P2X7 Receptor Channels Allow Direct Permeation of Nanometer-Sized Dyes

Browne, Liam E.; Compan, Vincent; Bragg, Laricia; North, Richard

doi:10.1523/jneurosci.2235-12.2013

Cited by 131 publications

(192 citation statements)

References 56 publications

(10 reference statements)

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“…Macroscopic current-voltage (I-V) relationships were obtained using voltage ramps in whole-cell recordings in which Na + was the primary internal cation and NMDG + was the primary external cation. In previous reports with related ionic conditions and protocols, the zero-current potential shifted in the positive direction in the continuous presence of ATP as the relative permeability of Na + to NMDG + changes (21)(22)(23). When Cd 2+ was applied to the C-C-C construct, we observed large increases in ATP-activated macroscopic current for both inward and outward limbs of the I-V relationship, with no detectable change in the zero-current potential (Fig.…”

Section: Leftsupporting

confidence: 61%

“…We also explored whether the conducting states stabilized by the C-C-C bridge might correspond to the dilated state of P2X receptors that gives rise to a change in the relative permeability of Na + to N-methyl-D-glucamine (NMDG + ) (21)(22)(23). Macroscopic current-voltage (I-V) relationships were obtained using voltage ramps in whole-cell recordings in which Na + was the primary internal cation and NMDG + was the primary external cation.…”

Section: Leftmentioning

confidence: 99%

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Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

Heymann

Dai

et al. 2013

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

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P2X receptor channels open in response to the binding of extracellular ATP, a property that is essential for purinergic sensory signaling. Apo and ATP-bound X-ray structures of the detergentsolubilized zebrafish P2X4 receptor provide a blueprint for receptor mechanisms but unexpectedly showed large crevices between subunits within the transmembrane (TM) domain of the ATPbound structure. Here we investigate both intersubunit and intrasubunit interactions between TM helices of P2X receptors in membranes using both computational and functional approaches. Our results suggest that intersubunit crevices found in the TM domain of the ATP-bound crystal structure are not present in membraneembedded receptors but substantiate helix interactions within individual subunits and identify a hot spot at the internal end of the pore where both the gating and permeation properties of P2X receptors can be tuned. We propose a model for the structure of the open state that has stabilizing intersubunit interactions and that is compatible with available structural constraints from functional channels in membrane environments.pore-opening mechanism | transmembrane intersubunit crevices P 2X receptor channels are a family of trimeric cation-selective channels that are activated by extracellular ATP (1, 2). These ligand-gated ion channels are expressed in many tissues, including the central and peripheral nervous systems and the immune system, where they play a range of important roles in sensory signaling and inflammation (1, 3).Recent X-ray structures of the zebrafish P2X4 (zfP2X4) receptor in apo and ATP-bound forms (Protein Data Bank ID codes 4DW0 and 4DW1, respectively) have revealed the molecular design of these proteins (2, 4, 5) and have provided valuable information on how ATP binding triggers the opening of the transmembrane (TM) pore ( Fig. 1 A and B). ATP binds to a cleft between subunits within the large extracellular domain, inducing cleft closure and an accompanying lateral flexing of the β-sheet connecting the extracellular domain to the TM domain (5). The apo structure of the zfP2X4 receptor reveals that the TM1 helix is positioned peripheral to the TM2 helix and that the TM2 helix occludes the pore at the central axis within the outer half of the membrane (4, 5). In accord with this structure, accessibility studies show that the TM2 helix lines the aqueous pore and that the residues forming the gate are positioned within the occlusion in the apo structure (6-8). Lateral fenestrations within the extracellular domain provide a path for ions to enter and exit the extracellular vestibule positioned above this TM2 occlusion, and these fenestrations are thought to change conformation in response to ATP binding (9, 10). The ATP-bound structure shows that pore opening involves widening of the extracellular vestibule and an iris-like expansion of the pore (5). Intersubunit interactions within the TM domain in the apo structure are limited to the gate region of TM2 (5), and thus the pore expansion observed in the ATP-bound struc...

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Section: Leftsupporting

confidence: 61%

Section: Leftmentioning

confidence: 99%

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

Heymann

Dai

et al. 2013

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

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“…If true, then the N terminus must be the site responsible for the different Pf % values of the two splice variants. Mutagenesis of the rat P2X7aR N terminus affects the conformational changes that accompany "pore dilation," a process by which a limiting constriction in the pore widens to the extent that large cations like NMDG ϩ show appreciable permeability (65,95,98). We suggest that dilation of the pore results in disruption of the putative Domain 2 Ca 2ϩ selectivity filter.…”

Section: Discussionmentioning

confidence: 90%

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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“…As for pannexin-1, Bhaskaracharya et al reported that neither carbenoxolone (up to 100 µM) nor 10 Panx peptide (100 µM), both of which are a pannexin-1 blocker, had any effects on ATP-induced uptake of ethidium, a pannexin-1-permeable dye, by hP2X7R-expressing HEK293 cells, 48) and Browne et al demonstrated that P2X7R channels allow direct permeation of nanometer-sized dyes including YP and ethidium in rP2X7R-expressing HEK293 cells. 49) Taken together, we consider that inhibitory effects of DMCs on BzATP/ATP-evoked YP uptake by HEK293T/hP2X7R and HEK293T/mP2X7R cells reflect the inhibition of agonist-evoked P2X7R activation, but not pannexin-1, in the cells.…”

Section: Discussionmentioning

confidence: 79%

Species Difference in Sensitivity of Human and Mouse P2X7 Receptors to Inhibitory Effects of Divalent Metal Cations

Fujiwara

Ohbori

Ohishi

et al. 2017

Biological & Pharmaceutical Bulletin

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P2X7 receptor (P2X7R), a purinergic receptor, is involved in pathophysiological events such as inflammation and cell death, and thus is an attractive target for therapeutic approaches. It is reported that divalent metal cations (DMCs) inhibit P2X7R activation and that there are species differences in their inhibitory effects. To extrapolate the findings in experimental animals to humans, these species differences have to be clarified, but species differences in the sensitivity of P2X7R to DMCs between man and mouse have not been demonstrated. Here we performed direct comparison of the inhibitory effects of DMCs on human and mouse P2X7R activation. Cell lines constitutively expressing human and mouse P2X7R were used, and their P2X7R activation was evaluated as means of YO-PRO-1 dye uptake. MgCl 2 , NiCl 2 , ZnCl 2 , CuCl 2 and CaCl 2 dose-dependently decreased agonist-induced YO-PRO-1 uptake via both human and mouse P2X7Rs. Apparent differences in the inhibitory profiles for NiCl 2 and CaCl 2 between them were found, and the IC 50 values of DMCs were in the order of CaCl 2 >MgCl 2 >NiCl 2 ≈ZnCl 2 >CuCl 2 for both human and mouse P2X7Rs. In this study, we demonstrate that human P2X7R exhibits different sensitivity to nickel and calcium compared with the case of the mouse one, while there is no species difference in the sensitivity of their P2X7Rs to magnesium, zinc and copper, suggesting that the effects of magnesium, zinc and copper on P2X7R-associated pathophysiological events in humans might be predicted from those in mice.Key words P2X7 receptor; divalent metal cation; species difference; IC 50 value ATP is an energy currency, and the finding of purinergic receptors (P2Rs) revealed another role of it as a signaling molecule. For instance, ATP acts as a neuro-and glio-transmitter in the neuronal system, and as a regulator of inflammatory response in immune system. 1-3) P2Rs are classified into two groups, ionotropic P2XRs and metabotropic P2YRs.4,5) Of P2XRs, P2X7R has unique characteristics, and its affinity for ATP is extremely low and its prolonged activation leads to formation of channels/pores. 6,7) In neuronal cells, extensive activation of P2X7Rs causes the death of neurons, 8,9) and induces microglial activation 10) and astrocytic engulfing activity. 11,12) In immune cells, ATP-induced P2X7R activation triggers Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation, followed by generation/ release of inflammatory cytokines.13-15) Kurashima et al. demonstrated that activation of P2X7R expressed by resident mast cells in the colon induces initiation of inflammation through the release of chemokines and cytokines, and exacerbation of inflammation through the recruitment of neutrophils, and robust release of cytokines in inflammatory bowel disease model mice and Crohn's disease patients.16) These findings strongly indicate that P2X7Rs are an attractive target for the development of therapeutic approaches for various diseases such as neurodegenerative disorders, inflammatory dise...

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P2X7 Receptor Channels Allow Direct Permeation of Nanometer-Sized Dyes

Cited by 131 publications

References 56 publications

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

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

Species Difference in Sensitivity of Human and Mouse P2X7 Receptors to Inhibitory Effects of Divalent Metal Cations

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