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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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Restricting motion by introducing double Cys mutants that form intersubunit disulfide bridges. (A) Locations of the five double Cys mutants created in this study: I, P89C/F291C; II, G93C/V95C; III, P90C/E91C; IV, S65C/D315C; V, E59C/Q321C. For clarity, bridges are only shown for one of the three subunit interfaces. (B) Western blot in the absence of reducing agent indicates that all five mutants form either trimers or dimers.
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fig8: Restricting motion by introducing double Cys mutants that form intersubunit disulfide bridges. (A) Locations of the five double Cys mutants created in this study: I, P89C/F291C; II, G93C/V95C; III, P90C/E91C; IV, S65C/D315C; V, E59C/Q321C. For clarity, bridges are only shown for one of the three subunit interfaces. (B) Western blot in the absence of reducing agent indicates that all five mutants form either trimers or dimers.

Mentions: To explore whether the central pathway undergoes a large conformational change upon ATP binding to create a wide route for extracellular ions to permeate, we engineered five pairs of double Cys mutants along the central pathway (Fig. 8 A), which based on the closed-state zfP2X4 structure would be predicted to form intersubunit disulfide bonds. Because P2X receptors are trimers, each Cys pair could give rise to a total of three disulfides. Such bridges should prevent a large motion because of conformational restraints caused by introducing intersubunit disulfide bridges. These double Cys mutants were expressed in HEK cells, and their ability to form disulfide bridges was assessed by SDS-PAGE in the absence of a reducing agent, followed by Western blotting. Under mild air-oxidizing conditions, the band for the wild-type rP2X2 migrated at the appropriate molecular weight for the monomeric species (∼53 kD), indicating that there is no intersubunit disulfide bridge formation in the parent construct (Fig. 8 B, lane WT). On the other hand, all double Cys mutants formed SDS-resistant trimers (∼150 kD) or dimers (∼100 kD), suggesting that intersubunit disulfides are formed, at least to some extent, in all the mutants (Fig. 8 B, lanes I–V).


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)

Restricting motion by introducing double Cys mutants that form intersubunit disulfide bridges. (A) Locations of the five double Cys mutants created in this study: I, P89C/F291C; II, G93C/V95C; III, P90C/E91C; IV, S65C/D315C; V, E59C/Q321C. For clarity, bridges are only shown for one of the three subunit interfaces. (B) Western blot in the absence of reducing agent indicates that all five mutants form either trimers or dimers.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig8: Restricting motion by introducing double Cys mutants that form intersubunit disulfide bridges. (A) Locations of the five double Cys mutants created in this study: I, P89C/F291C; II, G93C/V95C; III, P90C/E91C; IV, S65C/D315C; V, E59C/Q321C. For clarity, bridges are only shown for one of the three subunit interfaces. (B) Western blot in the absence of reducing agent indicates that all five mutants form either trimers or dimers.
Mentions: To explore whether the central pathway undergoes a large conformational change upon ATP binding to create a wide route for extracellular ions to permeate, we engineered five pairs of double Cys mutants along the central pathway (Fig. 8 A), which based on the closed-state zfP2X4 structure would be predicted to form intersubunit disulfide bonds. Because P2X receptors are trimers, each Cys pair could give rise to a total of three disulfides. Such bridges should prevent a large motion because of conformational restraints caused by introducing intersubunit disulfide bridges. These double Cys mutants were expressed in HEK cells, and their ability to form disulfide bridges was assessed by SDS-PAGE in the absence of a reducing agent, followed by Western blotting. Under mild air-oxidizing conditions, the band for the wild-type rP2X2 migrated at the appropriate molecular weight for the monomeric species (∼53 kD), indicating that there is no intersubunit disulfide bridge formation in the parent construct (Fig. 8 B, lane WT). On the other hand, all double Cys mutants formed SDS-resistant trimers (∼150 kD) or dimers (∼100 kD), suggesting that intersubunit disulfides are formed, at least to some extent, in all the mutants (Fig. 8 B, lanes I–V).

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