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Fig. S1. Evaluation of the X-ray structure (Protein Data Bank ID code 4DW1) and our model of the ATP-bound zebra fi sh P2X4 (zfP2X4) receptor against previous accessibility and bridging results. ( A ) Rates of Ag + modi fi cation of individual Cys residues (1, 2) mapped onto the transmembrane (TM) domain of the
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The opening of P2X receptor channels by extracellular ATP underlies purinergic signaling in many tissues. Here we use computational and functional approaches to study helix interactions within the transmembrane domain of P2X receptors. Our results suggest that the intersubunit crevices observed in the X-ray structure of detergent-solub...
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Context 1
... receptor channels are a family of trimeric cation-selective channels that are activated by extracellular ATP (1, 2). These ligand-gated ion channels are expressed in many tissues, including the central and peripheral nervous systems and the im- mune system, where they play a range of important roles in sensory signaling and in fl ammation (1, 3). Recent X-ray structures of the zebra fi sh P2X4 (zfP2X4) receptor in apo and ATP-bound forms (Protein Data Bank ID codes 4DW0 and 4DW1, respectively) have revealed the molecular design of these proteins (2, 4, 5) and have provided valuable information on how ATP binding triggers the opening of the transmembrane (TM) pore ( Fig. 1 A and B ). ATP binds to a cleft between subunits within the large extracellular domain, inducing cleft closure and an accompanying lateral fl exing of the β -sheet connecting the extracellular domain to the TM domain (5). The apo structure of the zfP2X4 receptor reveals that the TM1 helix is positioned peripheral to the TM2 helix and that the TM2 helix occludes the pore at the central axis within the outer half of the membrane (4, 5). In accord with this structure, accessibility studies show that the TM2 helix lines the aqueous pore and that the residues forming the gate are positioned within the occlusion in the apo structure (6 – 8). Lateral fenestrations within the extracellular domain provide a path for ions to enter and exit the extracellular vestibule positioned above this TM2 occlusion, and these fenestrations are thought to change conformation in response to ATP binding (9, 10). The ATP-bound structure shows that pore opening involves widening of the extracellular vestibule and an iris-like expansion of the pore (5). Intersubunit interactions within the TM domain in the apo structure are limited to the gate region of TM2 (5), and thus the pore expansion observed in the ATP-bound structure leave the three subunits es- sentially devoid of intersubunit interactions within the membrane (Fig. 1 A and B ). In effect, the lateral fenestrations that are positioned above the outer lea fl et of the membrane in the apo structure have expanded dramatically to encompass most of the TM domain. Many of the conformational rearrangements predicted from the X-ray structures of the zfP2X4 receptor are consistent with structural constraints obtained from functional studies on the protein in a membrane environment. For example, ATP binding to the cleft and subsequent cleft closure were predicted to occur based on proximity tethering (11), normal mode analysis (12), and both metal bridging and spectroscopic studies (13, 14). In addition, the accessibility of both methanethiolsulfonate compounds and metals (Ag + and Cd 2+ ) to cysteine residues introduced into TM1 and TM2 (6 – 8) is consistent with the expansion of the external pore predicted from the zfP2X4 structures (Fig. S1 A ). However, metal bridges engineered into the internal region of the TM2 helix (7, 8) suggest that the internal pore narrows as the channel opens, a feature that is not evident in the ATP-bound zfP2X4 structure (Fig. S1 B ). Moreover, the large crevices between subunits within the TM domain of the ATP-bound structure are unprecedented in membrane proteins and would be expected to destabilize the open state of the protein when it is embedded in a lipid membrane (15). One proposal is that lipids might occupy these crevices and serve to stabilize the structure of the open state (5). The goal of the present study was to investigate the structure of the open state of P2X receptors in a membrane environment using both computational and functional approaches and to assess the validity of the proposed mechanism of ATP activation and pore opening. Our results suggest that the absence of intersubunit interactions in the ATP-bound structure is not repre- sentative of the native structure but that intrasubunit interactions within the TM domain are faithfully captured in both apo and ATP-bound crystal structures of detergent-solubilized P2X receptors. Our results also demonstrate that the internal end of the pore plays a crucial role in tuning both the gating and permeation properties of P2X receptors. To evaluate how the large intersubunit crevices observed in the X-ray structure of the ATP-bound zfP2X4 receptor impact receptor function, we performed molecular dynamics (MD) simulations with the receptor incorporated into a hydrated 1,2-dimyr- istoyl-sn-glycero-3-phosphocholine (DMPC) bilayer, restraining the receptor and ATP to the X-ray structure while allowing sol- vent and lipids to move. At the outset of the simulation, the pore was heavily hydrated, compatible with the formation of an ion permeation pathway. However, over time, two lipids diffused into the intersubunit crevices and ultimately entered the pore (Fig. 1 C ). Shortly after the entrance of the fi rst lipid into the pore at ∼ 62.5 ns, the number of water molecules in the pore dropped sharply (Fig. 1 D ). The expulsion of water from the pore facilitated the entrance of the second lipid molecule. In the end, a 13-Å – deep section of the pore became devoid of water (Fig. 1 E ). We also ran a simulation in which the structural restraint on the receptor was maintained for the fi rst 12.2 ns to allow the lipids, water, and ions to equilibrate with the membrane-embedded receptor and then was released for the duration of the simulation. Again, we observed that two lipids entered the pore (the fi rst at ∼ 43.8 ns), resulting in dehydration of a large section of the pore. We also observed a pronounced hydrophobic mismatch between the receptor TM domain and the DMPC lipid bilayer in both the restrained and unrestrained MD simulations. Lipids in the lower lea fl et of the bilayer adjacent to the receptor moved toward the center of the bilayer (relative to the more distant lipids) to match the short hydrophobic thickness of the TM domain (Fig. 1 F ). The short hydrophobic thickness results from the relatively large tilt angle of the TM2 helices with respect to the membrane normal. It is worth noting that DMPC is a relatively short lipid with only 14 carbons on each acyl chain. Longer acyl chains in other common lipids would have increased further the extent of hydrophobic mismatch between the membrane and the TM domain. These simulations led us to conclude that the large intersubunit crevices observed in the ATP-bound structure of the detergent- solubilized zfP2X4 receptor are not compatible with ion conduction and thus likely represent a nonnative feature. In addition, the length of the TM domain in the X-ray structure may be in- consistent with the thickness of native lipid membranes. in P2X Receptors. To de fi ne interactions within individual subunits of the TM domain, we inspected the interface between TM1 and TM2 in the zfP2X4 X-ray structures to identify positions where state-dependent metal bridges might be engineered. In a previous study on a rat P2X2 (rP2X2) construct in which a native Cys in TM2 was mutated to threonine (C348T), we found that introducing the S345C mutation in TM2 resulted in an inhibitory metal bridge involving H33 in TM1 (7). This region of TM1 and TM2 was resolved in the structure of apo zfP2X4 spans G32 – L361 (corresponding to G30 – L353 in rP2X2); however, the fi rst TM1 residue resolved in the ATP-bound structure was R36, corresponding to R34 in rP2X2. To evaluate metal bridges involving this intracellular region of TM1 and TM2, we created models for metal coordination in both apo and ATP-bound zfP2X4. To include the position corresponding to H33 in P2X2, we grafted the fi rst turn of the TM1 helix from the apo structure onto the ATP-bound structure, extending the TM1 N terminus to G32 (G30 in rP2X2). We evaluated these zfP2X4 models to determine whether a state-dependent metal bridge for Cd 2+ could be formed at the ...Context 2
... receptor channels are a family of trimeric cation-selective channels that are activated by extracellular ATP (1, 2). These ligand-gated ion channels are expressed in many tissues, including the central and peripheral nervous systems and the im- mune system, where they play a range of important roles in sensory signaling and in fl ammation (1, 3). Recent X-ray structures of the zebra fi sh P2X4 (zfP2X4) receptor in apo and ATP-bound forms (Protein Data Bank ID codes 4DW0 and 4DW1, respectively) have revealed the molecular design of these proteins (2, 4, 5) and have provided valuable information on how ATP binding triggers the opening of the transmembrane (TM) pore ( Fig. 1 A and B ). ATP binds to a cleft between subunits within the large extracellular domain, inducing cleft closure and an accompanying lateral fl exing of the β -sheet connecting the extracellular domain to the TM domain (5). The apo structure of the zfP2X4 receptor reveals that the TM1 helix is positioned peripheral to the TM2 helix and that the TM2 helix occludes the pore at the central axis within the outer half of the membrane (4, 5). In accord with this structure, accessibility studies show that the TM2 helix lines the aqueous pore and that the residues forming the gate are positioned within the occlusion in the apo structure (6 – 8). Lateral fenestrations within the extracellular domain provide a path for ions to enter and exit the extracellular vestibule positioned above this TM2 occlusion, and these fenestrations are thought to change conformation in response to ATP binding (9, 10). The ATP-bound structure shows that pore opening involves widening of the extracellular vestibule and an iris-like expansion of the pore (5). Intersubunit interactions within the TM domain in the apo structure are limited to the gate region of TM2 (5), and thus the pore expansion observed in the ATP-bound structure leave the three subunits es- sentially devoid of intersubunit interactions within the membrane (Fig. 1 A and B ). In effect, the lateral fenestrations that are positioned above the outer lea fl et of the membrane in the apo structure have expanded dramatically to encompass most of the TM domain. Many of the conformational rearrangements predicted from the X-ray structures of the zfP2X4 receptor are consistent with structural constraints obtained from functional studies on the protein in a membrane environment. For example, ATP binding to the cleft and subsequent cleft closure were predicted to occur based on proximity tethering (11), normal mode analysis (12), and both metal bridging and spectroscopic studies (13, 14). In addition, the accessibility of both methanethiolsulfonate compounds and metals (Ag + and Cd 2+ ) to cysteine residues introduced into TM1 and TM2 (6 – 8) is consistent with the expansion of the external pore predicted from the zfP2X4 structures (Fig. S1 A ). However, metal bridges engineered into the internal region of the TM2 helix (7, 8) suggest that the internal pore narrows as the channel opens, a feature that is not evident in the ATP-bound zfP2X4 structure (Fig. S1 B ). Moreover, the large crevices between subunits within the TM domain of the ATP-bound structure are unprecedented in membrane proteins and would be expected to destabilize the open state of the protein when it is embedded in a lipid membrane (15). One proposal is that lipids might occupy these crevices and serve to stabilize the structure of the open state (5). The goal of the present study was to investigate the structure of the open state of P2X receptors in a membrane environment using both computational and functional approaches and to assess the validity of the proposed mechanism of ATP activation and pore opening. Our results suggest that the absence of intersubunit interactions in the ATP-bound structure is not repre- sentative of the native structure but that intrasubunit interactions within the TM domain are faithfully captured in both apo and ATP-bound crystal structures of detergent-solubilized P2X receptors. Our results also demonstrate that the internal end of the pore plays a crucial role in tuning both the gating and permeation properties of P2X receptors. To evaluate how the large intersubunit crevices observed in the X-ray structure of the ATP-bound zfP2X4 receptor impact receptor function, we performed molecular dynamics (MD) simulations with the receptor incorporated into a hydrated 1,2-dimyr- istoyl-sn-glycero-3-phosphocholine (DMPC) bilayer, restraining the receptor and ATP to the X-ray structure while allowing sol- vent and lipids to move. At the outset of the simulation, the pore was heavily hydrated, compatible with the formation of an ion permeation pathway. However, over time, two lipids diffused into the intersubunit crevices and ultimately entered the pore (Fig. 1 C ). Shortly after the entrance of the fi rst lipid into the pore at ∼ 62.5 ns, the number of water molecules in the pore dropped sharply (Fig. 1 D ). The expulsion of water from the pore facilitated the entrance of the second lipid molecule. In the end, a 13-Å – deep section of the pore became devoid of water (Fig. 1 E ). We also ran a simulation in which the structural restraint on the receptor was maintained for the fi rst 12.2 ns to allow the lipids, water, and ions to equilibrate with the membrane-embedded receptor and then was released for the duration of the simulation. Again, we observed that two lipids entered the pore (the fi rst at ∼ 43.8 ns), resulting in dehydration of a large section of the pore. We also observed a pronounced hydrophobic mismatch between the receptor TM domain and the DMPC lipid bilayer in both the restrained and unrestrained MD simulations. Lipids in the lower lea fl et of the bilayer adjacent to the receptor moved toward the center of the bilayer (relative to the more distant lipids) to match the short hydrophobic thickness of the TM domain (Fig. 1 F ). The short hydrophobic thickness results from the relatively large tilt angle of the TM2 helices with respect to the membrane normal. It is worth noting that DMPC is a relatively short lipid with only 14 carbons on each acyl chain. Longer acyl chains in other common lipids would have increased further the extent of hydrophobic mismatch between the membrane and the TM domain. These simulations led us to conclude that the large intersubunit crevices observed in the ATP-bound structure of the detergent- solubilized zfP2X4 receptor are not compatible with ion conduction and thus likely represent a nonnative feature. In addition, the length of the TM domain in the X-ray structure may be in- consistent with the thickness of native lipid membranes. in P2X Receptors. To de fi ne interactions within individual subunits of the TM domain, we inspected the interface between TM1 and TM2 in the zfP2X4 X-ray structures to identify positions where state-dependent metal bridges might be engineered. In a previous study on a rat P2X2 (rP2X2) construct in which a native Cys in TM2 was mutated to threonine (C348T), we found that introducing the S345C mutation in TM2 resulted in an inhibitory metal bridge involving H33 in TM1 (7). This region of TM1 and TM2 was resolved in the structure of apo zfP2X4 spans G32 – L361 (corresponding to G30 – L353 in rP2X2); however, the fi rst TM1 residue resolved in the ATP-bound structure was R36, corresponding to R34 in rP2X2. To evaluate metal bridges involving this intracellular region of TM1 and TM2, we created models for metal coordination in both apo and ATP-bound zfP2X4. To include the position corresponding to H33 in P2X2, we grafted the fi rst turn of the TM1 helix from the apo structure onto the ATP-bound structure, extending the TM1 N terminus to G32 (G30 in rP2X2). We evaluated these zfP2X4 models to determine whether a state-dependent metal bridge for Cd 2+ could be formed at the intrasubunit interface between TM1 and TM2 and found a promising candidate that forms when the N353C mutant (S345C in rP2X2) is introduced in a background that retains the native C356 in TM2 (C348 in rP2X2) and either His ...Context 3
... remain potentiated (Fig. 5 A ). In experiments with the H-C-C construct, we observed little evidence of recovery from Cd 2+ -mediated potentiation even 20 min after the removal of Cd 2+ (Fig. 5), demonstrating stable interaction of the metal with the closed channel. Using this protocol for the C-C-C construct, we also observed stable potentiation 15 min after removal of Cd 2+ . To investigate the mechanism of Cd 2+ potentiation further, we undertook single-channel recordings. Membrane patches in the outside-out con fi guration of the patch-clamp technique were isolated from cells expressing the C-C-C construct because these channels produced the most robust potentiation of ATP-activated currents by Cd 2+ . Analysis was conducted on three patches in which channel activity was observed in the presence of ATP but not in its absence. Two additional patches exhibited qualitatively similar results but were excluded from the analysis because in- frequent channel openings in the absence of ATP precluded the de fi nitive identi fi cation of ATP-activated currents. Channels activated by 0.3 μ M ATP fl uctuated rapidly among multiple current levels, with a predominant level of 3.1 ± 0.1 pA at − 120 mV (Fig. 6, Left ). The addition of 2 μ M Cd resulted in dramatic increases in both the open probability and unitary current amplitudes of the channel (Fig. 6, Right ). The predominant current amplitude in the single-channel current distribution increased approximately twofold in the presence of Cd 2+ , whereas individual burst events, de fi ned as channel openings not inter- rupted by a close event exceeding 50 ms in duration, increased 7.5 ± 1.8-fold. The maximum observed burst duration in ATP alone was 471 ms, whereas in the presence of Cd 2+ the longest measurable burst lasted 6,698 ms. The effect of Cd 2+ on burst duration is an underestimation, because bursts of channel activity during which a second channel also became active were excluded from the analysis, and such instances predominantly occurred during long bursts of channel activity. The observed effect of Cd 2+ on burst duration indicates that the bridge formed by Cd 2+ stabilizes open states. The effect of Cd 2+ on the unitary conductance of the channel is intriguing, because it suggests that the metal bridge either stabilizes channel conformations in- frequently visited by the channel in its absence or that the metal bridge leads to a conformational change that results in an ap- proximate doubling of the current at all channel sublevels. We also explored whether the conducting states stabilized by the C-C-C bridge might correspond to the dilated state of P2X receptors that gives rise to a change in the relative permeability of Na + to N -methyl- D -glucamine (NMDG + ) (21 – 23). Macroscopic current – voltage (I – V) relationships were obtained using voltage ramps in whole-cell recordings in which Na + was the primary internal cation and NMDG + was the primary external cation. In previous reports with related ionic conditions and protocols, the zero-current potential shifted in the positive direction in the continuous presence of ATP as the relative permeability of Na + to NMDG + changes (21 – 23). When Cd 2+ was applied to the C-C-C construct, we observed large increases in ATP-activated macroscopic current for both inward and outward limbs of the I – V relationship, with no detectable change in the zero-current potential (Fig. 7). These results indicate that the relative permeability of Na + :NMDG + for the rP2X2 receptor does not change when Cd 2+ forms a bridge between TM1 and TM2, and thus the state stabilized by the bridge does not correspond to the proposed dilated state of P2X receptor channels. A Structural Model for the Open State of zfP2X4 Receptors. The results presented thus far suggest that the intrasubunit interactions observed in both the apo and ATP-bound structures are remarkably consistent with our engineered metal bridges between TM1 and TM2. However, the ATP-bound structure of the zfP2X4 receptor is nonnative in that intersubunit interactions are largely absent within the TM domain. To generate a model for the open state that contains intersubunit interactions but retains intrasubunit helix – helix interactions, we developed a conformational search algorithm to explore alternate quaternary arrangements of the TM2 helices (see Methods , Fig. S2). TM1 then was introduced into each subunit using the same relative positioning with respect to TM2 as in the ATP-bound X-ray structure, and fi nally the extracellular ATP-binding domain was grafted after superimposing the TM domain. The resulting model was chosen because it is as close as possible to the X-ray structure of the ATP- bound zfP2X4 receptor but introduces additional intersubunit interactions to stabilize the TM domain. Consistent with both accessibility (6 – 8) and metal coordination (7, 8) data, our model features expansion of the external pore and narrowing of the internal pore (Fig. S1 B and C ). In this model, relative to the X-ray structure, the TM2 helices are translated 1.3 Å toward the central axis, the twist of the TM2 three-helix bundle is increased by 10°, and each TM2 helix is rotated 5° counterclockwise (as viewed from the top) around its helical axis, creating a much more tightly packed intersubunit interface (Fig. 8 A – C and Fig. S2). We performed MD simulations on this model, similar to those performed on the ATP- bound X-ray structure (Fig. 1), and observed that lipid molecules were excluded from the pore (Fig. 8 D ), which remained well- hydrated throughout the simulation (Fig. 8 D ). The addition of one turn of the helix in TM1 and a slight reduction in the tilt angle of TM2 result in a modest improvement in hydrophobic mismatch (Fig. S3 A ). The pore diameter of our model is some- what less than that of the X-ray structure, especially toward the internal end (Fig. S3 B and C ); however, it still is 4.7 Å wide at its narrowest point and therefore should support permeation of phys- iologically relevant cations such as Na + , K + , or Ca 2+ . Although we found that formation of a Cd 2+ bridge in the C-C-C construct does not alter the relative permeability of Na + to NMDG + , this channel clearly permeates NMDG + at voltages negative to − 70 mV (Fig. 7 A ). Corey – Pauling – Koltun (CPK) models of NMDG + can fi t into a box with dimensions of 5 × 6.4 × 12 Å (24), providing an upper limit on the dimensions necessary for permeation. The actual pore diameter required to support permeation would be lower, because the structures of both the organic cation and the protein will fl uctuate on a rapid timescale. Thus, it seems reasonable to conclude that the diameter of the pore in our open- state model is compatible with NMDG + permeation. In the simulation of the open-state model, the side chains of L351 alternate between two rotamers, one of which positions the side chain toward the inter-TM2 interface, and the other with the side chain projected into the pore. Interestingly, this residue is equivalent to V343 in the rP2X2 receptor, where substitution of Cys results in the formation of a stable Cd 2+ bridge that blocks the channel (7). This Cd 2+ bridge forms rapidly (rate constant = 10 M/s) in the presence of ATP, suggesting that bridging occurs in the highly populated open state, and requires Cys in all three subunits, implying that it forms at the central axis of the pore (7). L351 in the X-ray structure of the ATP-bound zfP2X4 receptor cannot coordinate Cd 2+ because of the larger diameter of the pore at this position (L351 C β – C β distances of 11.2 Å; Fig. S1 B ). However, in our open-state model L351C residues in the alter- native rotamer, together with two water molecules, can coordinate Cd 2+ in the triagonal bipyramidal geometry (Fig. 8 F ). The objective of the present study was to evaluate both intersubunit and intrasubunit interactions within the TM domain of P2X receptors using both computational and functional approaches. A key motivation for this work was the unexpected absence of intersubunit interactions in the recent X-ray structure of the ATP-bound zfP2X4 receptor, which raises the possibility that the structure is distorted (15) and brings into question the proposed mechanism of ATP activation. Indeed, our MD simulations suggest that lipid molecules can diffuse through the intersubunit crevices to occupy and dehydrate the ion permeation pathway (Fig. 1), a scenario that is incompatible with ion conduction. As noted previously (15), the predominance of hydro- carbon side chains in helical transmembrane domains implicates nonspeci fi c van der Waals and weak electrostatic interactions as the main intraprotein forces for tertiary and quaternary struc- tural stability. In native membranes, the hydrophobic environment provided by the acyl chains of lipid molecules enhances helix – helix packing and helps de fi ne the hydrophobic dimension. The lack of such a hydrophobic environment in the crystalline lattice is likely the reason for the poor intersubunit packing of the TM1 and TM2 helices and the apparent hydrophobic mismatch in the X-ray structure of the ATP-bound zfP2X4 receptor. Such distortions may be particularly problematic for membrane proteins such as P2X receptors, which contain large extracellular domains that provide most of the crystal contacts, together with small transmembrane domains that contain only six helices in the trimer (15). Although the intersubunit crevices are a striking feature of the ATP-bound X-ray structure, only relatively modest structural rearrangements are required to form an intersubunit interface within the membrane. In our open-state model of the zfP2X4 receptor, intersubunit interactions within two regions of the TM domain were created by small rotations and translation of the TM2 helix toward the central axis (Fig. 8). The fi rst interface is within the internal region of the TM domain, where residues on three ...Context 4
... state of P2X receptors that gives rise to a change in the relative permeability of Na + to N -methyl- D -glucamine (NMDG + ) (21 – 23). Macroscopic current – voltage (I – V) relationships were obtained using voltage ramps in whole-cell recordings in which Na + was the primary internal cation and NMDG + was the primary external cation. In previous reports with related ionic conditions and protocols, the zero-current potential shifted in the positive direction in the continuous presence of ATP as the relative permeability of Na + to NMDG + changes (21 – 23). When Cd 2+ was applied to the C-C-C construct, we observed large increases in ATP-activated macroscopic current for both inward and outward limbs of the I – V relationship, with no detectable change in the zero-current potential (Fig. 7). These results indicate that the relative permeability of Na + :NMDG + for the rP2X2 receptor does not change when Cd 2+ forms a bridge between TM1 and TM2, and thus the state stabilized by the bridge does not correspond to the proposed dilated state of P2X receptor channels. A Structural Model for the Open State of zfP2X4 Receptors. The results presented thus far suggest that the intrasubunit interactions observed in both the apo and ATP-bound structures are remarkably consistent with our engineered metal bridges between TM1 and TM2. However, the ATP-bound structure of the zfP2X4 receptor is nonnative in that intersubunit interactions are largely absent within the TM domain. To generate a model for the open state that contains intersubunit interactions but retains intrasubunit helix – helix interactions, we developed a conformational search algorithm to explore alternate quaternary arrangements of the TM2 helices (see Methods , Fig. S2). TM1 then was introduced into each subunit using the same relative positioning with respect to TM2 as in the ATP-bound X-ray structure, and fi nally the extracellular ATP-binding domain was grafted after superimposing the TM domain. The resulting model was chosen because it is as close as possible to the X-ray structure of the ATP- bound zfP2X4 receptor but introduces additional intersubunit interactions to stabilize the TM domain. Consistent with both accessibility (6 – 8) and metal coordination (7, 8) data, our model features expansion of the external pore and narrowing of the internal pore (Fig. S1 B and C ). In this model, relative to the X-ray structure, the TM2 helices are translated 1.3 Å toward the central axis, the twist of the TM2 three-helix bundle is increased by 10°, and each TM2 helix is rotated 5° counterclockwise (as viewed from the top) around its helical axis, creating a much more tightly packed intersubunit interface (Fig. 8 A – C and Fig. S2). We performed MD simulations on this model, similar to those performed on the ATP- bound X-ray structure (Fig. 1), and observed that lipid molecules were excluded from the pore (Fig. 8 D ), which remained well- hydrated throughout the simulation (Fig. 8 D ). The addition of one turn of the helix in TM1 and a slight reduction in the tilt angle of TM2 result in a modest improvement in hydrophobic mismatch (Fig. S3 A ). The pore diameter of our model is some- what less than that of the X-ray structure, especially toward the internal end (Fig. S3 B and C ); however, it still is 4.7 Å wide at its narrowest point and therefore should support permeation of phys- iologically relevant cations such as Na + , K + , or Ca 2+ . Although we found that formation of a Cd 2+ bridge in the C-C-C construct does not alter the relative permeability of Na + to NMDG + , this channel clearly permeates NMDG + at voltages negative to − 70 mV (Fig. 7 A ). Corey – Pauling – Koltun (CPK) models of NMDG + can fi t into a box with dimensions of 5 × 6.4 × 12 Å (24), providing an upper limit on the dimensions necessary for permeation. The actual pore diameter required to support permeation would be lower, because the structures of both the organic cation and the protein will fl uctuate on a rapid timescale. Thus, it seems reasonable to conclude that the diameter of the pore in our open- state model is compatible with NMDG + permeation. In the simulation of the open-state model, the side chains of L351 alternate between two rotamers, one of which positions the side chain toward the inter-TM2 interface, and the other with the side chain projected into the pore. Interestingly, this residue is equivalent to V343 in the rP2X2 receptor, where substitution of Cys results in the formation of a stable Cd 2+ bridge that blocks the channel (7). This Cd 2+ bridge forms rapidly (rate constant = 10 M/s) in the presence of ATP, suggesting that bridging occurs in the highly populated open state, and requires Cys in all three subunits, implying that it forms at the central axis of the pore (7). L351 in the X-ray structure of the ATP-bound zfP2X4 receptor cannot coordinate Cd 2+ because of the larger diameter of the pore at this position (L351 C β – C β distances of 11.2 Å; Fig. S1 B ). However, in our open-state model L351C residues in the alter- native rotamer, together with two water molecules, can coordinate Cd 2+ in the triagonal bipyramidal geometry (Fig. 8 F ). The objective of the present study was to evaluate both intersubunit and intrasubunit interactions within the TM domain of P2X receptors using both computational and functional approaches. A key motivation for this work was the unexpected absence of intersubunit interactions in the recent X-ray structure of the ATP-bound zfP2X4 receptor, which raises the possibility that the structure is distorted (15) and brings into question the proposed mechanism of ATP activation. Indeed, our MD simulations suggest that lipid molecules can diffuse through the intersubunit crevices to occupy and dehydrate the ion permeation pathway (Fig. 1), a scenario that is incompatible with ion conduction. As noted previously (15), the predominance of hydro- carbon side chains in helical transmembrane domains implicates nonspeci fi c van der Waals and weak electrostatic interactions as the main intraprotein forces for tertiary and quaternary struc- tural stability. In native membranes, the hydrophobic environment provided by the acyl chains of lipid molecules enhances helix – helix packing and helps de fi ne the hydrophobic dimension. The lack of such a hydrophobic environment in the crystalline lattice is likely the reason for the poor intersubunit packing of the TM1 and TM2 helices and the apparent hydrophobic mismatch in the X-ray structure of the ATP-bound zfP2X4 receptor. Such distortions may be particularly problematic for membrane proteins such as P2X receptors, which contain large extracellular domains that provide most of the crystal contacts, together with small transmembrane domains that contain only six helices in the trimer (15). Although the intersubunit crevices are a striking feature of the ATP-bound X-ray structure, only relatively modest structural rearrangements are required to form an intersubunit interface within the membrane. In our open-state model of the zfP2X4 receptor, intersubunit interactions within two regions of the TM domain were created by small rotations and translation of the TM2 helix toward the central axis (Fig. 8). The fi rst interface is within the internal region of the TM domain, where residues on three turns of the TM2 helix (L351, I355, and W358) from one subunit make contact with L346, A347, and V354 on the adjacent TM2 helix. The second is within the external end of the TM domain, where Y45 in TM1 contacts L340 in TM2 of the adjacent subunit. These contacts do not completely seal off the pore from the surrounding lipid membrane but diminish the crevices to openings or portals (Fig. 8 C ), similar to what has been seen in the KcsA potassium channel (25) and the NavAb voltage-activated sodium channel (26). In addition, our model exhibits a modest improvement in hydrophobic mismatch (Fig. S3 A ). We also investigated the intrasubunit interactions depicted in both the apo and ATP-bound X-ray structures (Fig. 2), and in this case we identi fi ed metal bridges that are fully compatible with those structures. Our results demonstrate that S345C and C348 in TM2 and H/C33 in TM1 form a robust intrasubunit Cd 2+ bridge that stabilizes the open state of the rP2X2 receptor (Figs. 3 – 6). The X-ray structures position the equivalent of S345C and C348 on one face of the TM2 helix where they can coordinate Cd 2+ in either the apo or ATP-bound states (Fig. 2), consistent with our functional results demonstrating that Cd 2+ remains stably associated with the channel in the closed state (Fig. 5). Those structures also show that ATP binding triggers a relative rotation of the TM1/TM2 interface so that residue 33 in TM1 also can participate in coordinating the metal. Thus, our metal-bridging results substantiate intrasubunit helix interactions and the intrasubunit motions between TM1 and TM2 upon ATP binding that are depicted in the X-ray structures of the zfP2X4 receptor. When considered together with the modest modi fi cations that are required to decrease the size of the intersubunit crevices, these results support the proposed structural mechanism by which ATP binding leads to an iris-like opening of P2X receptor channels (5). In our structural model for the open state, this structural rear- rangement would also be accompanied by a narrowing of the internal pore to create intersubunit interactions between the TM2 helices (Fig. 8). The metal bridges that we engineered between TM1 and TM2 also identify an internal region of the TM domain that is particularly sensitive to modi fi cation, where the activity of the channel can be tuned readily (Figs. 3 – 6). The relative motions between TM1 and TM2 in this region are not very large (Fig. 2), but occupancy of the engineered metal-binding site has profound consequences for both the gating and conduction properties of P2X receptors. In the case of the C-C-C bridge, ...Similar publications
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... The online version of this article includes the following figure supplement(s) for figure 2: To explore whether ions might permeate through the lateral fenestrations, we decided to investigate the accessibility of an introduced Cys residue to thiol-reactive methanethiosulfonate (MTS) compounds. The rP2X2 receptor channel has been extensively used for accessibility studies because it slowly desensitizes and, therefore, is well-suited for examining changes in accessibility between open and closed states (Jiang et al., 2001;Li et al., 2008;Li et al., 2010;Kawate et al., 2011;Heymann et al., 2013). In addition, the rP2X2-3 T construct, wherein three native Cys residues were substituted with Thr residues, is insensitive to MTS compounds unless additional Cys residues are introduced and have been extensively used for accessibility studies (Li et al., 2008;Li et al., 2010;Kawate et al., 2011;Heymann et al., 2013). ...
... The rP2X2 receptor channel has been extensively used for accessibility studies because it slowly desensitizes and, therefore, is well-suited for examining changes in accessibility between open and closed states (Jiang et al., 2001;Li et al., 2008;Li et al., 2010;Kawate et al., 2011;Heymann et al., 2013). In addition, the rP2X2-3 T construct, wherein three native Cys residues were substituted with Thr residues, is insensitive to MTS compounds unless additional Cys residues are introduced and have been extensively used for accessibility studies (Li et al., 2008;Li et al., 2010;Kawate et al., 2011;Heymann et al., 2013). We began by introducing a Cys into rP2X2-3 T at the position equivalent to E11 in hP2X3 (E17 in the rP2X2), reasoning that if this acidic residue lines the lateral fenestrations in P2X2 receptor channels, a Cys introduced at this position would be accessible to thiol-reactive compounds. ...
P2X receptor channels are trimeric ATP-activated ion channels expressed in neuronal and non-neuronal cells that are attractive therapeutic targets for human disorders. Seven subtypes of P2X receptor channels have been identified in mammals that can form both homomeric and heteromeric channels. P2X1-4 and P2X7 receptor channels are cation-selective, whereas P2X5 has been reported to have both cation and anion permeability. P2X receptor channel structures reveal that each subunit is comprised of two transmembrane helices, with both N-and C-termini on the intracellular side of the membrane and a large extracellular domain that contains the ATP binding sites at subunit interfaces. Recent structures of ATP-bound P2X receptors with the activation gate open reveal the unanticipated presence of a cytoplasmic cap over the central ion permeation pathway, leaving lateral fenestrations that may be largely buried within the membrane as potential pathways for ions to permeate the intracellular end of the pore. In the present study, we identify a critical residue within the intracellular lateral fenestrations that is readily accessible to thiol-reactive compounds from both sides of the membrane and where substitutions influence the relative permeability of the channel to cations and anions. Taken together, our results demonstrate that ions can enter or exit the internal pore through lateral fenestrations that play a critical role in determining the ion selectivity of P2X receptor channels.
... The intracellular N-and C-termini of P2X receptors are linked to a large extracellular ligand-binding domain (with 10 conserved cysteine residues) using two transmembrane-spanning helices (TM1 and TM2) (143)(144)(145). TM1 assists with channel gating, while TM2 lines the ion pore (146). ...
Regardless of improved biological insights and therapeutic advances, cancer is consuming multiple lives worldwide. Cancer is a complex disease with diverse cellular, metabolic, and physiological parameters as its hallmarks. This instigates a need to uncover the latest therapeutic targets to advance the treatment of cancer patients. Purines are building blocks of nucleic acids but also function as metabolic intermediates and messengers, as part of a signaling pathway known as purinergic signaling. Purinergic signaling comprises primarily adenosine triphosphate (ATP) and adenosine (ADO), their analogous membrane receptors, and a set of ectonucleotidases, and has both short- and long-term (trophic) effects. Cells release ATP and ADO to modulate cellular function in an autocrine or paracrine manner by activating membrane-localized purinergic receptors (purinoceptors, P1 and P2). P1 receptors are selective for ADO and have four recognized subtypes—A1, A2A, A2B, and A3. Purines and pyrimidines activate P2 receptors, and the P2X subtype is ligand-gated ion channel receptors. P2X has seven subtypes (P2X1–7) and forms homo- and heterotrimers. The P2Y subtype is a G protein-coupled receptor with eight subtypes (P2Y1/2/4/6/11/12/13/14). ATP, its derivatives, and purinoceptors are widely distributed in all cell types for cellular communication, and any imbalance compromises the homeostasis of the cell. Neurotransmission, neuromodulation, and secretion employ fast purinergic signaling, while trophic purinergic signaling regulates cell metabolism, proliferation, differentiation, survival, migration, invasion, and immune response during tumor progression. Thus, purinergic signaling is a prospective therapeutic target in cancer and therapy resistance.
... To explore whether ions might permeate through the lateral fenestrations, we decided to investigate the accessibility of an introduced Cys residue to thiol reactive methanethiosulfonate (MTS) compounds. The rP2X2 receptor channel has been extensively used for accessibility studies because it slowly desensitizes and therefore is well-suited for examining changes in accessibility between open and closed states (Li, Chang et al. 2008, Li, Kawate et al. 2010, Kawate, Robertson et al. 2011, Heymann, Dai et al. 2013). In addition, the rP2X2-3T construct, wherein three native Cys residues were substituted with Thr residues, is insensitive to MTS compounds unless additional Cys residues are introduced and has been extensively used for accessibility studies (Li, Chang et al. 2008, Li, Kawate et al. 2010, Kawate, Robertson et al. 2011, Heymann, Dai et al. 2013. ...
... The rP2X2 receptor channel has been extensively used for accessibility studies because it slowly desensitizes and therefore is well-suited for examining changes in accessibility between open and closed states (Li, Chang et al. 2008, Li, Kawate et al. 2010, Kawate, Robertson et al. 2011, Heymann, Dai et al. 2013). In addition, the rP2X2-3T construct, wherein three native Cys residues were substituted with Thr residues, is insensitive to MTS compounds unless additional Cys residues are introduced and has been extensively used for accessibility studies (Li, Chang et al. 2008, Li, Kawate et al. 2010, Kawate, Robertson et al. 2011, Heymann, Dai et al. 2013. We began by introducing a Cys into rP2X2-3T at the position equivalent E11 in hP2X3 (E17 in the rP2X2), reasoning that if this acidic residue lines the lateral fenestrations in P2X2 receptor channels, introducing a Cys at this position would be accessible to thiol-reactive compounds. ...
P2X receptor channels are trimeric ATP-activated ion channels expressed in neuronal and nonneuronal cells that are attractive therapeutic targets for human disorders. Seven subtypes of P2X receptor channels have been identified in mammals that can form both homomeric and heteromeric channels. P2X1-4 and P2X7 receptor channels are cation selective, whereas P2X5 has been reported to have both cation and anion permeability. P2X receptor channel structures reveal that each subunit is comprised of two transmembrane helices, with both N-and C-termini on the intracellular side of the membrane, and a large extracellular domain that contains the ATP binding sites at subunit interfaces. Recent structures of ATP-bound P2X receptors with the activation gate open reveal the unanticipated presence of a cytoplasmic cap over the central ion permeation pathway, leaving lateral fenestrations that may be largely buried within the membrane as potential pathways for ions to permeate the intracellular end of the pore. In the present study we identify a critical residue within the intracellular lateral fenestrations that is readily accessible to thiol reactive compounds from both sides of the membrane and where substitutions influence the relative permeability of the channel to cations and anions. Taken together, our results demonstrate that ions can enter or exit the internal pore through lateral fenestrations that play a critical role in determining the ion selectivity of P2X receptor channels.
... Despite the known dependencies of P2X4 function on metal cation binding, the structural mechanisms by which common ions like Mg 2þ and K þ affect the channel function are less understood. All-atom molecular dynamics (MD) simulations have been used to investigate aspects of P2X4 function, and generally encompassed short nanosecondlevel unbiased MD simulations or enhanced sampling MD simulations to probe P2X4 activation (32)(33)(34)(35)(36)(37)(38)(39). More recently, an MD study has elucidated mechanisms of sodium and chloride conductance through zfP2X4 and human P2X3 (40). ...
... The two-and one-ATP-bound open-state zfP2X4 systems were generated by removing ATP from the three-ATP-bound crystal structure. To facilitate comparison with published zfP2X4 simulation data from Heymann et al. (33), we followed their model-building protocol by using the default options in the CHARMM-GUI protocol (42), except that we used a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. We additionally evaluated the assignment of protons at pH 7.4 using PROPKA (43,44), which confirmed that the default protonation states of residues near the bound ions were appropriate. ...
... Nonetheless, our simulations indicate that bound Mg 2þ limits the collapse of open state to a closed form, which is consistent with experiments using hP2X3 (29) and Gulf Coast P2X (63). Our simulations exhibited a modest constriction of the pore domain (see Figs. 2 B and 3 C) relative to the crystallized open state with bound Mg 2þ , which is concurrent with a simulation study from Heymann et al. (33). Nonetheless, subsequent studies may consider building homology models of the capping domain based on the hP2X3 structure (29), although the relatively low sequence identity (z41%) may introduce considerable uncertainty into the modeling. ...
The P2X4 receptor plays a prominent role in cellular responses to extracellular ATP. Through classical all-atom molecular dynamicsMD simulations totaling 24 μs we have investigated how metal-complexed ATP stabilizes the channel’s open state and prevents its closing. We have identified two metal-binding sites, (Mg²⁺) and potassium (K⁺), one at the intersection of the three subunits in the ectodomain (MBS1) and the second one near the ATP binding site (MBS2), similar to those characterized in Gulf coast P2X. Our data indicate that when Mg²⁺ and K⁺ ions are complexed with ATP, the channel is locked into an open state. Interestingly, irrespective of the number of bound ATP molecules, Mg²⁺ ions bound to the MBS2 resisted collapse of the open state protein to a closed state by stabilizing the ATP-protein interactions. However, when Mg²⁺ in the MBS2 was replaced with K⁺ ions, as might be expected when in equilibrium with an extracellular solution, the interactions between the subunits were weakened and the pore collapsed. This collapse was apparent when fewer than two ATP were bound to MBS2 in the presence of K⁺. Therefore, the different capacities of common cations to stabilize the channel may underlie a mechanism governing P2X4 channel gating in physiological systems. This study therefore provides structural insights into the differential modulation of ATP activation of P2X4 by Mg²⁺ and K⁺.
... 48 MD simulations were also used to verify the ATP-bound structure of zfP2X4, suggesting the existence of the potentially nonphysiological intersubunit gap in the TM domain in the experimental structure. 49 Notably, computational structural biology has also been widely used for the molecular docking between P2X receptors and chemical compounds, 50 Overall, considering the importance of P2X receptors as drug targets, additional structural information on the interactions between P2X receptors and subtype-specific compounds are necessary for future drug discovery. ...
P2X receptors are ATP-gated trimeric nonselective cation channels that are important for various physiological and pathological processes, including synaptic transmission, pain perception, immune regulation and apoptosis. Accordingly, they attract a wide range of interest as drug targets, such as those for chronic cough, neuropathic pain and depression. After the zebrafish P2X4 receptor structure was reported in 2009, various other P2X receptor structures have been reported, extending our understanding of the molecular mechanisms of P2X receptors. This review article describes the recent progress on understanding the structures and mechanisms of P2X receptors, especially of the mechanisms underlying ATP binding and conformational changes during the gating cycle. In addition, since several antagonists for different P2X subtypes have entered into clinical trials, this review also summarizes the binding sites and regulatory mechanisms of these antagonists, which may contribute to new strategies of targeting P2X receptors for drug discovery.
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... If these gaps exist when the channel is in its native environment, they would likely be occupied by bilayer lipid molecules, which might then be expected to occlude the channel pore. In fact, evidence has been presented that these gaps are not present in membrane-integrated [23], suggesting that at least this feature of the crystal structures is not representative of the native structure of the receptor. ...
The P2X4 receptor (P2X4R) is an ATP-gated cation channel. Here, we used fast-scan atomic force microscopy (AFM) to visualize changes in the structure and mobility of individual P2X4Rs in response to activation. P2X4Rs were purified from detergent extracts of transfected cells and integrated into lipid bilayers. Activation resulted in a rapid (2 s) and substantial (10-20 nm2 ) increase in the cross-sectional area of the extracellular region of the receptor and a corresponding decrease in receptor mobility. Both effects were blocked by the P2X4R antagonist 5-BDBD. Addition of cholesterol to the bilayer reduced receptor mobility, although the ATP-induced reduction in mobility was still observed. We suggest that the observed responses to activation may have functional consequences for purinergic signalling.
... Tryptophan and cysteine scanning studies of both TM1 and TM2 revealed that the upper regions of each segment undergo substantial rearrangement and adopt α-helical secondary structures during channel gating [15][16][17]. Furthermore, interactions between the TM1 region of one subunit and the TM2 region of another subunit have been shown to regulate the gating and ion conductance of P2X4Rs, specifically the lower ends of the TM segments [18]. Studies have suggested that homomeric P2X4Rs contain a putative ethanol activity site formed by residues located at the ectodomain-TM interfaces [19], aspartic acid at position 331, and methionine at 336 located in the upper part of the TM2 segment [12], and two tryptophan residues at position 50 at TM1-ectodomain interface and position 46 in the TM1 segment [12]. ...
... To generate ATP-concentration curves, oocytes were exposed to 0.05-100 µM ATP range for 5 sec followed by 5-15 min washout. The effect of ethanol on P2X4R function is more robust and reliable when tested in the presence of sub-maximal concentrations of ATP (typically EC [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] [19,20]. Therefore, ethanol was co-applied with EC 10 ATP. ...
Mouse models of alcohol use disorder (AUD) revealed purinergic P2X4 receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)–ectodomain interface and within the TM1 segment contribute to the formation of an ethanol action pocket in P2X4Rs. In the present study, we tested the hypothesis that there are more residues in TM1 and TM2 segments that are important for the ethanol sensitivity of P2X4Rs. Using site-directed mutagenesis and two electrode voltage-clamp electrophysiology in Xenopus oocytes, we found that arginine at position 33 (R33) in the TM1 segment plays a role in the ethanol sensitivity of P2X4Rs. Molecular models in both closed and open states provided evidence for interactions between R33 and aspartic acid at position 354 (D354) of the neighboring TM2 segment. The loss of ethanol sensitivity in mixtures of wild-type (WT) and reciprocal single mutants, R33D:WT and D354R:WT, versus the WT-like response in R33D-D354R:WT double mutant provided further support for this interaction. Additional findings indicated that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility/stability during channel opening. Collectively, these findings identified new activity sites and suggest the importance of TM1-TM2 interaction for the function and ethanol sensitivity of P2X4Rs.
... Tryptophan scanning studies of both TM1 and TM2 revealed that the upper regions of each segment undergo substantial rearrangement and adopt α-helical secondary structures during channel gating [15][16][17]. Furthermore, interactions between the TM1 region of one subunit and TM2 region of another subunit have been shown to regulate the gating and ion conductance of P2X4Rs, specifically, the lower ends of the TM segments [18,19]. Studies have suggested that homomeric P2X4Rs contain a putative ethanol activity site formed by residues located at the ectodomain-TM interfaces [20], aspartic acid at position 331 and methionine at 336 located in the upper part of the TM2 segment [12], and two tryptophan residues at position 50 at TM1-ectodomain interface and position 46 in the TM1 segment [12]. ...
Mouse models of alcohol use disorder (AUD) revealed a subtype of purinergic receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)-ectodomain interface and within TM1 segment contribute to the formation of an ethanol action pocket in P2X4Rs. In the present study, we tested the hypothesis that there are more residues in TM segments, which are important for ethanol sensitivity of P2X4Rs. Using site-directed mutagenesis and two-electrode voltage-clamp electrophysiology in Xenopus oocytes, we found that arginine at position 33 (R33) in the TM1 segment plays a role in ethanol sensitivity of P2X4Rs. Molecular models in both closed and open states provided evidence for interactions between R33 and aspartic acid at position 354 (D354) of the neighboring TM2 segment. Further work with mixtures of wild-type (WT) and reciprocal single (R33D:WT, D354R:WT) and double (R33D-D354R:WT) mutants confirmed the importance of this interaction for ethanol sensitivity, ivermectin action and channel function. Additionally, our findings suggest that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility during channel opening. Collectively, these findings identified new activity sites, and suggest the importance of TM1-TM2 interaction for channel function and ethanol sensitivity of P2X4Rs.
... The 4DW1 structure presents intersubunit crevices that should not be present in membra- ne-embedded receptors. 52 Previous MD simulations have demonstrated that lipid molecules can enter the channel through these crevices. When building our model for P2X 4 , we pur- posely chose to superpose the structure of P2X 4 with that of P2X 3 using residues located in the upper part of the transmembrane domains. ...
P2X receptors are a group of trimeric cationic channels that activate by ATP. They perform critical roles in the membranes of mammalian cells and their improper functioning is associated with numerous diseases. Despite the vast amount of research devoted to them, several aspects of their operation are currently unclear. Among these remain the causes of their charge selectivity. We present the results of molecular dynamics simulations which shed light on this issue for the case of P2X4 channels. We examined in detail the behavior of Na+ and Cl- ions inside the receptor. The examination reveals that charge discrimination occurs in two stages. First, cations bear precedence over anions to enter the extracellular vestibule. Next, cations at the extracellular vestibule are more likely to cross the pore than anions in an equivalent position. In this manner, a thorough but straightforward analysis of computational simulations suggests a stepwise mechanism, without a unique determinant factor.
... Ces mouvements entrainent alors une augmentation du diamètre du pore, de l'ordre de 3 Å. Ces données structurales obtenues en 2012, ont été nuancées en 2013 par une étude in silico qui, par le biais de la création d'un site de métalation au cadmium, propose que l'hélice TM2 subit une rotation de 5° dans le sens antihoraire et un déplacement léger de 1,3 Å par rapport à la structure cristallographique(Heymann et al., 2013). Enfin, la structure cristallographique de 2016 montre que l'hélice TM2 de hP2X3 effectue une rotation de 15°(Figure 19.C). ...
Les récepteurs P2X, activés par l’ATP extracellulaire et cations non-sélectifs, sont impliqués dans de nombreux rôles physiopathologiques. Le manque de sélectivité de molécules pharmacologiques est un inconvénient majeur pour leur étude. La résolution de leurs structures cristallographiques a permis de les comprendre à l’échelle moléculaire, cependant les mécanismes impliqués dans les transitions allostériques restent mal compris. Au laboratoire, deux outils, dérivés d’azobenzène, permettant l’activation des récepteurs P2X en absence d’ATP et par la lumière ont été développés. L’utilisation de ces outils ont permis l’étude de la transition allostérique de l’état ouvert à l’état désensibilisé, mettant en avant une zone de régulation efficace dans les espaces transmembranaires. De plus, leur utilisation a permis l’investigation biophysique d’une mutation présente sur P2X2 humain, responsable d’une surdité non-syndromatique. Cette mutation entraine un rétrécissement du pore, impactant le passage de gros cations impliqués dans le processus d’audition. Enfin, la relation entre le diamètre du pore ionique et le passage de gros cations a été établi.
