Ion permeation pathway within the internal pore of P2X receptor channels
Curation statements for this article:-
Curated by eLife
eLife assessment:
This study provides valuable insight into the molecular mechanism of ion selectivity in the broader family of ATP-gated P2X receptors. The experimental data are of high quality, the evidence supporting the conclusions is convincing, and the work will be of broad interest to biophysicists working on ion channel selectivity.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Neuroscience (eLife)
Abstract
P2X receptor channels are trimeric ATP-activated ion channels expressed in neuronal and non-neuronal cells that are attractive therapeutic targets for human disorders. Seven subtypes of P2X receptor channels have been identified in mammals that can form both homomeric and heteromeric channels. P2X1–4 and P2X7 receptor channels are cation-selective, whereas P2X5 has been reported to have both cation and anion permeability. P2X receptor channel structures reveal that each subunit is comprised of two transmembrane helices, with both N-and C-termini on the intracellular side of the membrane and a large extracellular domain that contains the ATP binding sites at subunit interfaces. Recent structures of ATP-bound P2X receptors with the activation gate open reveal the unanticipated presence of a cytoplasmic cap over the central ion permeation pathway, leaving lateral fenestrations that may be largely buried within the membrane as potential pathways for ions to permeate the intracellular end of the pore. In the present study, we identify a critical residue within the intracellular lateral fenestrations that is readily accessible to thiol-reactive compounds from both sides of the membrane and where substitutions influence the relative permeability of the channel to cations and anions. Taken together, our results demonstrate that ions can enter or exit the internal pore through lateral fenestrations that play a critical role in determining the ion selectivity of P2X receptor channels.
Article activity feed
-
-
eLife assessment:
This study provides valuable insight into the molecular mechanism of ion selectivity in the broader family of ATP-gated P2X receptors. The experimental data are of high quality, the evidence supporting the conclusions is convincing, and the work will be of broad interest to biophysicists working on ion channel selectivity.
-
Reviewer #1 (Public Review):
In this work, the authors investigated the mechanism by which ions are selected in ATP-gated P2X receptor channels using patch-clamp electrophysiology. P2X receptors are known to be cation-selective channels, but one of them (the P2X5) also displays anion permeability through a molecular mechanism that is unclear. Here, the authors identify in P2X2 a glutamate residue (E17) which plays a critical role in determining ion selectivity. This residue is localized in the intracellular side of the permeating pathway and is part of three large intracellular lateral fenestrations that are thought to be potential exit/entry pathways for ions. The authors elegantly show that when the side chain of E17 was substituted for cysteine, it became accessible to water-soluble, thiol-reactive methanethiosulfonate (MTS) …
Reviewer #1 (Public Review):
In this work, the authors investigated the mechanism by which ions are selected in ATP-gated P2X receptor channels using patch-clamp electrophysiology. P2X receptors are known to be cation-selective channels, but one of them (the P2X5) also displays anion permeability through a molecular mechanism that is unclear. Here, the authors identify in P2X2 a glutamate residue (E17) which plays a critical role in determining ion selectivity. This residue is localized in the intracellular side of the permeating pathway and is part of three large intracellular lateral fenestrations that are thought to be potential exit/entry pathways for ions. The authors elegantly show that when the side chain of E17 was substituted for cysteine, it became accessible to water-soluble, thiol-reactive methanethiosulfonate (MTS) derivatives that were applied from both sides of the membrane. By mutating E17 into lysine, which reverts the charge, they show that mutated channels displayed increased anion permeability, although channels still remained largely cation selective. However, reverting the charge in the mouse P2X5 (K17E and K17D), they provide evidence for a complete ion selectivity switch (that is mutated P2X5 became cation selective). Therefore, although the mechanism by which P2X2 selects cation versus anion still remains incompletely understood, it seems that K17 is a key determinant for P2X5 anion permeability.
The conclusions of this paper are well supported by data. The work should advance our understanding of ion selectivity in P2X receptors and will likely provide the foundation for further studies.
-
Reviewer #2 (Public Review):
The study by Tam and colleagues addresses the ion-conducting pathway and selectivity of P2X receptor channels. Recent structures of ATP-bound P2X receptors with the activation gate open revealed the presence of a cytoplasmic cap over the central ion permeation pathway. This prompts the authors to examine if lateral fenestrations are potential pathways for ions to permeate the intracellular end of the channel pore, even although they appear to be largely buried within the membrane. Based on sequence alignment, the authors identified a critical residue E17 within the intracellular lateral fenestrations and found that it is accessible to two thiol reactive reagents. Importantly, mutations of E17 also affect the relative permeability of the channels to cations and anions. The work thus solves an ion-conducting …
Reviewer #2 (Public Review):
The study by Tam and colleagues addresses the ion-conducting pathway and selectivity of P2X receptor channels. Recent structures of ATP-bound P2X receptors with the activation gate open revealed the presence of a cytoplasmic cap over the central ion permeation pathway. This prompts the authors to examine if lateral fenestrations are potential pathways for ions to permeate the intracellular end of the channel pore, even although they appear to be largely buried within the membrane. Based on sequence alignment, the authors identified a critical residue E17 within the intracellular lateral fenestrations and found that it is accessible to two thiol reactive reagents. Importantly, mutations of E17 also affect the relative permeability of the channels to cations and anions. The work thus solves an ion-conducting mystery of the physiologically important P2X receptor channels. It demonstrates that lateral fenestrations are part of the internal pore of P2X channels and play a critical role in determining ion selectivity.
The structural and sequence analysis is performed carefully, and the electrophysiological experiments are carried out beautifully. Although the data largely seem to support the conclusions, statistical analysis is required to strengthen the claims. Cysteine accessibility experiments may have alternative interpretations; thus, the rigor can be further improved to include the reversibility of the block by treating it with reducing agents.
-
Reviewer #3 (Public Review):
P2X channels are homomeric or heteromeric, non-selective cation channels that are gated by extracellular ATP. They are found in many tissues and are implicated in bodily functions including digestion and urination, and other processes such as pain and immune response. Recent atomic resolution structures of P2X3 and P2X7 have captured the principal gating states likely conserved within this channel family. Among novel structural features that were identified was a cytoplasmic cap that appears to stabilize the intracellular region of the pore in the open state. This cap is not present in the closed and inactivated states. From these data, it has been proposed that the intracellular side of a conductive P2X pore is formed by a cytoplasmic-exposed portion of a larger, membrane-embedded fenestration, a somewhat …
Reviewer #3 (Public Review):
P2X channels are homomeric or heteromeric, non-selective cation channels that are gated by extracellular ATP. They are found in many tissues and are implicated in bodily functions including digestion and urination, and other processes such as pain and immune response. Recent atomic resolution structures of P2X3 and P2X7 have captured the principal gating states likely conserved within this channel family. Among novel structural features that were identified was a cytoplasmic cap that appears to stabilize the intracellular region of the pore in the open state. This cap is not present in the closed and inactivated states. From these data, it has been proposed that the intracellular side of a conductive P2X pore is formed by a cytoplasmic-exposed portion of a larger, membrane-embedded fenestration, a somewhat unusual characteristic for ion channels. In this manuscript, the authors delineated the region of the fenestration that is likely exposed to the cytoplasm and identify a residue that is negatively charged in P2X1-4 and P2X7 but positively charged in P2X5-6. They suggest that not only could this residue line the ion pore, but also it may contribute to differences in cation-to-anion permeability previously observed between these P2X subfamilies. They demonstrate by electrophysiology that E17 lines the ion pore through a series of classical MTS blocking experiments. They further demonstrate that the charge of this residue confers partial or strong cation to anion permeability in rP2X2 and mP2X5, respectively. This is an elegant investigation of the internal pore of P2X channels and the experiments presented in this work are of high quality.
-
