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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 potential in the lateral fenestrations may contribute to selecting cations over anions. Sagittal sections of the electrostatic potential surface calculated using the APBS tools (Baker et al., 2001). (A) zfP2X4 with supplemented side chains. Homology models of rP2X2 (B) and hP2X5 (C). 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.
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fig10: Electrostatic potential in the lateral fenestrations may contribute to selecting cations over anions. Sagittal sections of the electrostatic potential surface calculated using the APBS tools (Baker et al., 2001). (A) zfP2X4 with supplemented side chains. Homology models of rP2X2 (B) and hP2X5 (C). 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.

Mentions: In identifying the lateral fenestration as the permeation pathway for ions to enter or exit the pore, it becomes interesting to consider whether this pathway may contribute to the ion selectivity of P2X receptor channels. Our Poisson-Boltzmann calculations reveal that the electrostatic interaction energy along this short (∼15-Å) pathway is relatively favorable for Na+ permeation (ΔΔGint = −1 to +1 kcal mol−1), but not for Cl− (ΔΔGint > +10 kcal mol−1) (Fig. 3), suggesting that the overall negative potential in this area may contribute to selecting cations over anions. Interestingly, the P2X5 receptor has been reported to also be permeable to anions (pCl−/pNa+ = ∼0.5; Ruppelt et al., 2001; Bo et al., 2003), and the electrostatic potential in a hP2X5 receptor homology model is significantly positive around the lateral fenestration (∼+10 kcal mol−1; Fig. 10), supporting the hypothesis that the electrostatics along the lateral fenestration plays a role in ion selection.


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 potential in the lateral fenestrations may contribute to selecting cations over anions. Sagittal sections of the electrostatic potential surface calculated using the APBS tools (Baker et al., 2001). (A) zfP2X4 with supplemented side chains. Homology models of rP2X2 (B) and hP2X5 (C). 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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig10: Electrostatic potential in the lateral fenestrations may contribute to selecting cations over anions. Sagittal sections of the electrostatic potential surface calculated using the APBS tools (Baker et al., 2001). (A) zfP2X4 with supplemented side chains. Homology models of rP2X2 (B) and hP2X5 (C). 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.
Mentions: In identifying the lateral fenestration as the permeation pathway for ions to enter or exit the pore, it becomes interesting to consider whether this pathway may contribute to the ion selectivity of P2X receptor channels. Our Poisson-Boltzmann calculations reveal that the electrostatic interaction energy along this short (∼15-Å) pathway is relatively favorable for Na+ permeation (ΔΔGint = −1 to +1 kcal mol−1), but not for Cl− (ΔΔGint > +10 kcal mol−1) (Fig. 3), suggesting that the overall negative potential in this area may contribute to selecting cations over anions. Interestingly, the P2X5 receptor has been reported to also be permeable to anions (pCl−/pNa+ = ∼0.5; Ruppelt et al., 2001; Bo et al., 2003), and the electrostatic potential in a hP2X5 receptor homology model is significantly positive around the lateral fenestration (∼+10 kcal mol−1; Fig. 10), supporting the hypothesis that the electrostatics along the lateral fenestration plays a role in ion selection.

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