, we represented the amino acids by Cα atoms (Hinsen, 1998). NMA
using this approximation was shown to give a fair description of protein flexibility (Bahar,
1999; Bahar & Rader, 2005; Tama & Brooks, 2002). We used NMA to reveal inherent
motions of the rP2X2 receptor homology model based on the closed-state zfP2X4 structure.
We computed and selected the first ten non-trivial normal modes, which revealed structural
changes of the protein (Figure 1A and Supplementary Figure 1). Mild pore openings for
modes 7, 9 and 11 were detected, but apparently not sufficient to allow ion flux
(Supplementary Figure 2), suggesting that although these modes could, in principle, be
involved in gating of the rP2X2 receptor, none described the transition in full.
Because no clear pore opening was observed in all tested modes, we decided to
perturb the modes by the presence of ATP. This approach has already been successfully
applied to another LGIC (Taly et al, 2006), and approximates changes that occur on protein

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dynamics upon ligand binding (Ming & Wall, 2005; Mitternacht & Berezovsky, 2011; Zheng
et al, 2005). We docked ATP into the binding site, according to our recent affinity labeling
data (Jiang et al, 2011), and computed NMA in the presence of ATP. We found that changes
in frequency of the non-trivial modes due to ATP ranged from 1 to 26%. Interestingly, mode
10, which corresponds to an asymmetric motion of the three heads out of the membrane
plane, displayed the largest frequency difference, suggesting that the presence of ATP
substantially modified the energy needed to explore this mode (Figure 1A and Supplementary
Figure 3). These calculations thus suggest that the domains surrounding the ATP-binding site
experience significant mobility that may be related to ATP binding, and potentially to P2X
gating.
Interestingly, the distance separating Cα atoms from residues His120 to His213 that
were initially found to be too far apart to create a Zn2+-coordinating site, shortened following
mode 10 exploration from 15.5 to 12.2 Å (Figure 1B and C). More importantly, we succeeded
in forming a Zn2+-binding site through these histidine residues, and the distance and angle
measured between Zn2+ and the coordinating atom NE2 of the histidine residues were close to
those obtained from the analysis of 111 crystal structures of Zn2+-binding proteins available in
the Protein Data Bank (PDB) (Alberts et al, 1998). Therefore, these results suggest that mode
10 is able to form the native Zn2+-potentiating site in the rP2X2 receptor, by getting residues
His120 and His213 together.

Zinc activates the T339S mutant. Although previous studies (Nagaya et al, 2005; Tittle &
Hume, 2008; Tittle et al, 2007) have shown that zinc potentiates ATP currents, there is no
direct evidence supporting the hypothesis that close apposition of residues His120 and His213
gates the ion channel. We thus asked whether zinc could open the ion channel, by its own, in
the absence of ATP. In human embryonic kidney (HEK)-293 cells expressing the wild-type

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(WT) rP2X2 receptor, extracellular zinc failed to produce detectable currents as assayed by
whole-cell (Figure 2B) and single-channel (Supplementary Figure 4) patch-clamp
electrophysiology. We decided to introduce the T339S mutation, which is known to confer to
the channel significant spontaneous openings in the absence of ATP (Cao et al, 2007), with
the hope that the mutation would be able to reduce the energy barrier for the receptor to reach
the open state. Outstandingly, Zn2+ produced significant currents that were concentration
dependent with maximal current (58 ± 22 pA/pF, n = 5) representing about 4% of that evoked
by ATP (Figure 2A and B). Currents were specific to Zn2+ binding, and not due to
contamination by trace amounts of ATP because the double mutant K69A/T339S, in which
the ATP-binding site was disrupted (Cao et al, 2007), still responded to Zn2+, with a
concentration-response curve and maximal current that were very similar to those measured
for the T339S mutant (33 ± 12 pA/pF, n = 5) (Figure 2B). Furthermore, these currents were
specific to the presence of both residues His120 and H213 because the triple mutant
H120A/H213A/T339S and corresponding single histidine mutants H120A/T339S and
H213A/T339S, in which the native zinc-potentiating site was abolished (Nagaya et al, 2005),
were not activated by Zn2+ anymore, while ATP current density remained robust (674 ± 270
pA/pF, n = 4; 432 ± 106, n = 6; 473 ± 51, n = 4 for H120A/H213A/T339S, H120A/T339S and
H213A/T339S, respectively) (Figure 2B). These results thus demonstrate that coordination of
Zn2+ ions by the pair of histidines His120 and His213 gate the T339S mutant channel.
To further gain insights into the mechanism underlying zinc activation, we made
single-channel recordings. In outside-out patches excised from HEK cells expressing the
T339S mutant, a low concentration of Zn2+ (10 μM) further increased the nominal open
probability (NPo) of single-channels that were spontaneously open (Figure 2C and E).
However, there was no change of the unitary conductance that was, in fact, similar to that
determined previously for the WT receptor (Cao et al, 2007; Jiang et al, 2011) in the presence

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of ATP (Figure 2E). Distributions of the spontaneous open times were best fitted with the sum
of two exponential components, whereas those analyzed in the presence of zinc showed both
a large increase of the proportion of the slowest component —with no change of time
constants— and the occurrence of a third, slower component (Figure 2D and Supplementary
Table 2). This resulted in an increase of the mean open-time from 1.2 to 2.2 ms, suggesting
that zinc further stabilizes the spontaneous open-channel state(s) in the T339S mutant.
Analysis of the shut-time distributions revealed, in contrast, a decrease of the mean shut-time
from 84.8 to 51.6 ms, suggesting that zinc increases pore opening frequency. Because some
patches contained more than one active channel as evidenced by the presence of double
openings that represented no more than 2% of total transitions (see Materials and Methods),
absolute values of shut-time constants may not be meaningful. Taken together, these data
clearly show that i) the opening probability of the channel increases when zinc ions are bound
to His120 and His213, ii) the motion bringing these two residues together is coupled to pore
opening, and iii) this motion can occur without ATP binding.

Jaw tightening gates the ion channel. To further explore the dynamic motion of the ATP-
binding site during gating, we decided to transfer the native zinc-potentiating site to another
place of the jaw with the hope that the divalent cation would also bridge the engineered
histidines in the activated state(s). To select all possible pairwise positions, we computed a
matrix representing the relative movement of the protein between initial and final models
explored in mode 10, and selected pairs of residues in which their relative movement
measured from their respective Cα atoms was greater than 2.5 Å, and the distance separating
these Cα atoms was less than 13 Å in the final model, compatible with a putative zinc-
coordinating site (Alberts et al, 1998). Interestingly, we found that two previously
unidentified regions of the jaw, named loops A and B corresponding respectively to a

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segment of the β5-β6 loop from the head domain and the β7-α2 loop linking the head to the
right flipper, satisfied these geometrical constrains, suggesting that these loops may get close
enough together (Figure 1C).
To test experimentally this theoretical prediction, we first abolished the native zinc-
potentiating site in the rP2X2 receptor channel as reported previously (Nagaya et al, 2005).
We used 20 μM zinc, because higher concentrations inhibit the activity of the receptor in
which the zinc-potentiation site was removed (Tittle & Hume, 2008). As expected, no
potentiation by Zn2+ of a low concentration of ATP that elicited ~10% of the maximal
response (EC10) was observed in the double mutant H120A/H213A, while strong potentiation
was detected in the WT rP2X2 receptor (Figure 3A). In addition, ATP sensitivity and Hill
coefficient determined for the double mutant H120A/H213A remained similar to those found
for the WT receptor (Supplementary Table 1). Second, we mutated in this background,
referred to below as *, the selected residues from loops A and B into histidines. All mutants
yielded robust ATP currents and EC50 values that did not exceed 4-times the value obtained
for the background * (Supplementary Table 1), suggesting that these mutations did not alter
substantially the ability of ATP to bind to, and gate the receptor channel.
Interestingly, when paired with the T170H* mutation, the histidine mutants in which
loop A was investigated showed a gradual increase of zinc potentiation, resulting in a more
than 4-fold increase of the ATP response for the pair S116H/T170H*, whereas a weak or no
potentiation was recorded for the pairs including the G169H* mutation (Figure 3A). In the
control experiments, in which the single histidine mutants were tested, virtually no
potentiation was detected, except for S116H* and T170H*, for which a weak, but
reproducible, zinc potentiation was recorded (Figure 3A). Because of these remaining
potentiations, we considered the possibility that the engineered zinc-binding site may be
formed exclusively from one side of the binding jaw, including unidentified endogenous

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residues. To address this issue, we sought to determine in our molecular model which
endogenous residues carrying possible zinc-coordinating side chains were closely positioned
to residues Ser116 and Thr170 (Figure 3C). We found that replacement of Asp168 with
alanine from one side of the binding jaw (i.e. loop B) in the S116H* mutant abolished the
remaining potentiation (Figure 3B and C). Similar results were obtained when H152A from
the other side (i.e. the head domain) was introduced in the T170H* mutant (Figure 3B and C),
suggesting that the remaining potentiation resulted only from a closure of the two lips of the
jaw. Surprisingly, only small (~0.2-fold) zinc potentiations were observed when D168A and
H152A were individually introduced in the double histidine mutant S116H/T170H* (Figure
3B), suggesting that both Asp168 and His152 side chains are necessary, but not sufficient to
mediate the strong zinc potentiation observed in the S116H/T170H* mutant receptor (~4-
fold). In support to this, the engineered zinc-binding site was reconstituted in the tightened
jaw model, including the endogenous residue His152. Note, however, that Asp168 that was
not constrained in modeling, did not directly contact zinc, but was located sufficiently close
(~4 Å) to the engineered zinc-binding site (Figure 3D). Altogether, these data argue against
the possibility that potentiations observed in both single and double histidine mutants are due
to zinc-binding sites formed exclusively from one side of the binding jaw; instead, they
strongly suggest that both sides of the binding jaw are needed to form the engineered
potentiation site.
Introduction of mutations S116H/T170H* in the T339S mutant enabled zinc to gate
the ion channel in the absence of ATP, as revealed by both whole-cell (Figure 3E) and single-
channel recordings (Figure 3F). Compared to the T339S mutant, S116H/T170H/T339S*
mutant displayed a decrease of the Zn2+-gated current density (7 ± 2 pA/pF, n = 6), but a
marked increase of about 10-fold in Zn2+ sensitivity (Figure 3E). Detailed analysis of the
open-time distributions showed a similar trend to that observed for the T339S mutant. Indeed,

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distributions of the spontaneous open times were best fitted by the sum of two exponential
components, whereas those analyzed in the presence of zinc showed an increase of the
proportion of the slowest component —with little change of time constants— and the
occurrence of a third, slower component (Figure 3G and Supplementary Table 2). Similarly,
there was an overall increase of both the mean open-time (from 1.1 to 1.9 ms) and NPo, and
no change of the unitary conductance (Figure 3H). Analysis of the shut-time distribution
revealed a decrease of the mean shut-time from 84.5 to 38.7 ms, suggesting that zinc increases
pore opening frequency of the S116H/T170H/T339S* mutant channel, as observed for the
T339S mutant. Taken together, these results indicate that i) zinc gates the engineered channels
by stabilizing the open channel state(s), and ii) the distance separating the Cα atoms from
residues Ser116/His152 to Asp168/Thr170, along with that separating Cα atoms from
residues His120 to His213, must shorten in the activated state(s) to be compatible with zinc-
coordinating sites (Figure 3D). We conclude from these experiments that tightening of the
two lips of the ATP-binding jaw favors pore opening.

Agonist binding favors jaw tightening, whereas binding of competitive antagonist
inhibits gating induced by this movement. We have shown that in the absence of ATP,
Zn2+-induced jaw tightening favors pore opening. To challenge the hypothesis that ATP also
favors tightening, we tested if ATP was able to potentiate zinc currents through an allosteric
mechanism. Validation of this hypothesis is important, because it remains possible that in the
presence of ATP, Zn2+ may directly help the agonist to bind to the receptor and this,
consequently, may produce longer residence time for ATP at its binding site. In that case,
ATP by itself may induce another protein movement that is not related to jaw tightening. To
address this issue, we engineered receptors, in which ATP and zinc were not able to mediate
their effect on the same subunit interface. We thus co-expressed K69A/T339S mutant that is

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defective in ATP binding with H213A/T339S mutant that is not activated by zinc. The
resulting trimeric heteromers contain K69A/T339S and H213A/T339S subunits with a
stoichiometry of either 1:2 or 2:1 (Figure 4A). As shown in Figure 4A, in these heteromers,
ATP gating and zinc-induced activation cannot be mediated simultaneously at a common
binding jaw. We reasoned that if ATP favors zinc activation, this would mean that ATP
binding in one interface bearing the H213A mutation must favor, through an allosteric
conformational change, zinc-induced tightening of another interface, which carries the K69A
mutation (Figure 4A). This is, in fact, what we observed (Figure 4B). First, evidence
supporting the assembly of heteromeric channels came from the fact that both ATP sensitivity
and Hill coefficient determined in cells expressing both K69A/T339S and H213A/T339S
mutants decreased compared with those determined in cells expressing only the mutant
H213A/T339S (Supplementary Figure 5). Second, we found that a very low concentration of
ATP that produced currents that represented 19 ± 4% of those evoked by zinc alone strongly
potentiated zinc responses in cells expressing the heteromers. In contrast, no detectable
potentiation of zinc currents by ATP was observed in cells expressing only the homomers
K69A/T339S —no zinc-evoked inward current was detected in cells expressing the
H213A/T339S mutant—(Figure 4B and C). Overall, this suggests that ATP binding favors
jaw tightening.
If this hypothesis is correct, then application of TNP-ATP, which is a known
competitive P2X2 antagonist (Cao et al, 2007; Trujillo et al, 2006), should inhibit zinc
activation that reported jaw tightening. Because it has been shown that TNP-ATP also acted
as a partial agonist on the T339S mutant (Cao et al, 2007), we chose a concentration at which
the antagonist effect is predominant over the agonist effect. In these conditions, we found
indeed that TNP-ATP largely inhibited zinc currents in cells expressing either the T339S or

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the S116H/T170H/T339S* mutant (Figure 5), suggesting that TNP-ATP prevents zinc gating
induced by jaw tightening.

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DISCUSSION
In this paper, we present a model of P2X receptor gating based on geometrical
constrains established by zinc bridging at native and engineered binding sites in the
ectodomain. We demonstrate that agonist binding tightens the open jaw forming the ATP-
binding site and this motion is coupled to channel opening, whereas binding of the
competitive antagonist TNP-ATP prevents gating induced by this movement.
Our data revealed the inherent molecular dynamic of the binding site jaw. This
conclusion was reached essentially by the use of the T339S mutation, which produces
channels displaying significant spontaneous openings (Cao et al, 2007). It should be noted
that other spontaneously active mutants have been identified in LGICs (Changeux &
Edelstein, 1998). These mutants, usually carrying mutations in the pore region such as L247T
in the α7 nicotinic acetylcholine receptor, have provided valuable insights into the mechanism
of receptor activation (Bertrand et al, 1992; Palma et al, 1999). In the present study, we
propose that the T339S mutation reveals the inherent movement of the intact binding pocket
for the following reasons. First, the conductance of mutated channels activated by zinc was
not different from that of the WT receptor activated by ATP determined previously (Jiang et
al, 2011) (Figures 2E and 3H), suggesting that the open state(s) of the T339S ion channel
stabilized by zinc are similar to those of the WT receptor normally induced by ATP. Second,
it has been shown that the Thr339 residue, which is located in TM2, contributes to the gate of
the channel (Cao et al, 2009; Kracun et al, 2010; Li et al, 2008; Li et al, 2010; Migita et al,
2001) and, in agreement with this hypothesis, its homologous residue is positioned at the
narrowest part of the closed channel in the zfP2X4 crystal structure (Kawate et al, 2009).
Mutating this residue, which corresponds to a rather minor side chain alteration (Thr to Ser),
may thus simply change locally the structure of the gate, leading to the occurrence of
spontaneous openings. Third, because the Thr339 residue is separated by more than 50 Å