Adeline Martz

no abstract

ATP-gated purinergic P2X7 receptors are crucial ion channels involved in inflammation. They sense abnormal ATP release during stress or injury and are considered promising clinical targets for therapeutic intervention. However, despite their predominant expression in immune cells such as microglia, there is limited information on P2X7 membrane expression and regulation during inflammation at the single-molecule level, necessitating new labeling approaches to visualize P2X7 in native cells. Here, we present X7-uP , an unbiased, affinity-guided P2X7 chemical labeling reagent that selectively biotinylates endogenous P2X7 in BV2 cells, a murine microglia model, allowing subsequent labeling with streptavidin-Alexa 647 tailored for super-resolution imaging. We uncovered a nanoscale microglial P2X7 redistribution mechanism where evenly spaced individual receptors in quiescent cells undergo upregulation and clustering in response to the pro-inflammatory agent lipopolysaccharide and ATP, leading to synergistic interleukin-1β release. Our method thus offers a new approach to revealing endogenous P2X7 expression at the single-molecule level.

PIEZO proteins are unusually large, mechanically-activated trimeric ion channels. The central pore features structural similarities with the pore of other trimeric ion channels, including purinergic P2X receptors, for which optical control of channel gating has been previously achieved with photoswitchable azobenzenes. Extension of these chemical optogenetics methods to mechanically-activated ion channels would provide tools for specific manipulation of pore activity alternative to non-specific mechanical stimulations. Here we report a light-gated mouse PIEZO1 channel, in which an azobenzene-based photoswitch covalently tethered to an engineered cysteine, Y2464C, localized at the extracellular apex of the transmembrane helix 38, rapidly triggers channel gating upon 365-nm-light irradiation. We provide evidence that this light-gated channel recapitulates mechanically-activated PIEZO1 functional properties, and show that light-induced molecular motions are similar to those evoked mechanically. These results push the limits of azobenzene-based methods to unusually large ion channels and provide a simple stimulation means to specifically interrogate PIEZO1 function.

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as “macropore” formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities

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 N -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.

P2X receptors function by opening a transmembrane pore in response to extracellular ATP. Recent crystal structures solved in apo and ATP-bound states revealed molecular motions of the extracellular domain following agonist binding. However, the mechanism of pore opening still remains controversial. Here we use photo-switchable cross-linkers as ‘molecular tweezers’ to monitor a series of inter-residue distances in the transmembrane domain of the P2X2 receptor during activation. These experimentally based structural constraints combined with computational studies provide high-resolution models of the channel in the open and closed states. We show that the extent of the outer pore expansion is significantly reduced compared to the ATP-bound structure. Our data further reveal that the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion. These results provide new insight into the gating mechanism of P2X receptors and establish a versatile strategy applicable to other membrane proteins.

The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus.

no abstract

ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent “tethering” reaction, covalent bonds between a synthesized ATP-derived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATP-binding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.

BACKGROUND AND PURPOSE Flavonoids, important plant pigments, have been shown to allosterically modulate brain GABA A receptors (GABA A Rs). We previously reported that trans ‐6,4′‐dimethoxyretrochalcone (Rc‐OMe), a hydrolytic derivative of the corresponding flavylium salt, displayed nanomolar affinity for the benzodiazepine binding site of GABA A Rs. Here, we evaluate the functional modulations of Rc‐OMe, along with two other synthetic derivatives trans ‐6‐bromo‐4′‐methoxyretrochalcone (Rc‐Br) and 4,3′‐dimethoxychalcone (Ch‐OMe) on GABA A Rs. EXPERIMENTAL APPROACH Whole‐cell patch‐clamp recordings were made to determine the effects of these derivatives on GABA A Rs expressed in HEK‐293 cells and in hippocampal CA1 pyramidal and thalamic neurones from rat brain. KEY RESULTS Rc‐OMe strongly potentiated GABA‐evoked currents at recombinant α 1–4 β 2 γ 2s and α 4 β 3 δ receptors but much less at α 1 β 2 and α 4 β 3 . Rc‐Br and Ch‐OMe potentiated GABA‐evoked currents at α 1 β 2 γ 2s . The potentiation by Rc‐OMe was only reduced at α 1 H101Rβ 2 γ 2s and α 1 β 2 N265Sγ 2s , mutations known to abolish the potentiation by diazepam and loreclezole respectively. The modulation of Rc‐OMe and pentobarbital as well as by Rc‐OMe and the neurosteroid 3α,21‐dihydroxy‐5α‐pregnan‐20‐one was supra‐additive. Rc‐OMe modulation exhibited no apparent voltage‐dependence, but was markedly dependent on GABA concentration. In neurones, Rc‐Br slowed the decay of spontaneous inhibitory postsynaptic currents and both Rc‐OMe and Rc‐Br positively modulated synaptic and extrasynaptic diazepam‐insensitive GABA A Rs. CONCLUSIONS AND IMPLICATIONS The trans ‐retrochalcones are powerful positive allosteric modulators of synaptic and extrasynaptic GABA A Rs. These novel modulators act through an original mode, thus making them putative drug candidates in the treatment of GABA A ‐related disorders in vivo .