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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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Accessibility of MTS reagents along the lateral fenestration. (A) The mutated residues are shown in stick representation (blue, carbon atoms; red, oxygen; dark blue, nitrogen), and the remaining region is shown in cartoon representations (gray). A transmembrane pore-lining residue, T336, is shown in orange. Representative residues are labeled. (B–E) Accessibility of two different-size MTS reagents in the presence of ATP. Superimposed scaled current traces recorded in response to ATP without (black) and with (blue) MTSET or MTS-TPAE applications (blue bars) show no modification for Q52C (B) and S326C (C), and modification for I328C (D) and T336C (E). (F) TR-MTSEA is accessible to I328C in the open state but not in the closed state. Exposing the channels to TR-MTSEA for 10 s in the closed state did not modify the ATP-induced current, whereas a 6-s exposure in the presence of ATP modified the current.
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fig4: Accessibility of MTS reagents along the lateral fenestration. (A) The mutated residues are shown in stick representation (blue, carbon atoms; red, oxygen; dark blue, nitrogen), and the remaining region is shown in cartoon representations (gray). A transmembrane pore-lining residue, T336, is shown in orange. Representative residues are labeled. (B–E) Accessibility of two different-size MTS reagents in the presence of ATP. Superimposed scaled current traces recorded in response to ATP without (black) and with (blue) MTSET or MTS-TPAE applications (blue bars) show no modification for Q52C (B) and S326C (C), and modification for I328C (D) and T336C (E). (F) TR-MTSEA is accessible to I328C in the open state but not in the closed state. Exposing the channels to TR-MTSEA for 10 s in the closed state did not modify the ATP-induced current, whereas a 6-s exposure in the presence of ATP modified the current.

Mentions: The lateral fenestrations consist of two strands connecting the two membrane-spanning α helices within each subunit to the large extracellular ligand-binding region. In contrast to the long and narrow central pathway, these lateral fenestrations are relatively wide and shallow in the closed-state structure (Fig. 1 B). We set out to explore whether these lateral fenestrations serve as ion permeation pathways by substituting Cys residues at each of the 17 residues that make up the walls of the fenestrations (Fig. 4 A), and tested whether their reaction with MTS reagents has detectable effects on ATP-activated currents. In the closed-state structure, these residues exhibit extensive solvent exposure and therefore should react with MTS reagents. However, if the wide and shallow features of the lateral fenestrations are maintained in the open state, one might imagine that MTS reagents would not have a dramatic effect on ion conduction, but might alter gating of the channel because these strands connect the ligand-binding region to the pore of the channel. For these experiments, we externally applied 2-(trimethylammonium)ethyl MTS (MTSET; mol wt: 278 D) and 2-(tripentylammonium)ethyl MTS (MTS-TPAE; mol wt: 447 D), two different-size reagents that are water soluble, membrane impermeable, and positively charged. Because both reagents react relatively rapidly with T336C within the gate region of rP2X2 in the presence of ATP (∼104 M−1s−), and cause reductions of ATP-induced currents (Fig. 4 E) (Li et al., 2008, 2010), the extracellular pathway to the pore must be wide enough for these MTS reagents to access the ion permeation pathway.


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)

Accessibility of MTS reagents along the lateral fenestration. (A) The mutated residues are shown in stick representation (blue, carbon atoms; red, oxygen; dark blue, nitrogen), and the remaining region is shown in cartoon representations (gray). A transmembrane pore-lining residue, T336, is shown in orange. Representative residues are labeled. (B–E) Accessibility of two different-size MTS reagents in the presence of ATP. Superimposed scaled current traces recorded in response to ATP without (black) and with (blue) MTSET or MTS-TPAE applications (blue bars) show no modification for Q52C (B) and S326C (C), and modification for I328C (D) and T336C (E). (F) TR-MTSEA is accessible to I328C in the open state but not in the closed state. Exposing the channels to TR-MTSEA for 10 s in the closed state did not modify the ATP-induced current, whereas a 6-s exposure in the presence of ATP modified the current.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Accessibility of MTS reagents along the lateral fenestration. (A) The mutated residues are shown in stick representation (blue, carbon atoms; red, oxygen; dark blue, nitrogen), and the remaining region is shown in cartoon representations (gray). A transmembrane pore-lining residue, T336, is shown in orange. Representative residues are labeled. (B–E) Accessibility of two different-size MTS reagents in the presence of ATP. Superimposed scaled current traces recorded in response to ATP without (black) and with (blue) MTSET or MTS-TPAE applications (blue bars) show no modification for Q52C (B) and S326C (C), and modification for I328C (D) and T336C (E). (F) TR-MTSEA is accessible to I328C in the open state but not in the closed state. Exposing the channels to TR-MTSEA for 10 s in the closed state did not modify the ATP-induced current, whereas a 6-s exposure in the presence of ATP modified the current.
Mentions: The lateral fenestrations consist of two strands connecting the two membrane-spanning α helices within each subunit to the large extracellular ligand-binding region. In contrast to the long and narrow central pathway, these lateral fenestrations are relatively wide and shallow in the closed-state structure (Fig. 1 B). We set out to explore whether these lateral fenestrations serve as ion permeation pathways by substituting Cys residues at each of the 17 residues that make up the walls of the fenestrations (Fig. 4 A), and tested whether their reaction with MTS reagents has detectable effects on ATP-activated currents. In the closed-state structure, these residues exhibit extensive solvent exposure and therefore should react with MTS reagents. However, if the wide and shallow features of the lateral fenestrations are maintained in the open state, one might imagine that MTS reagents would not have a dramatic effect on ion conduction, but might alter gating of the channel because these strands connect the ligand-binding region to the pore of the channel. For these experiments, we externally applied 2-(trimethylammonium)ethyl MTS (MTSET; mol wt: 278 D) and 2-(tripentylammonium)ethyl MTS (MTS-TPAE; mol wt: 447 D), two different-size reagents that are water soluble, membrane impermeable, and positively charged. Because both reagents react relatively rapidly with T336C within the gate region of rP2X2 in the presence of ATP (∼104 M−1s−), and cause reductions of ATP-induced currents (Fig. 4 E) (Li et al., 2008, 2010), the extracellular pathway to the pore must be wide enough for these MTS reagents to access the ion permeation pathway.

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