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

Article · March 2012with66 Reads
DOI: 10.1038/emboj.2012.75 · Source: PubMed
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.
6 Figures
Tightening of the ATP-binding sites induces the opening of P2X receptor
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
Running title: Inherent dynamics of the ATP sites in P2X receptor
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
Keywords: purinergic receptors/ ligand-gated ion channels/ normal mode analysis/ zinc-
binding sites/ allosteric mechanism
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
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
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.
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
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
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
(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
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
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
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,
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
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
the S116H/T170H/T339S* mutant (Figure 5), suggesting that TNP-ATP prevents zinc gating
induced by jaw tightening.
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
from residues investigated in the present study, it is likely that T339S mutation has little
impact on the structure of the binding pocket. Therefore, these arguments favor, as stated
earlier (Cao et al, 2007) that the T339S mutation alters primarily the open-shut equilibrium by
substantially destabilizing the closed channel, and thus, allows extracellular zinc to control
gating of the ion channel without ATP.
We present evidence that our engineered zinc-binding site requires not only the
presence of the engineered histidines S116H and T170H, but also of endogenous aspartate
and histidine residues (Asp168 and His152). This is not surprising because most of zinc-
binding sites in crystallized proteins are tetrahedral, in which three or four coordinating atoms
are provided by the protein and the fourth site is usually occupied by water (Alberts et al,
1998). At present, we do not know the atomic details of zinc coordination spheres of both
native and engineered sites, but analysis of 111 crystal structures of Zn2+-binding proteins
revealed a nearly universal motif, in which one of the histidine provided by the first primary
coordinating shell makes an H-bond to an oxygen atom from a protein residue (e.g. aspartate
or glutamate) or a water molecule (Alberts et al, 1998). This interaction is believed to increase
the basicity and ligand strength of the histidine and arrange it correctly for interaction with the
metal. In this context, it is thus possible that Asp168 directly interacts with one of the
engineered histidines, which in turn interacts more favorably with zinc. Alternatively, Asp168
may directly interact with zinc. In addition, it is possible that residue Asp136, which has
recently been proposed to contribute to the native zinc-potentiating site (Friday & Hume,
2008), plays a role similar to Asp168, because this residue is located in the head domain, in
relative close proximity to residue H120. Regardless of these details, our data clearly show
that zinc activation cannot result from alternative zinc-binding sites provided by residues
located exclusively in one lip of the binding jaw, but instead requires bridging of the two lips.
It should be noted that the theoretical molecular motions obtained by NMA presented
in this study include some approximations that were due to the absence of water and
membrane, and also to a simplified force field restricted to Cα atoms. These approximations
imply that models may include deviations in the precise positioning of atoms. In the present
study, NMA did not detect significant pore opening. The reason for this is unclear, but one
explanation would be that the force field used in the modeling process overestimates the
interaction of the three TM2 α-helices at the level of the gate, and this, in turn constitutes a
barrier that locks the pore structure in a closed state. Another explanation is that gating results
from a combination of different modes including mode 10. In this respect, mode 7 is of
particular interest because it corresponds to a global twist of the receptor, which results in a
decrease of the angle between TM2 α-helices and the membrane plane normal
(Supplementary Figure 1), a hypothesis that is fully consistent with recent experimental
studies (Kracun et al, 2010; Li et al, 2010), which showed through engineering metal bridges
that pore opening is caused by the straightening of these helices.
We provide strong evidence that tightening of the ATP-binding jaw correlates with the
opening of the ion channel. Considering that the rP2X2 homology model likely represents the
resting closed state of the receptor, as previously proposed for the zfP2X4 crystal structure
(Kawate et al, 2009), this indicates that the head and dorsal fin domains would need to move
closer to each other to reach the open state(s), by a relative movement of more than 3 Å. Our
data also show that movement of the jaw occurs in the absence of ATP, or even in receptors
in which the ATP-binding sites have been disrupted by the K69A mutation. This suggests that
binding of ATP is not an absolute prerequisite to the modification of the binding site during
gating, supporting our hypothesis that the movement of the jaw is an inherent property of the
rP2X2 receptor.
Does ATP actually induce tightening of the binding jaws in a way similar to that
produced by zinc? We provide evidence that binding of ATP allosterically favors Zn2+
activation, essentially by tightening residues His120 and His213 around the metal (Figure 4).
This prompts us to suggest that the mechanism by which ATP tightens these residues is
similar to that involved in zinc activation (Figure 6). However, we do not eliminate the
possibility that, in addition, a direct interaction between zinc and part of the ATP molecule
may —at least partially— account for the potentiation mechanism of ATP response observed
in the WT P2X2 receptor, because the position homologous to His120 in the P2X7 receptor
has been shown to form a covalent bound with ADP-ribose (Adriouch et al, 2008) and
therefore is potentially close to the ATP-binding pocket.
We show that binding of the competitive antagonist TNP-ATP blocks pore opening
upon zinc-induced jaw tightening. One straightforward interpretation would be that binding of
TNP-ATP blocks movement of jaw tightening as a foot-in-the door. Alternatively, binding of
the antagonist may decouple jaw closure movement to pore opening, in which jaw closure
still occurs but the subsequent conformation change cannot be spread to initiate pore opening.
Additional experiments are needed to discriminate between these two mechanisms.
Taken together, these data pave the way for a plausible activation mechanism of P2X
receptors. We propose that following ATP binding the head and dorsal fin domains move
closer to each other, resulting in the tightening of the open jaw (Figure 6 and Supplementary
movie). This new conformational state, which binds zinc at native residues His120 and
His213, favors the opening of the ion channel. Because recent studies have suggested that
P2X gating requires a structural change of the three pore-forming TM2 α-helices (Browne et
al, 2011; Kracun et al, 2010; Li et al, 2008; Li et al, 2010), this indicates that there must exist
a molecular link between jaws tightening and pore opening. The entire molecular pathway by
which this allosteric conformational change is spread to the channel is not yet completely
understood, but likely involves a structural change of the subunit interface, as previously
proposed (Jiang et al, 2003; Jiang et al, 2010; Marquez-Klaka et al, 2007; Nagaya et al, 2005).
In conclusion, we demonstrate that tightening of the ATP-binding sites correlates
precisely with channel opening in the P2X2 receptor. This original mechanism that may likely
be shared by other members of the mammals P2X receptors family is reminiscent of those
found in other LGICs, in which closing of lobes (Sobolevsky et al, 2009) or loops (Hibbs &
Gouaux, 2011) around bound agonist has been reported to initiate pore opening.
Homology models. The all-heavy-atom structure of the zfP2X4 receptor (PDB ID code
3H9V) was taken as the template to construct the initial rP2X2 model with the program
MODELLER (Sali & Blundell, 1993). The resulting model was energy-minimized by using
the program CHARMM (Brooks & Karplus, 1983) with the all-atom param22 parameter set;
the solvent was represented implicitly with the GBSW model. For zinc-binding site
reconstructions, a distance constrain (2.06 Å) was added between zinc and the NE2 atom of
Normal Mode Analysis. The gating mechanism was studied by NMA, as previously
described (Taly et al, 2005), with a Cα atoms elastic-network model (Tirion, 1996), which
represents the protein as a network of residues linked by springs (with a 8 Å cutoff). To
examine the normal modes, the positions of the Cα atoms were modified along the normal
mode vectors. The new positions of the Cα atoms were introduced, while retaining the
positions of the other atoms. The position of those atoms were then adjusted by energy
minimization using decreasing harmonic constraints with the program CHARMM (Brooks &
Karplus, 1983). The magnitude of the amplification of each mode is one of the variables of
the study. It was set by modifying the energy applied for the exploration such as to attain the
following objectives: First, for the initial analysis of all modes, the energy applied was
adjusted such that the two structures would have an RMSD of ~2 Å. This arbitrary RMSD
value was chosen because it produced realistic structures with all modes and the changes were
large enough to be analyzed by inspection of the end points; second, for the reconstruction of
the zinc-binding site, the structure along mode 10 with the lowest His120-Cβ/His213-Cβ
distance was selected (Supplementary Figure 1).
Pore Analysis. The size of the pore was measured by using the program HOLE(Smart et al,
1996) with a radius of 4 Å for each Cα atom and the Connolly algorithm (Connolly, 1983).
ATP Probe. To identify normal modes likely to participate in the gating mechanism, we
studied the effect of the perturbation introduced by ATP. Given the simple model we were
using, we added atoms to the protein Cα model so as to locally modify the elastic network
(Ming & Wall, 2005; Mitternacht & Berezovsky, 2011; Taly et al, 2006; Zheng et al, 2005).
The experiment-compatible position of ATP (Jiang et al, 2011) was projected on 3 interfaces.
The probes were then modeled by retaining three atoms of ATP. This number was chosen to
be similar to the coarse-graining of amino acids. The atoms were the gamma phosphate, the O
from the furane, and C8 from adenine. The resulting modification of the network mimics the
effect of binding a ligand or introducing a mutation.
Complementary DNA construction and site-directed mutagenesis. The pcDNA-based
expression plasmids, mutagenesis and sequencing procedure have been described previously
(Jiang et al, 2010).
Cell culture and transfection. HEK-293 cells were cultured and transiently transfected with
the rP2X2 constructs (0.01-2 μg) and a green fluorescent protein cDNA construct (0.3 μg), as
described previously(Jiang et al, 2010). For the co-expression of K69A/T339S and
H213A/T339S subunits described in Figure 4, HEK-293 cells were transfected with 1 µg
cDNAs in a 1:1 ratio.
Chemicals. ZnCl2 solution and most of other drugs were purchased from Sigma (St Louis,
MO, USA). TNP-ATP was purchased from Invitrogen (Eugen, Oregon, USA).
Whole-cell recordings. Data were acquired with a patch-clamp amplifier (HEKA EPC 10)
using PATCHMASTER software (HEKA Co.). Patch pipettes (3-5 MΩ) contained (in mM):
140 KCl, 5 MgCl2, 5 EGTA, 10 HEPES, pH 7.3. External solution contained (in mM): 140
NaCl, 2.8 KCl, 2 CaCl2, 2 MgCl2, 10 glucose, 10 HEPES, pH 7.3. The holding potential was -
60 mV. Dose-response relationship experiments and drug applications were carried out as
described previously (Jiang et al, 2011).
Single-channel recordings. Single-channel recordings using outside-out configuration were
made from HEK-293 cells at room temperature 24-72 h after transfection. Recording pipettes
were coated with Sylgard 184 (Dow Corning Co.) and fire polished to yield resistances of 6-
20 MΩ. The holding potential was -120 mV. The extracellular solution contained (in mM):
147 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 13 glucose, pH 7.3. The intracellular
solution contained (in mM): 147 NaF, 10 HEPES, and 10 EGTA, pH 7.3. The osmolarity was
300 mosmol.kg-1 for all the solutions.
Data were sampled at 10 kHz and low-pass filtered at 2.9 kHz. For off-line analysis, data
were refiltered to give a cascaded filter cutoff frequency of 2 kHz. Channel events were
detected by using the 50% threshold criterion and idealized with TAC (Bruxton co.) as
described previously (Ding & Sachs, 1999; Jiang et al, 2011). Nominal open probability
(NPo) was determined with TACFit (Bruxton co.), and was defined as the fraction of time for
which the channels are open, where N is the number of active channels in the patch and Po is
the single-channel open probability. For obtaining dwell-time distributions of open- and shut-
times, only patches in which individual channel openings could be clearly resolved (multiple-
opening events represented less than 2% of total events) were subjected to further analysis as
described previously (Jiang et al, 2011). The open- and shut-time histograms were fitted by
the sum of exponentials with an imposed appropriated time resolution of 215 µs using
We thank Prof. Maurice Goeldner, Drs Alexandre Specht and Valérie Taly for critical reading
of the manuscript. This work was supported by the ANR Grant 06-0050-01, the CNRS
through the programme d’incitation à la mobilité d’équipe (PIME), the Ministère de la
Recherche and has been funded in part by the ic-FRC (www.icfrc.fr). R.J. is a recipient of a
fellowship from the China Scholarship Council.
Author contribution
R.J., O.C. and D.L. performed cell culture, transfection, and whole-cell patch-clamp
electrophysiology and analyzed data. R.J. and D.L. preformed single-channel recordings. A.T.
performed molecular modeling. A.M. and O.C. made mutations. T.G. designed research,
supervised the project and wrote the manuscript with feedback from R.J. and A.T. All authors
discussed the results and commented on the manuscript.
Conflict of Interest
The authors declare that they have no conflict of interest.
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Figure 1. Exploration of the rP2X2 receptor flexibility by NMA allows formation of the
native potentiating zinc-binding site. (A) The rP2X2 receptor model is shown in cartoon
representation and colored by subunits. Three views are presented: lateral to the membrane
plane (left), top view from the extracellular side (right-top) and bottom view from the
intracellular side (right-bottom). Yellow arrows represent protein displacement captured by
mode 10. Movements superior to 3 Å are colored orange and those inferior to 1 Å are omitted
for clarity. Close-up view of the binding site jaw shown in the previous panel before (B) and
after (C) exploration of mode 10. The distance separating the Cα atoms from residues His120
to His213 known to be involved in zinc potentiation (Nagaya et al, 2005) as well as the
location of the ATP site (Jiang et al, 2011) are also shown. Inset in C: Zoom-in view of zinc
coordination showing the mean distance and angle measured between the divalent cation and
coordinating atoms NE2 from the two histidines. Exploration of mode 10 also reveals that
loops A and B, colored in blue, get closer together.
Figure 2. Zn2+ ions gate the rP2X2 T339S mutant receptor through residues H120 and H213.
(A) Representative traces of Zn2+- (with indicated concentrations) and ATP- (30 µM,
saturating) evoked currents recorded from the same cell expressing the T339S mutant. (B)
Zinc dose-response curves for T339S and K69A/T339S mutant receptors (EC50 = 221 ± 47
μM, nH = 1.2 ± 0.2, n = 5 for T339S; EC50 = 273 ± 89 μM, nH = 1.2 ± 0.1, n = 5 for
K69A/T339S). No response was detected in cells expressing either the WT rP2X2,
H120A/H213A/T339S, H120A/T339S or H213A/T339S mutant receptor. (C) Single-channel
currents recorded from an outside-out patch excised from HEK cell expressing the T339S
mutant (openings downward) showing the increase of spontaneous channel activities by Zn2+
ions. (D) Distributions of open- and shut-times (pooled data from 7 patches). Data recorded in
the absence (up) or presence (bottom) of Zn2+ were fitted by the sum of exponentials (for
details see Supplementary Table 2). (E) Histograms (based on data from panel D) showing
the increase of NPo and the unaffected conductance of channels activated by Zn2+ (indicated
in orange). Pooled data in this and all other figures represent mean ± s.e.m.
Figure 3. Engineering of novel Zn2+-binding sites supports the tightening model of the
binding site jaw. (A) Histogram showing ATP (EC~10) evoked responses potentiated by Zn2+
(20 µM) in cells expressing the indicated mutants (n = 4-7). The H120A/H213A background
is symbolized by *. Inset: representative traces showing the protocol used for establishing the
histogram. (B) Histogram revealing the contribution of the endogenous residues Asp168 and
His152 to the engineered Zn2+ potentiating site in the S116H/T170H* mutant. Data for
S116H* and T170H* were taken from panel A. (C) Open jaw model of the WT rP2X2
receptor highlighting the identified residues from loops A and B establishing zinc bridges. (D)
Tightened jaw model of the S116H/T170H* mutant following mode 10 exploration
highlighting reconstitution of the engineered Zn2+-binding site. The distance and angle
measured between zinc ion (orange sphere) and coordinating atoms NE2 from histidines are
2.3, 2.2, 2.3 Å and 95°, 137° and 128°. Note that the distance measured between Zn2+ and one
oxygen of the carboxylate from Asp168 is 3.9 Å. (E) Zinc dose-response curves for the
S116H/T170H/T339S* mutant (EC50 = 27 ± 3 μM, nH = 1.2 ± 0.1, n = 6). The dashed curve
for T339S mutant was taken from Figure 2B. (F) Single-channel currents recorded from an
outside-out patch excised from HEK expressing the S116H/T170H/T339S* mutant (openings
downward) showing the increase of spontaneous channel activities by Zn2+ ions. (G)
Distributions of open- and shut-times (pooled data from 6 patches). Data recorded in the
absence (up) or presence (bottom) of Zn2+ were fitted by the sum of exponentials (for details
see Supplementary Table 2). (H) Histograms (based on data from panel G) showing the
increase of NPo and the unaffected conductance of channels activated by Zn2+ (indicated in
Figure 4. ATP binding favors jaw tightening. (A) Schematic representation of the
heteromeric expression of K69A/T339S and H213A/T339S mutants (see text for details). (B)
Representative traces of Zn2+ (200 µM) evoked currents potentiated by 0.1 µM ATP recorded
from cells expressing either the heteromer (black), the K69A/T339S homomer (blue) or the
H213A/T339S homomer (orange). (C) Pooled data for each of the three constructions shown in
panel B (n = 4-7). Potentiation was defined as the ratio of currents recorded in the presence of
both ATP and zinc to those recorded in the presence of zinc alone. N.D. Not determined, because
no Zn2+ inward current was detected in cells expressing the mutant H213A/T339S.
Figure 5. The competitive antagonist TNP-ATP inhibits Zn2+-gated currents through
receptors containing either the native or the engineered Zn2+-binding site. (A) Representative
traces showing Zn2+ (200 µM) evoked currents recorded in the absence (indicated as control,
light traces) or presence (heavy traces) of TNP-ATP (10 µM) in cells expressing either the
T339S (green trace) or the S116H/T170H/T339S* mutant (brown trace). (B) Pooled data of
TNP-ATP inhibition of zinc current for each construction shown in panel A (n = 5-8).
Figure 6. Schematic representation of a plausible activation mechanism of P2X receptor.
Agonist binding at the subunit interface tightens the open jaws forming ATP-binding sites, a
movement that is mechanically coupled to channel opening. Zn2+ further stabilizes the
conformation of closed jaws, through coordination of a pair of histidines that are located at
opposite lips of the jaw.
    • Furthermore, we found high closeness residues to be superimposed with high betweenness residues in the extracellular region. Considering that high closeness residues are generally evolutionary conserved and located in the active site[35,50], it is likely that, following ATP binding, the residues in the orthosteric site first transmit a signal to the central extracellular core of the channel, subsequently transmit a signal (with an allosteric and cooperative mechanism) to the transmembrane domain, which leads to channel opening, according to previous reports about the allosteric nature of P2Xs receptors gating[51,52]. The conclusion that the allosteric pocket of P2X7R is close to its orthosteric site is also in accordance with previous reports, showing that competitive antagonists block the P2X7 receptor without blocking the ATP binding site[28,46,[53][54][55]. Selective P2X7 antagonists have been docked to structural models of hP2X7R, and for sake of comparison to a model of hP2X4R.
    [Show abstract] [Hide abstract] ABSTRACT: Diabetic retinopathy (DR) is the most frequent complication of diabetes and one of leading causes of blindness worldwide. Early phases of DR are characterized by retinal pericyte loss mainly related to concurrent inflammatory process. Recently, an important link between P2X7 receptor (P2X7R) and inflammation has been demonstrated indicating this receptor as potential pharmacological target in DR. Here we first carried out an in silico molecular modeling study in order to characterize the allosteric pocket in P2X7R, and identify a suitable P2X7R antagonist through molecular docking. JNJ47965567 was identified as the hit compound in docking calculations, as well as for its absorption, distribution, metabolism and excretion (ADME) profile. As an in vitro model of early diabetic retinopathy, human retinal pericytes were exposed to high glucose (25 mM, 48 h) that caused a significant (p<0.05) release of IL-1β and LDH. The block of P2X7R by JNJ47965567 significantly (p<0.05) reverted the damage elicited by high glucose, detected as IL-1β and LDH release. Overall, our findings suggest that the P2X7R represents an attractive pharmacological target to manage the early phase of diabetic retinopathy, and the compound JNJ47965567 is a good template to discover other P2X7R selective antagonists.
    Full-text · Article · May 2017
    • Because the EC 50 values and channel assembly were proven to be comparable to the WT channels in some mutants (Fig. 3), the decreased current density in those mutations might attribute to altered channel gating. Previous studies demonstrated that the head, the DF, and the LF domains have participated in nearly every essential or constituent element of P2X channel gating (20)(21)(22)24,39,42), including channel desensitization (24). Given that the changed salt-bridge possibly resulted in the conformation changes in β2,3 and its physically coupled head and LF domains, the decreased current density in those mutants may attribute to the combination of the affecting factors mentioned above.
    [Show abstract] [Hide abstract] ABSTRACT: Significant progress has been made in understanding roles of crucial residues/motifs in the channel function of P2X receptors during the pre-structure era. The recent structural determination of P2X receptors allows us to reevaluate the role of those residues/motifs. Residues R309 and D85 (rat P2X4 numbering) are highly conserved throughout the P2X family and were involved in loss-of-function polymorphism in human P2X receptors. Previous studies proposed that they participated in direct ATP-binding. However, the crystal structure of P2X demonstrated that those two residues form an intersubunit salt-bridge located far away from the ATP-binding site. Therefore, it is necessary to reevaluate the role of this salt-bridge in P2X receptors. Here we suggest the crucial role of this structural element both in protein stability and in channel gating rather than direct ATP interaction and channel assembly. Combining mutagenesis, charge swap, and disulfide crosslinking, we revealed the stringent requirement of this salt-bridge in normal P2X4 channel function. This salt-bridge may contribute to stabilizing the bending conformation of the β2,3-sheet which is structurally coupled with this salt-bridge and the α2-helix. Strongly kinked β2,3 is essential for domain-domain interactions between head domain, dorsal fin domain, right flipper domain, and the loop β7,8 in P2X4 receptors. Disulfide crosslinking with directions opposing or along the bending angle of the β2,3-sheet towards the α2-helix led to loss-of-function and gain-of-function of P2X4 receptors, respectively. Further insertion of amino acids with bulky side chains into the linker between the β2,3-sheet or the conformational change of the α2 helix, interfering with the kinked conformation of β2,3, led to loss-of-function of P2X4 receptors. All these findings provided new insights in understanding the contribution of the salt-bridge D85-R309 and its structurally coupled β2, 3-sheet to the function of P2X receptors.
    Full-text · Article · Feb 2016
    • There are three major subunit–subunit interfaces: body-to-body, head-to-body and left-flipper-to-dorsal-fin[5]. Through extensive cysteine substitution mutagenesis, many pairs of residues in the extracellular domain were demonstrated to lie close to one another across the intersubunit interfacesThese pairs play important roles in ion channel functions, such as ATP binding and ATP induced conformational changes, providing a zinc binding site and stabilizing the conformation of the open channel conformation state[7][8][9][10][11][12][13]. All of these interfaces are exclusively involved in body-to-body, head-to-dorsal-fin and body-to-left-flipper interactions.
    [Show abstract] [Hide abstract] ABSTRACT: P2X receptors are trimeric ATP-activated non-selective cation channels. The ATP binding pocket is positioned between two neighboring subunits. Accompanying ligand binding, subunit-subunit contacts are most likely involved in receptor function and drive a conformational change to open the ion permeation pathway. In this way, we sought to determine the function of side chains of the zebrafish P2X4 receptor ectodomain left-flipper-to-dorsal-fin interface residues in ligand binding. By Combining site-directed mutagenesis and electrophysiology methods, we showed that cysteine substitutions of I212, S215, Y216 and L217 resulted in decreased sensitivity to ATP. In addition, the ATP induced current at L217C was completely inhibited by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)), indicating a role for this residue in ATP action. Deletion of residues 285-293 from the zebrafish P2X4 receptor abolished channel function. However, insertion of the same sequence frame into a homologous position of the rat P2X6 receptor did not rescue channel function, suggesting that these residues are necessary but not sufficient for achieving the correct ATP-induced conformation.
    Full-text · Article · Aug 2014
    • We also found that AR1 rather than AR2 and 3 can promote the inherent downward motion of head domain (Fig. 5A). A combination of 10-ns MD simulations of and PCA analysis of snapshots from simulations revealed that AR1-induced a downward movement of the head domain, upward motion of dorsal fin domain and a subsequent closing of ATP-binding site jaw (Fig. 5A), consistent with previous observations[31,26]. Meanwhile, it has been well established that those allosteric changes are associated with downstream opening-related allosteric changes, such as the radial expansions of extracellular vestibule and the final iris-like channel opening [26,33].
    [Show abstract] [Hide abstract] ABSTRACT: P2X receptors are ATP-gated ion channels involved in many physiological functions, and determination of ATP-recognition (AR) of P2X receptors will promote the development of new therapeutic agents for pain, inflammation, bladder dysfunction and osteoporosis. Recent crystal structures of the zebrafish P2X4 (zfP2X4) receptor reveal a large ATP-binding pocket (ABP) located at the subunit interface of zfP2X4 receptors, which is occupied by a conspicuous cluster of basic residues to recognize triphosphate moiety of ATP. Using the engineered affinity labeling and molecular modeling, at least three sites (S1, S2 and S3) within ABP have been identified that are able to recognize the adenine ring of ATP, implying the existence of at least three distinct AR modes in ABP. The open crystal structure of zfP2X4 confirms one of three AR modes (named AR1), in which the adenine ring of ATP is buried into site S1 while the triphosphate moiety interacts with clustered basic residues. Why architecture of ABP favors AR1 not the other two AR modes still remains unexplored. Here, we examine the potential role of inherent dynamics of head domain, a domain involved in ABP formation, in AR determinant of P2X4 receptors. In silico docking and binding free energy calculation revealed comparable characters of three distinct AR modes. Inherent dynamics of head domain, especially the downward motion favors the preference of ABP for AR1 rather than AR2 and AR3. Along with the downward motion of head domain, the closing movement of loop139-146 and loop169-183, and structural rearrangements of K70, K72, R298 and R143 enabled ABP to discriminate AR1 from other AR modes. Our observations suggest the essential role of head domain dynamics in determining AR of P2X4 receptors, allowing evaluation of new strategies aimed at developing specific blockers/allosteric modulators by preventing the dynamics of head domain associated with both AR and channel activation of P2X4 receptors.
    Full-text · Article · May 2014
    • The ATP binding site is ∼40 Å from the membrane-spanning segments, which constitute the ionic pore of P2X receptors. The recent ATP-bound crystal structure, and previous studies utilizing normal mode analysis (Du et al., 2012; Jiang et al., 2012a ), metalbridging experiments (Jiang et al., 2012a), electron microscopy (Roberts et al., 2012), and voltage-clamp fluorometry (Lorinczi et al., 2012), have now revealed a plausible activation mechanism that can be dissected into five steps (Figure 4): binding of ATP 4− (Li et al., 2013) to a pocket located at the interface between each subunit (first step) leads to the tightening of the head domain relative to the dorsal fin (second step). Because the ribose and adenine base interact hydrophobically with L217 and I232 (chain B), which are part of the dorsal fin, closure of the binding " jaw " induces the upward movement of the dorsal fin.
    [Show abstract] [Hide abstract] ABSTRACT: P2X receptors are ATP-gated non-selective cation channels involved in many different physiological processes, such as synaptic transmission, inflammation, and neuropathic pain. They form homo- or heterotrimeric complexes and contain three ATP-binding sites in their extracellular domain. The recent determination of X-ray structures of a P2X receptor solved in two states, a resting closed state and an ATP-bound, open-channel state, has provided unprecedented information not only regarding the three-dimensional shape of the receptor, but also on putative conformational changes that couple ATP binding to channel opening. These data provide a structural template for interpreting the huge amount of functional, mutagenesis, and biochemical data collected during more than fifteen years. In particular, the interfacial location of the ATP binding site and ATP orientation have been successfully confirmed by these structural studies. It appears that ATP binds to inter-subunit cavities shaped like open jaws, whose tightening induces the opening of the ion channel. These structural data thus represent a firm basis for understanding the activation mechanism of P2X receptors.
    Full-text · Article · Dec 2013
    • Binding of agonist is supposed to induce channel opening by a closing movement of the head domain that is further propagated to the TM domains (Hattori and Gouaux, 2012; Jiang et al., 2012; Lörinczi et al., 2012). Dose response curves for ATP yield generally Hill-slopes greater than one (Bean, 1992; Brake et al., 1994) indicating that more than one agonist molecule binds before channel opening occurs.
    [Show abstract] [Hide abstract] ABSTRACT: Transcripts and/or proteins of P2X receptor (P2XR) subunits have been found in virtually all mammalian tissues. Generally more than one of the seven known P2X subunits have been identified in a given cell type. Six of the seven cloned P2X subunits can efficiently form functional homotrimeric ion channels in recombinant expression systems. This is in contrast to other ligand-gated ion channel families, such as the Cys-loop or glutamate receptors, where homomeric assemblies seem to represent the exception rather than the rule. P2XR mediated responses recorded from native tissues rarely match exactly the biophysical and pharmacological properties of heterologously expressed homomeric P2XRs. Heterotrimerization of P2X subunits is likely to account for this observed diversity. While the existence of heterotrimeric P2X2/3Rs and their role in physiological processes is well established, the composition of most other P2XR heteromers and/or the interplay between distinct trimeric receptor complexes in native tissues is not clear. After a description of P2XR assembly and the structure of the intersubunit ATP-binding site, this review summarizes the distribution of P2XR subunits in selected mammalian cell types and the biochemically and/or functionally characterized heteromeric P2XRs that have been observed upon heterologous co-expression of P2XR subunits. We further provide examples where the postulated heteromeric P2XRs have been suggested to occur in native tissues and an overview of the currently available pharmacological tools that have been used to discriminate between homo- and heteromeric P2XRs.
    Full-text · Article · Dec 2013
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Investigate the structure of Dynamin-Related Proteins involved in mitochondrial membrane fusion through molecular modelling and dynamics atomistic simulation
September 2012 · Channels (Austin, Tex.) · Impact Factor: 2.32
    The molecular mechanism underlying channel opening in response to agonist binding remains a challenging issue in neuroscience. In this regard, many efforts have been recently undertaken in ATP-gated P2X receptors. Among those efforts, we have provided evidence in the P2X2 receptor that tightening of ATP sites upon agonist binding induces opening of the ion channel. Here we extend our analysis... [Show full abstract]
    March 2010 · Journal of Biological Chemistry · Impact Factor: 4.57
      The recent crystal structure of the ATP-gated P2X4 receptor revealed a static view of its architecture, but the molecular mechanisms underlying the P2X channels activation are still unknown. By using a P2X2 model based on the x-ray structure, we sought salt bridges formed between charged residues located in a region that directly connects putative ATP-binding sites to the ion channel. To... [Show full abstract]
      May 2011 · Proceedings of the National Academy of Sciences · Impact Factor: 9.67
        ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the... [Show full abstract]
        November 2012 · Trends in Biochemical Sciences · Impact Factor: 11.23
          P2X receptors are nonselective cation channels gated by extracellular ATP. They represent new therapeutic targets, and they form channels with a unique trimeric architecture. In 2009, the first crystal structure of a P2X receptor was reported, in which the receptor was in an ATP-free, closed channel state. However, our view recently changed when a second crystal structure was reported, in... [Show full abstract]
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