Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel

Habermacher, Chloé; Martz, Adeline; Calimet, Nicolas; Lemoine, Damien; Peverini, Laurie; Specht, Alexandre; Cecchini, Marco; Grütter, Thomas

doi:10.7554/elife.11050

Cited by 31 publications

(45 citation statements)

References 69 publications

(132 reference statements)

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“…To increase seal resistance and patch stability, fluoride ions (F − ) were used as internal counterions to NMDG + cations (Materials and Methods). We transiently transfected human embryonic kidney (HEK-293) cells with plasmids encoding the rat P2X2-3T, which is a cysteine-less mutated receptor that retains wild-type P2X2 functionality (35) but displays increased single-channel conductance (36). In a first series of experiments, we used excised outside-out patches that contained multiple channels and observed robust inward NMDG + currents that developed rapidly following fast perfusion of 3 μM ATP, while holding the voltage to -120 mV (activation time constant τ NMDG+ = 203 ± 47 ms, n = 9 patches; Fig.…”

Section: Resultsmentioning

confidence: 99%

On the permeation of large organic cations through the pore of ATP-gated P2X receptors

Harkat

Peverini

Cerdan

et al. 2017

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

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Pore dilation is thought to be a hallmark of purinergic P2X receptors. The most commonly held view of this unusual process posits that under prolonged ATP exposure the ion pore expands in a striking manner from an initial small-cation conductive state to a dilated state, which allows the passage of larger synthetic cations, such as -methyl-d-glucamine (NMDG). However, this mechanism is controversial, and the identity of the natural large permeating cations remains elusive. Here, we provide evidence that, contrary to the time-dependent pore dilation model, ATP binding opens an NMDG-permeable channel within milliseconds, with a conductance that remains stable over time. We show that the time course of NMDG permeability superimposes that of Na and demonstrate that the molecular motions leading to the permeation of NMDG are very similar to those that drive Na flow. We found, however, that NMDG "percolates" 10 times slower than Na in the open state, likely due to a conformational and orientational selection of permeating molecules. We further uncover that several P2X receptors, including those able to desensitize, are permeable not only to NMDG but also to spermidine, a large natural cation involved in ion channel modulation, revealing a previously unrecognized P2X-mediated signaling. Altogether, our data do not support a time-dependent dilation of the pore on its own but rather reveal that the open pore of P2X receptors is wide enough to allow the permeation of large organic cations, including natural ones. This permeation mechanism has considerable physiological significance.

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Section: Resultsmentioning

confidence: 99%

On the permeation of large organic cations through the pore of ATP-gated P2X receptors

Harkat

Peverini

Cerdan

et al. 2017

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

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“…To date, there are no structures of a P2X receptor in the desensitized state and currently available structures of the zebra fish P2X 4 receptor (zfP2X 4 ) in apo and open state conformations do not visualize cytoplasmic residues 27–29 . There is also concern that the available structure of zfP2X 4 bound to ATP 27 may not represent a physiologic state because the truncated crystallization construct, lacking both terminal domains, might distort pore architecture 12,30–32 . A recent NMR study suggests that TNP-ATP inhibits activation by closing the extracellular fenestrations to ion access, rather than by stabilizing a closed-pore conformation 33 .…”

Section: Introductionmentioning

confidence: 99%

X-ray structures define human P2X3 receptor gating cycle and antagonist action

Mansoor

Lü

Oosterheert

et al. 2016

Nature

224

398

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Summary P2X receptors are trimeric, non-selective cation channels activated by ATP that play important roles in cardiovascular, neuronal and immune systems. Despite their central function in human physiology and as potential targets of therapeutic agents, there are no structures of human P2X receptors. Mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structure of the pore-forming transmembrane domains remain unclear. We report x-ray crystal structures of human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/desensitized and antagonist-bound closed states. The open state structure harbors an intracellular motif we term the “cytoplasmic cap”, that stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. Competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements underpinning P2X receptor gating and provide a foundation for development of new pharmacologic agents.

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“…Recent work has targeted GIRK[29], TRPV1[30,31], TRPA1[32], P2X[33,34] and ASIC[33,35] channels. Of special note, two papers[33,34] on P2X channels employ a variant of the chemical optogenetic concept to create “molecular tweezers”: two subunits of the channel are tethered together with azobenzene via covalent cysteine-maleimide bonds at key positions in the channel, allowing the photoswitching azobenzene to open and close the pore.…”

Section: Other Ion Channels and Promising Targetsmentioning

confidence: 99%