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Ion access pathway to the transmembrane pore in P2X receptor channels.

Kawate T, Robertson JL, Li M, Silberberg SD, Swartz KJ - J. Gen. Physiol. (2011)

Bottom Line: P2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain.The extracellular region also contains a void at the central axis, providing a second potential pathway.The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Porter Neuroscience Research Center, Molecular Physiology and Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. kawatet@­ninds.nih.gov

ABSTRACT
P2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain. The x-ray structure of the P2X4 receptor from zebrafish (zfP2X4) receptor reveals that the extracellular vestibule above the gate opens to the outside through lateral fenestrations, providing a potential pathway for ions to enter and exit the pore. The extracellular region also contains a void at the central axis, providing a second potential pathway. To investigate the energetics of each potential ion permeation pathway, we calculated the electrostatic free energy by solving the Poisson-Boltzmann equation along each of these pathways in the zfP2X4 crystal structure and a homology model of rat P2X2 (rP2X2). We found that the lateral fenestrations are energetically favorable for monovalent cations even in the closed-state structure, whereas the central pathway presents strong electrostatic barriers that would require structural rearrangements to allow for ion accessibility. To probe ion accessibility along these pathways in the rP2X2 receptor, we investigated the modification of introduced Cys residues by methanethiosulfonate (MTS) reagents and constrained structural changes by introducing disulfide bridges. Our results show that MTS reagents can permeate the lateral fenestrations, and that these become larger after ATP binding. Although relatively small MTS reagents can access residues in one of the vestibules within the central pathway, no reactive positions were identified in the upper region of this pathway, and disulfide bridges that constrain movements in that region do not prevent ion conduction. Collectively, these results suggest that ions access the pore using the lateral fenestrations, and that these breathe as the channel opens. The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.

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Electrostatic interaction free energy of ions along the central pathways in zfP2X4 and rP2X2. Results for zfP2X4 are shown in A and B, whereas those for rP2X2 are shown in C and D. (A and C) ΔΔGint of Na+ (black), Ca2+ (green), and Cl− (blue), calculated along the central pathway, with the transmembrane region depicted with the yellow slab and the center of the membrane at Z = 0 Å. The pore radius profile is shown in the bar graph on top. (B and D) ΔΔGint of Na+ along the central pathway of the original closed model (black), and an artificially opened pore widened to 5-Å radius from the molecular threefold symmetry axis (red). The surface representations of the original closed (white) and the forced open (red) conformations are shown.
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fig2: Electrostatic interaction free energy of ions along the central pathways in zfP2X4 and rP2X2. Results for zfP2X4 are shown in A and B, whereas those for rP2X2 are shown in C and D. (A and C) ΔΔGint of Na+ (black), Ca2+ (green), and Cl− (blue), calculated along the central pathway, with the transmembrane region depicted with the yellow slab and the center of the membrane at Z = 0 Å. The pore radius profile is shown in the bar graph on top. (B and D) ΔΔGint of Na+ along the central pathway of the original closed model (black), and an artificially opened pore widened to 5-Å radius from the molecular threefold symmetry axis (red). The surface representations of the original closed (white) and the forced open (red) conformations are shown.

Mentions: To assess the energetics of ions along the two pathways, we calculated the electrostatic free energy of transferring Na+ or Ca2+ from aqueous solution to the central pore of zfP2X4. The electrostatic free energy profile, shown in Fig. 2 A, includes both the static field arising from the protein charges, as well as the reaction field resulting from the different dielectric regions. In this closed-state structure, the interaction energy of cations is prohibitive near the extracellular entrance of the central pathway (55 Å < Z < 85 Å). In contrast, the center of the extracellular domain (30 Å < Z < 55 Å) is extremely favorable for both Na+ and Ca2+, whereas the energy of Cl− is repulsive at each point along the central axis. Both of these observations result from a strongly electronegative vestibule, lined by six negative residues (E98 and D99), which corresponds to the Gd3+-binding site in the zfP2X4 structure. A simple expanded pore model was created to probe the degree of widening needed to reduce the electrostatic reaction field barrier and assess the static charge contribution to ion-interaction energies at the central axis. This was done by imposing a repulsive cylindrical potential restraint at the center of the pore and carrying out energy minimizations of the structure while successively increasing the cylinder radius. The resultant model has a pore that is 10 Å in diameter and maintains the overall distribution of residues lining the pore in the closed structure. In this model, the electrostatic free energy for Na+ is favorable along most of the central pathway (Fig. 2 B), making it a possible pathway for cation permeation in zfP2X4. However, when we performed the same calculations for the rP2X2 homology model, there were still significant electrostatic barriers along the pore that occur because of the lack of acidic residues in the sequence of this homologue (Fig. 2, C and D). This suggests that although the central pathway is a favorable option in zfP2X4, the sequence variation in rP2X2 creates an unfavorable electrostatic environment that is unlikely to support cations along the central axis.


Ion access pathway to the transmembrane pore in P2X receptor channels.

Kawate T, Robertson JL, Li M, Silberberg SD, Swartz KJ - J. Gen. Physiol. (2011)

Electrostatic interaction free energy of ions along the central pathways in zfP2X4 and rP2X2. Results for zfP2X4 are shown in A and B, whereas those for rP2X2 are shown in C and D. (A and C) ΔΔGint of Na+ (black), Ca2+ (green), and Cl− (blue), calculated along the central pathway, with the transmembrane region depicted with the yellow slab and the center of the membrane at Z = 0 Å. The pore radius profile is shown in the bar graph on top. (B and D) ΔΔGint of Na+ along the central pathway of the original closed model (black), and an artificially opened pore widened to 5-Å radius from the molecular threefold symmetry axis (red). The surface representations of the original closed (white) and the forced open (red) conformations are shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105519&req=5

fig2: Electrostatic interaction free energy of ions along the central pathways in zfP2X4 and rP2X2. Results for zfP2X4 are shown in A and B, whereas those for rP2X2 are shown in C and D. (A and C) ΔΔGint of Na+ (black), Ca2+ (green), and Cl− (blue), calculated along the central pathway, with the transmembrane region depicted with the yellow slab and the center of the membrane at Z = 0 Å. The pore radius profile is shown in the bar graph on top. (B and D) ΔΔGint of Na+ along the central pathway of the original closed model (black), and an artificially opened pore widened to 5-Å radius from the molecular threefold symmetry axis (red). The surface representations of the original closed (white) and the forced open (red) conformations are shown.
Mentions: To assess the energetics of ions along the two pathways, we calculated the electrostatic free energy of transferring Na+ or Ca2+ from aqueous solution to the central pore of zfP2X4. The electrostatic free energy profile, shown in Fig. 2 A, includes both the static field arising from the protein charges, as well as the reaction field resulting from the different dielectric regions. In this closed-state structure, the interaction energy of cations is prohibitive near the extracellular entrance of the central pathway (55 Å < Z < 85 Å). In contrast, the center of the extracellular domain (30 Å < Z < 55 Å) is extremely favorable for both Na+ and Ca2+, whereas the energy of Cl− is repulsive at each point along the central axis. Both of these observations result from a strongly electronegative vestibule, lined by six negative residues (E98 and D99), which corresponds to the Gd3+-binding site in the zfP2X4 structure. A simple expanded pore model was created to probe the degree of widening needed to reduce the electrostatic reaction field barrier and assess the static charge contribution to ion-interaction energies at the central axis. This was done by imposing a repulsive cylindrical potential restraint at the center of the pore and carrying out energy minimizations of the structure while successively increasing the cylinder radius. The resultant model has a pore that is 10 Å in diameter and maintains the overall distribution of residues lining the pore in the closed structure. In this model, the electrostatic free energy for Na+ is favorable along most of the central pathway (Fig. 2 B), making it a possible pathway for cation permeation in zfP2X4. However, when we performed the same calculations for the rP2X2 homology model, there were still significant electrostatic barriers along the pore that occur because of the lack of acidic residues in the sequence of this homologue (Fig. 2, C and D). This suggests that although the central pathway is a favorable option in zfP2X4, the sequence variation in rP2X2 creates an unfavorable electrostatic environment that is unlikely to support cations along the central axis.

Bottom Line: P2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain.The extracellular region also contains a void at the central axis, providing a second potential pathway.The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Porter Neuroscience Research Center, Molecular Physiology and Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. kawatet@­ninds.nih.gov

ABSTRACT
P2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain. The x-ray structure of the P2X4 receptor from zebrafish (zfP2X4) receptor reveals that the extracellular vestibule above the gate opens to the outside through lateral fenestrations, providing a potential pathway for ions to enter and exit the pore. The extracellular region also contains a void at the central axis, providing a second potential pathway. To investigate the energetics of each potential ion permeation pathway, we calculated the electrostatic free energy by solving the Poisson-Boltzmann equation along each of these pathways in the zfP2X4 crystal structure and a homology model of rat P2X2 (rP2X2). We found that the lateral fenestrations are energetically favorable for monovalent cations even in the closed-state structure, whereas the central pathway presents strong electrostatic barriers that would require structural rearrangements to allow for ion accessibility. To probe ion accessibility along these pathways in the rP2X2 receptor, we investigated the modification of introduced Cys residues by methanethiosulfonate (MTS) reagents and constrained structural changes by introducing disulfide bridges. Our results show that MTS reagents can permeate the lateral fenestrations, and that these become larger after ATP binding. Although relatively small MTS reagents can access residues in one of the vestibules within the central pathway, no reactive positions were identified in the upper region of this pathway, and disulfide bridges that constrain movements in that region do not prevent ion conduction. Collectively, these results suggest that ions access the pore using the lateral fenestrations, and that these breathe as the channel opens. The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.

Show MeSH
Related in: MedlinePlus