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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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I317C and H319C in the central vestibule are accessible to MTSET but not to MTS-TPAE. Superimposed scaled current traces on the left show that extracellular application of MTS-TPAE (blue bar) has little effect on currents evoked by ATP (black bar). Extracellular application of MTSET, even after the application of MTS-TPAE, irreversibly modified currents evoked by ATP (right traces). (A) Deactivation of I317C mutant became faster after MTSET application. (B) ATP-induced current of H319C was potentiated by MTSET application. ATP was applied every 3 min. The control traces (black traces) in A and B are the same in each of the three panels. (C) Quantitative comparisons of current modifications by MTS reagents. Averaged current modifications by MTSET (left) or MTS-TPAE (right) at each Cys mutant (n = 3–9). Error bars are SEM. Orange bar highlights H319C, a position where MTSET causes pronounced current potentiation. Although I317C showed little change in current amplitude after MTSET application, the MTS reagent accelerated current deactivation after ATP removal; therefore, the residue is highlighted with an asterisk to indicate MTS reactivity.
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fig7: I317C and H319C in the central vestibule are accessible to MTSET but not to MTS-TPAE. Superimposed scaled current traces on the left show that extracellular application of MTS-TPAE (blue bar) has little effect on currents evoked by ATP (black bar). Extracellular application of MTSET, even after the application of MTS-TPAE, irreversibly modified currents evoked by ATP (right traces). (A) Deactivation of I317C mutant became faster after MTSET application. (B) ATP-induced current of H319C was potentiated by MTSET application. ATP was applied every 3 min. The control traces (black traces) in A and B are the same in each of the three panels. (C) Quantitative comparisons of current modifications by MTS reagents. Averaged current modifications by MTSET (left) or MTS-TPAE (right) at each Cys mutant (n = 3–9). Error bars are SEM. Orange bar highlights H319C, a position where MTSET causes pronounced current potentiation. Although I317C showed little change in current amplitude after MTSET application, the MTS reagent accelerated current deactivation after ATP removal; therefore, the residue is highlighted with an asterisk to indicate MTS reactivity.

Mentions: To explore whether the central pathway might serve as a permeation pathway, we introduced Cys residues at 26 positions (Fig. 6 A) corresponding to residues at the central threefold axis in the zfP2X4 structure and tested for modification by MTS reagents. For 24 of these positions, both MTSET and MTS-TPAE had no effect on ATP-activated currents (Figs. 6, B–E, and 7 C). In the acidic central vestibule along the central pathway, two mutants (I317C and H319C) were modified by MTSET, but not by the larger MTS-TPAE. For I317C, MTSET increased the rate of deactivation (Fig. 7 A, middle traces), whereas for H319C, MTSET potentiated the ATP-induced current by ∼40% and slowed the rate of current deactivation (Fig. 7 B, middle traces). In the case of I317C, subsequent application of ATP 3 min after the removal of MTSET resulted in ATP-activated currents that retained rapid deactivation, suggesting that the initial effects of MTSET are the result of covalent modification of I317C (Fig. 7 A, right traces). In the case of H319C, the potentiation of ATP-activated currents by MTSET did not recover after the removal of the MTS reagent, and a subsequent application of MTSET in the presence of ATP was without effect (Fig. 7 B, right traces), indicating covalent modification of H319C by MTSET. The rate of MTSET modification at H319C was ∼3.5 × 103 M−1s−1 (n = 5), consistent with this residue being solvent exposed in the presence of ATP. In both cases, preapplication of MTS-TPAE did not affect the MTSET-dependent current modification (Fig. 7, A and B, left and middle traces), suggesting that the larger MTS-TPAE cannot access these residues.


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)

I317C and H319C in the central vestibule are accessible to MTSET but not to MTS-TPAE. Superimposed scaled current traces on the left show that extracellular application of MTS-TPAE (blue bar) has little effect on currents evoked by ATP (black bar). Extracellular application of MTSET, even after the application of MTS-TPAE, irreversibly modified currents evoked by ATP (right traces). (A) Deactivation of I317C mutant became faster after MTSET application. (B) ATP-induced current of H319C was potentiated by MTSET application. ATP was applied every 3 min. The control traces (black traces) in A and B are the same in each of the three panels. (C) Quantitative comparisons of current modifications by MTS reagents. Averaged current modifications by MTSET (left) or MTS-TPAE (right) at each Cys mutant (n = 3–9). Error bars are SEM. Orange bar highlights H319C, a position where MTSET causes pronounced current potentiation. Although I317C showed little change in current amplitude after MTSET application, the MTS reagent accelerated current deactivation after ATP removal; therefore, the residue is highlighted with an asterisk to indicate MTS reactivity.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105519&req=5

fig7: I317C and H319C in the central vestibule are accessible to MTSET but not to MTS-TPAE. Superimposed scaled current traces on the left show that extracellular application of MTS-TPAE (blue bar) has little effect on currents evoked by ATP (black bar). Extracellular application of MTSET, even after the application of MTS-TPAE, irreversibly modified currents evoked by ATP (right traces). (A) Deactivation of I317C mutant became faster after MTSET application. (B) ATP-induced current of H319C was potentiated by MTSET application. ATP was applied every 3 min. The control traces (black traces) in A and B are the same in each of the three panels. (C) Quantitative comparisons of current modifications by MTS reagents. Averaged current modifications by MTSET (left) or MTS-TPAE (right) at each Cys mutant (n = 3–9). Error bars are SEM. Orange bar highlights H319C, a position where MTSET causes pronounced current potentiation. Although I317C showed little change in current amplitude after MTSET application, the MTS reagent accelerated current deactivation after ATP removal; therefore, the residue is highlighted with an asterisk to indicate MTS reactivity.
Mentions: To explore whether the central pathway might serve as a permeation pathway, we introduced Cys residues at 26 positions (Fig. 6 A) corresponding to residues at the central threefold axis in the zfP2X4 structure and tested for modification by MTS reagents. For 24 of these positions, both MTSET and MTS-TPAE had no effect on ATP-activated currents (Figs. 6, B–E, and 7 C). In the acidic central vestibule along the central pathway, two mutants (I317C and H319C) were modified by MTSET, but not by the larger MTS-TPAE. For I317C, MTSET increased the rate of deactivation (Fig. 7 A, middle traces), whereas for H319C, MTSET potentiated the ATP-induced current by ∼40% and slowed the rate of current deactivation (Fig. 7 B, middle traces). In the case of I317C, subsequent application of ATP 3 min after the removal of MTSET resulted in ATP-activated currents that retained rapid deactivation, suggesting that the initial effects of MTSET are the result of covalent modification of I317C (Fig. 7 A, right traces). In the case of H319C, the potentiation of ATP-activated currents by MTSET did not recover after the removal of the MTS reagent, and a subsequent application of MTSET in the presence of ATP was without effect (Fig. 7 B, right traces), indicating covalent modification of H319C by MTSET. The rate of MTSET modification at H319C was ∼3.5 × 103 M−1s−1 (n = 5), consistent with this residue being solvent exposed in the presence of ATP. In both cases, preapplication of MTS-TPAE did not affect the MTSET-dependent current modification (Fig. 7, A and B, left and middle traces), suggesting that the larger MTS-TPAE cannot access these residues.

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