The P2X7 receptor from Xenopus laevis: formation of a large pore in Xenopus oocytes

Paukert, Martin; Hidayat, Susanti; Gründer, Stefan

doi:10.1016/s0014-5793(02)02324-4

Cited by 30 publications

(24 citation statements)

References 22 publications

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“…Transfected cells responded with robust inward currents (membrane holding potential ϭ Ϫ60 mV) and changes in fura-2 fluorescence to applications of BzATP (100 -600 M) with one exception: we failed to record agonist-gated current or a change in fura-2 fluorescence in response to BzATP (up to and including 1 mM) and ATP (6 mM) in HEK293 cells transfected with the gene encoding the Xenopus laevis P2X7aR. This was surprising because injection of frog P2X7aR cRNA results in large ATPgated currents in a Xenopus oocyte expression system (79 (Fig. 3, A and B).…”

Section: Resultsmentioning

confidence: 83%

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

Liang¹,

Samways²,

Wolf³

et al. 2015

Journal of Biological Chemistry

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Background: Ca 2ϩ triggers many of the actions of extracellular ATP. Results: We measured the Ca 2ϩ component of the ATP-gated non-selective cation current of P2X7 receptors. Conclusion: Ca 2ϩ flux varied with the species of origin, the splice variant, and the agonist concentration. Significance: Our results suggest that domains that lie outside of the pore regulate the Ca 2ϩ flux of P2X7 receptors.

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Section: Resultsmentioning

confidence: 83%

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

Liang¹,

Samways²,

Wolf³

et al. 2015

Journal of Biological Chemistry

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“…First, P2X 7 receptor mediated NMDG ϩ permeability increases and YOPRO1 uptake have been observed in oocytes that lack native Panx-1 (2,31,32). In contrast, a recent study concluded that these properties could only be measured when Panx-1 was coexpressed (18).…”

Section: Resultsmentioning

confidence: 99%

Patch–clamp coordinated spectroscopy shows P2X ₂ receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

Chaumont

Khakh

2008

Proc. Natl. Acad. Sci. U.S.A.

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ATP-gated P2X receptors display ion permeability increases within seconds of receptor activation as the channels enter the I 2 state, which is permeable to organic cations and dye molecules. The mechanisms underlying this important behavior are not completely understood. In one model, the I 2 state is thought to be due to opening of Pannexin-1 (Panx-1) channels, and, in the second, it is thought to be an intrinsic P2X property. We tested both models by measuring ion and dye permeability and used a patch-clamp coordinated spectroscopy approach to measure conformational changes. Our data show that Panx-1 channels make no detectable contribution to the P2X 2 receptor I2 state. However, P2X 2 receptors display permeability dynamics, which are correlated with conformational changes in the cytosolic domain remote from the selectivity filter itself. Finally, the data illustrate the utility of a new approach, using tetracysteine tags and biarsenical fluorophores to measure site-specific conformational changes in membrane proteins. (Fig. 1A), which is permeable to cations, such as Na ϩ and Ca 2ϩ (7,8). In P2X 2 , P2X 4 , and P2X 7 receptors after seconds of activation by ATP, the channel undergoes additional seconds time-scale changes that increase permeability to organic cations, such as N-methyl-D-glucamine (NMDG) ϩ , and dyes, such as YOPRO1 (2,(4)(5)(6)9). This second open state is referred to as I 2 (Fig. 1 A). The first reports of the I 2 state permeable to large molecules go back 30 years, when ATP was shown to permeabilize cells (10). The mechanisms that underlie opening to the I 2 state remain unclear, even though it is a trigger for a variety of pathophysiological processes, including blebbing, microvesicle shedding, release of signaling molecules, permeablization, and cell death (7,11). In addition to the obvious relevance to ATP signaling, a better understanding of the I 2 state would also advance our understanding of the diversity of mechanisms used by ion channels. This is because ion permeability and/or selectivity filter dynamics are not unique to P2X but also occur for other channels (12, 13). In particular, the organic cation and dye permeable I 2 state of TRP channels (14, 15) shares similarity to P2X 2 receptors. A key open question is how the organic cation and dye permeable I 2 state arises.Two models exist to explain the I 2 state for P2X receptors. The first model proposes that P2X 7 -mediated dye uptake (16, 17) occurs via Pannexin-1 (Panx-1) channels, leading to the suggestion that the I 2 state is due to Panx-1 channels rather than P2X (11,18). In this study, we call this the ''Panx-1 model'' (Fig. 1 A). The second model posits that the I 2 state is an intrinsic property of P2X receptors involving slow conformational changes that allow organic cations to flow (2-6, 19). We call this the ''gating model'' (Fig. 1 A). We tested both models under a common set of recording conditions. We also used a patch-clamp coordinated imaging approach to directly measure protein conformational changes. Results...

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“…Intermolecular models argue that pore formation occurs by successive acquisition of subunits to existing oligomeric structures (Cockcroft and Gomperts, 1979). While recent studies suggest that pannexin1 is required for P2X 7 R pore formation (Locovei et al, 2007), there are also reports that injection of P2X 7 R mRNA alone is sufficient to allow generation of pores in oocytes (Paukert et al, 2002). Intramolecular models propose that subtle structural changes, possibly in the selectivity filter, contribute to channel-pore transition (Lester and Karschin, 2000).…”

Section: P2x7rg345y-egfp: a Valuable Tool For Separating Ion Channel mentioning

confidence: 99%

The P2X₇Receptor Drives Microglial Activation and Proliferation: A Trophic Role for P2X₇R Pore

et al. 2009

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Microglial activation is an integral part of neuroinflammation associated with many neurodegenerative conditions. Interestingly, a number of neurodegenerative conditions exhibit enhanced P2X 7 receptor (P2X 7 R) expression in the neuroinflammatory foci where activated microglia are a coexisting feature. Whether P2X 7 R overexpression is driving microglial activation or, conversely, P2X 7 R overexpression is a consequence of microglial activation is not known. We report that overexpression alone of a purinergic P2X 7 R, in the absence of pathological insults, is sufficient to drive the activation and proliferation of microglia in rat primary hippocampal cultures. The trophic responses observed in microglia were found to be P2X 7 R specific as the P2X 7 R antagonist, oxidized ATP (oxATP), was effective in markedly attenuating microgliosis. oxATP treatment of primary hippocampal cultures expressing exogenous P2X 7 Rs resulted in a significant decrease in the number of activated microglia. P2X 7 R is unusual in exhibiting two conductance states, a cation channel and a plasma membrane pore, and there are no pharmacological agents capable of cleanly discriminating between these two states. We used a point mutant of P2X 7 R (P2X7RG345Y) with intact channel function but ablated pore-forming capacity to establish that the trophic effects of increased P2X 7 R expression are exclusively mediated by the pore conductance. Collectively, and contrary to previous reports describing P2X 7 R as a "death receptor," we provide evidence for a novel trophic role for P2X 7 R pore in microglia.

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The P2X₇ receptor from Xenopus laevis: formation of a large pore in Xenopus oocytes

Cited by 30 publications

References 22 publications

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

Patch–clamp coordinated spectroscopy shows P2X ₂ receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

The P2X₇Receptor Drives Microglial Activation and Proliferation: A Trophic Role for P2X₇R Pore

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The P2X7 receptor from Xenopus laevis: formation of a large pore in Xenopus oocytes

Cited by 30 publications

References 22 publications

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

Patch–clamp coordinated spectroscopy shows P2X 2 receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

The P2X7Receptor Drives Microglial Activation and Proliferation: A Trophic Role for P2X7R Pore

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The P2X₇ receptor from Xenopus laevis: formation of a large pore in Xenopus oocytes

Patch–clamp coordinated spectroscopy shows P2X ₂ receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

The P2X₇Receptor Drives Microglial Activation and Proliferation: A Trophic Role for P2X₇R Pore