Limits...
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.

Show MeSH

Related in: MedlinePlus

Disulfide bridge formation in the central pathway preserves channel activity. Two short pulses of ATP applications (1 s; black arrows) with 2-min intervals showed inward currents without significant rundown. External DTT application (5-min duration) reduced the current amplitude of the mutant pair I and potentiated mutant pair V (red asterisks).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig9: Disulfide bridge formation in the central pathway preserves channel activity. Two short pulses of ATP applications (1 s; black arrows) with 2-min intervals showed inward currents without significant rundown. External DTT application (5-min duration) reduced the current amplitude of the mutant pair I and potentiated mutant pair V (red asterisks).

Mentions: Under identical air-oxidizing conditions, we performed whole cell patch clamp recordings from HEK cells expressing each pair of double Cys mutants. We chose to apply ATP (10 µM for the pairs I and III–V, and 30 µM for the pair II) for relatively short durations (1 s) at 2-min intervals because under these conditions, we observed relatively little rundown upon repeated applications of ATP (Fig. 9, left and middle traces). Notably, when cells were perfused with 10 mM DTT for 5 min to reduce disulfides, the top pair along the central pathway (Fig. 9, I) showed substantial current inhibition (∼70%), whereas currents in three other pairs (II–IV) were unchanged. The data for bridge I indicate that the channel activity detected for the outer pair of Cys mutants under oxidizing conditions must be mediated, at least in part, by the Cys mutants whose subunits were covalently cross-linked by intersubunit disulfides. Thus, we conclude that restricting the subunit–subunit movements at the top of the central pathway is compatible with ion permeation through the rP2X2 channel, supporting the idea that a large opening of the central pathway is unlikely. The interpretation of the three bridges where DTT has no effect (Fig. 9, II–IV) is ambiguous because, as pointed out in the previous studies on K+ channels (Kobertz et al., 2000), it is unclear whether the currents we observed are mediated by the population of disulfide cross-linked channels. We tried to drive disulfide bridge formation to completion by treating HEK cells with 0.3% H2O2 for 10 min, but no significant increase in disulfide formation was obtained (not depicted). In the case of the bottom pair (V), we observed large current potentiation when the cell was exposed to DTT, implying that the disulfide bond impairs a conformational change involved in channel opening. This bridge is actually positioned at the top of the lateral fenestration, and these results are therefore consistent with experiments with TR-MTSEA, which suggest that the lateral fenestrations become larger when the channel opens.


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)

Disulfide bridge formation in the central pathway preserves channel activity. Two short pulses of ATP applications (1 s; black arrows) with 2-min intervals showed inward currents without significant rundown. External DTT application (5-min duration) reduced the current amplitude of the mutant pair I and potentiated mutant pair V (red asterisks).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig9: Disulfide bridge formation in the central pathway preserves channel activity. Two short pulses of ATP applications (1 s; black arrows) with 2-min intervals showed inward currents without significant rundown. External DTT application (5-min duration) reduced the current amplitude of the mutant pair I and potentiated mutant pair V (red asterisks).
Mentions: Under identical air-oxidizing conditions, we performed whole cell patch clamp recordings from HEK cells expressing each pair of double Cys mutants. We chose to apply ATP (10 µM for the pairs I and III–V, and 30 µM for the pair II) for relatively short durations (1 s) at 2-min intervals because under these conditions, we observed relatively little rundown upon repeated applications of ATP (Fig. 9, left and middle traces). Notably, when cells were perfused with 10 mM DTT for 5 min to reduce disulfides, the top pair along the central pathway (Fig. 9, I) showed substantial current inhibition (∼70%), whereas currents in three other pairs (II–IV) were unchanged. The data for bridge I indicate that the channel activity detected for the outer pair of Cys mutants under oxidizing conditions must be mediated, at least in part, by the Cys mutants whose subunits were covalently cross-linked by intersubunit disulfides. Thus, we conclude that restricting the subunit–subunit movements at the top of the central pathway is compatible with ion permeation through the rP2X2 channel, supporting the idea that a large opening of the central pathway is unlikely. The interpretation of the three bridges where DTT has no effect (Fig. 9, II–IV) is ambiguous because, as pointed out in the previous studies on K+ channels (Kobertz et al., 2000), it is unclear whether the currents we observed are mediated by the population of disulfide cross-linked channels. We tried to drive disulfide bridge formation to completion by treating HEK cells with 0.3% H2O2 for 10 min, but no significant increase in disulfide formation was obtained (not depicted). In the case of the bottom pair (V), we observed large current potentiation when the cell was exposed to DTT, implying that the disulfide bond impairs a conformational change involved in channel opening. This bridge is actually positioned at the top of the lateral fenestration, and these results are therefore consistent with experiments with TR-MTSEA, which suggest that the lateral fenestrations become larger when the channel opens.

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