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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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Two potential pathways for extracellular ions to access the transmembrane pore in P2X receptors. (A) Surface representation of the zfP2X4 receptor model in the closed state. Each color represents one of the three subunits. (B) A sagittal section of the closed-state model reveals two potential ion access pathways (central and lateral pathways; blue dashed arrows). Electrostatic potential surface calculated using the APBS tools (Baker et al., 2001) shows two acidic vestibules along these potential ion access pathways (upper and central vestibules). The surface is colored based on the potential, contoured from −6 kcal mol−1 (red) to +6 kcal mol−1 (blue). White denotes 0 kcal mol−1. To complement the electrostatic contributions of the missing side chains in the crystal structure because of weak electron density, the side chains were added back to the model, and their geometry was optimized using the MODELLER software (Eswar et al., 2006). Structural representations in all figures were generated using PyMOL (Schrödinger, LLC).
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fig1: Two potential pathways for extracellular ions to access the transmembrane pore in P2X receptors. (A) Surface representation of the zfP2X4 receptor model in the closed state. Each color represents one of the three subunits. (B) A sagittal section of the closed-state model reveals two potential ion access pathways (central and lateral pathways; blue dashed arrows). Electrostatic potential surface calculated using the APBS tools (Baker et al., 2001) shows two acidic vestibules along these potential ion access pathways (upper and central vestibules). The surface is colored based on the potential, contoured from −6 kcal mol−1 (red) to +6 kcal mol−1 (blue). White denotes 0 kcal mol−1. To complement the electrostatic contributions of the missing side chains in the crystal structure because of weak electron density, the side chains were added back to the model, and their geometry was optimized using the MODELLER software (Eswar et al., 2006). Structural representations in all figures were generated using PyMOL (Schrödinger, LLC).

Mentions: In this study, we investigated the pathway through which ions traverse the extracellular domain of P2X receptors to enter or exit the transmembrane pore. The x-ray structure of zfP2X4 reveals that two potential pathways exist. The first pathway is through three lateral fenestrations that connect the extracellular solution to an extracellular vestibule just above the occluded gate region of the pore (Fig. 1, A and B). Although these lateral fenestrations have dimensions of ∼8 × 10 Å in the zfP2X4 receptor structure, several important side chains were not modeled because of the poor quality of electron density maps in this region (N54, D59, T60, Q329, I335, and I336 in zfP2X4). The inclusion of all side chains in the zfP2X4 structure suggests that these fenestrations may be somewhat narrower. The second possible pathway for ions to access the pore is along the central threefold axis of the zfP2X4 structure (Fig. 1, A and B). Two conspicuous electronegative cavities are present between the outermost end of the extracellular region and the extracellular vestibule in zfP2X4, which are predicted to form favorable sites for cation binding. Although the diameter of the pathways connecting these chambers with each other and the extracellular solution at the top of the receptor would not support rapid ion throughput, they might expand after the binding of ATP. There is precedence in other ion channels for both types of potential permeation pathways. Ions must traverse an extended (∼85 Å) pore that projects into the cytoplasm in inward-rectifier potassium (Kir) channels (Nishida and MacKinnon, 2002; Nishida et al., 2007; Tao et al., 2009), and ions are thought to use four lateral fenestrations between the transmembrane pore and the cytoplasmic “hanging gondola” in voltage-activated potassium (Kv) channels (Kobertz and Miller, 1999; Long et al., 2007), and in corresponding regions in CNG channels (Johnson and Zagotta, 2005). To probe the ion access pathway in the extracellular region of a P2X receptor, we performed Cys-scanning mutagenesis and accessibility studies using methanethiosulfonate (MTS) reagents. In combination with disulfide cross-linking experiments and electrostatic energy calculations, our data suggest that ions do not use the central pathway, but likely permeate through the three lateral fenestrations located between the extracellular region and the transmembrane pore.


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)

Two potential pathways for extracellular ions to access the transmembrane pore in P2X receptors. (A) Surface representation of the zfP2X4 receptor model in the closed state. Each color represents one of the three subunits. (B) A sagittal section of the closed-state model reveals two potential ion access pathways (central and lateral pathways; blue dashed arrows). Electrostatic potential surface calculated using the APBS tools (Baker et al., 2001) shows two acidic vestibules along these potential ion access pathways (upper and central vestibules). The surface is colored based on the potential, contoured from −6 kcal mol−1 (red) to +6 kcal mol−1 (blue). White denotes 0 kcal mol−1. To complement the electrostatic contributions of the missing side chains in the crystal structure because of weak electron density, the side chains were added back to the model, and their geometry was optimized using the MODELLER software (Eswar et al., 2006). Structural representations in all figures were generated using PyMOL (Schrödinger, LLC).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Two potential pathways for extracellular ions to access the transmembrane pore in P2X receptors. (A) Surface representation of the zfP2X4 receptor model in the closed state. Each color represents one of the three subunits. (B) A sagittal section of the closed-state model reveals two potential ion access pathways (central and lateral pathways; blue dashed arrows). Electrostatic potential surface calculated using the APBS tools (Baker et al., 2001) shows two acidic vestibules along these potential ion access pathways (upper and central vestibules). The surface is colored based on the potential, contoured from −6 kcal mol−1 (red) to +6 kcal mol−1 (blue). White denotes 0 kcal mol−1. To complement the electrostatic contributions of the missing side chains in the crystal structure because of weak electron density, the side chains were added back to the model, and their geometry was optimized using the MODELLER software (Eswar et al., 2006). Structural representations in all figures were generated using PyMOL (Schrödinger, LLC).
Mentions: In this study, we investigated the pathway through which ions traverse the extracellular domain of P2X receptors to enter or exit the transmembrane pore. The x-ray structure of zfP2X4 reveals that two potential pathways exist. The first pathway is through three lateral fenestrations that connect the extracellular solution to an extracellular vestibule just above the occluded gate region of the pore (Fig. 1, A and B). Although these lateral fenestrations have dimensions of ∼8 × 10 Å in the zfP2X4 receptor structure, several important side chains were not modeled because of the poor quality of electron density maps in this region (N54, D59, T60, Q329, I335, and I336 in zfP2X4). The inclusion of all side chains in the zfP2X4 structure suggests that these fenestrations may be somewhat narrower. The second possible pathway for ions to access the pore is along the central threefold axis of the zfP2X4 structure (Fig. 1, A and B). Two conspicuous electronegative cavities are present between the outermost end of the extracellular region and the extracellular vestibule in zfP2X4, which are predicted to form favorable sites for cation binding. Although the diameter of the pathways connecting these chambers with each other and the extracellular solution at the top of the receptor would not support rapid ion throughput, they might expand after the binding of ATP. There is precedence in other ion channels for both types of potential permeation pathways. Ions must traverse an extended (∼85 Å) pore that projects into the cytoplasm in inward-rectifier potassium (Kir) channels (Nishida and MacKinnon, 2002; Nishida et al., 2007; Tao et al., 2009), and ions are thought to use four lateral fenestrations between the transmembrane pore and the cytoplasmic “hanging gondola” in voltage-activated potassium (Kv) channels (Kobertz and Miller, 1999; Long et al., 2007), and in corresponding regions in CNG channels (Johnson and Zagotta, 2005). To probe the ion access pathway in the extracellular region of a P2X receptor, we performed Cys-scanning mutagenesis and accessibility studies using methanethiosulfonate (MTS) reagents. In combination with disulfide cross-linking experiments and electrostatic energy calculations, our data suggest that ions do not use the central pathway, but likely permeate through the three lateral fenestrations located between the extracellular region and the transmembrane pore.

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