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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 lateral fenestrations in zfP2X4 and rP2X2. Results for zfP2X4 are shown in A, whereas those for rP2X2 are shown in B. The ion-accessible positions are shown as orange spheres near the extracellular membrane interface around Z = 15–25 Å (left). Free energy plots (ΔΔGint) for cross sections through the lateral fenestrations (Z = 16, 18, and 20 Å) are shown on the right for Na+ (top) and Cl− (bottom). Black squares indicate ΔΔGint < −2 Kcal mol−.
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fig3: Electrostatic interaction free energy of ions along the lateral fenestrations in zfP2X4 and rP2X2. Results for zfP2X4 are shown in A, whereas those for rP2X2 are shown in B. The ion-accessible positions are shown as orange spheres near the extracellular membrane interface around Z = 15–25 Å (left). Free energy plots (ΔΔGint) for cross sections through the lateral fenestrations (Z = 16, 18, and 20 Å) are shown on the right for Na+ (top) and Cl− (bottom). Black squares indicate ΔΔGint < −2 Kcal mol−.

Mentions: The electrostatic accessibility of the lateral fenestrations was also examined in the zfP2X4 structure and rP2X2 model (Fig. 3, A and B). We found that for both of these closed structures, there was a clear pathway connecting the outside solution to the central pore that was electrostatically accessible by Na+ (<1 kcal mol−1). Notably, Cl− was not energetically stable along these pathways. Small openings of the windows would be sufficient to remove any small electrostatic barriers created by the protein reaction field and provide an energetically favorable pathway for cations. These calculations suggest that it is electrostatically possible for cations to move via the lateral fenestrations, even in the closed-state structure of both zfP2X4 and rP2X2.


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 lateral fenestrations in zfP2X4 and rP2X2. Results for zfP2X4 are shown in A, whereas those for rP2X2 are shown in B. The ion-accessible positions are shown as orange spheres near the extracellular membrane interface around Z = 15–25 Å (left). Free energy plots (ΔΔGint) for cross sections through the lateral fenestrations (Z = 16, 18, and 20 Å) are shown on the right for Na+ (top) and Cl− (bottom). Black squares indicate ΔΔGint < −2 Kcal mol−.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Electrostatic interaction free energy of ions along the lateral fenestrations in zfP2X4 and rP2X2. Results for zfP2X4 are shown in A, whereas those for rP2X2 are shown in B. The ion-accessible positions are shown as orange spheres near the extracellular membrane interface around Z = 15–25 Å (left). Free energy plots (ΔΔGint) for cross sections through the lateral fenestrations (Z = 16, 18, and 20 Å) are shown on the right for Na+ (top) and Cl− (bottom). Black squares indicate ΔΔGint < −2 Kcal mol−.
Mentions: The electrostatic accessibility of the lateral fenestrations was also examined in the zfP2X4 structure and rP2X2 model (Fig. 3, A and B). We found that for both of these closed structures, there was a clear pathway connecting the outside solution to the central pore that was electrostatically accessible by Na+ (<1 kcal mol−1). Notably, Cl− was not energetically stable along these pathways. Small openings of the windows would be sufficient to remove any small electrostatic barriers created by the protein reaction field and provide an energetically favorable pathway for cations. These calculations suggest that it is electrostatically possible for cations to move via the lateral fenestrations, even in the closed-state structure of both zfP2X4 and rP2X2.

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