Biology subjects

Corringer, P.-J.

Publications and source records attributed to Corringer, P.-J..

11 recordsLinked to original sources

bioRxiv2026

An optonanobody for reversible photoactivation of recombinant and native α7 nicotinic

Photopharmacology which enables the precise optical control of endogenous receptor activity, represents a powerful approach in neuroscience. However, photochromic diffusible ligands often exhibit limited subtype specificity, while alternative strategies for controlling specific receptor subtypes require genetic modification. Here, to achieve high subtype selectivity without the need of receptor engineering, we introduce a genetically independent strategy for optical control of endogenous receptors based on highly selective and chemically-defined photoswitchable nanobodies. By covalently coupling a light-sensitive azobenzene agonist to a high-affinity nanobody targeting 7 nicotinic acetylcholine receptor (nAChR), we engineered MalAzoCh-C4, an optonanobody that confers reversible, light-dependent activation of native 7 receptors. In Xenopus oocytes, MalAzoCh-C4 enables wavelength-controlled modulation of recombinant 7 receptors, with enhanced activity in trans configuration. In acute hippocampal slices, application of MalAzoCh-C4 produces robust photocontrol of endogenous 7 nAChRs in interneurons, sufficient to modulate action potential firing. This strategy combines nanobody specificity with the temporal resolution of photopharmacology, establishing optonanobodies as a platform for control of native neuronal receptors.

neuroscience↗

bioRxiv2024

Biochemical, biophysical, and structural investigations of two mutants (C154Y and R312H) of the human Kir2.1 channel involved in the Andersen-Tawil syndrome.

Inwardly rectifying potassium (Kir) channels play a pivotal role in physiology by establishing, maintaining, and regulating the resting membrane potential of the cells, particularly contributing to the cellular repolarization of many excitable cells. Dysfunction in Kir2.1 channels is implicated in several chronic and debilitating human diseases for which there are currently no effective treatments. Specifically, Kir2.1-R312H and Kir2.1-C154Y mutations are associated with Andersen-Tawil syndrome (ATS) in humans. We have investigated the impact of these two mutants in the trafficking of the channel to the cell membrane and function in Xenopus laevis oocytes. Despite both mutations being successfully trafficked to the cell membrane and capable of binding PIP2 (phosphatidylinositol-4,5- bisphosphate), the main modulator for channel activity, they resulted in defective channels that do not display K+ current, albeit through different molecular mechanisms. Co-expression studies showed that R312H and C154Y are expressed and associated with the WT subunits. While WT subunits could rescue R312H dysfunction, the presence of a unique C154Y subunit disrupts the function of the entire complex, which is a typical feature of mutations with a dominant-negative effect. Molecular dynamics simulations showed that Kir2.1-C154Y mutation induces a loss in the structural plasticity of the selectivity filter, impairing the K+ flow. In addition, the cryo-EM structure of the Kir2.1-R312H mutant has been reconstructed. This study identified the molecular mechanisms by which two ATS-causing mutations impact Kir2.1 channel function and provide valuable insights that can guide potential strategies for the development of future therapeutic interventions for ATS.

biophysics↗

bioRxiv2023

The interpeduncular nucleus blunts the rewarding effect of nicotine

Nicotine, by stimulating ventral tegmental area (VTA) dopaminergic neurons, has a rewarding effect that drives tobacco consumption. In turn, the interpeduncular nucleus (IPN) is thought to become activated at high nicotine doses to restrict drug intake. However, the dynamics of the IPN response to nicotine and its impact on the rewarding effect of the drug remain unknown. To address this issue, we have developed a genetically-modified mouse model, in which a "suicide" antagonist of nicotinic acetylcholine receptors (nAChRs) selectively attaches to a designer {beta}4 nAChR subunit. By locally infusing this antagonist in the IPN, we achieved pharmacologically-specific and sustained antagonism of nAChRs containing the {beta}4 subunit. By combining this chemogenetic method with in vivo electrophysiology, we show that even at low doses, nicotine activates and inhibits two different populations of IPN neurons, and that {beta}4-containing nAChRs are only involved in the activation response. Furthermore, blocking the response to nicotine selectively in the IPN increased both the sensitivity of the VTA to the drug and its rewarding effect in a conditioned place preference paradigm. These findings indicate that the IPN is engaged across a large range of nicotine doses and acts as a regulatory brake on the nicotine reward circuit.

neuroscience↗

bioRxiv2023

Mapping the molecular motions of 5-HT3 serotonin-gated channel by Voltage-Clamp Fluorometry

The serotonin-gated ion channel (5-HT3R) mediates excitatory neuronal communication in the gut and the brain. It is the target for setrons, a class of competitive antagonists widely used as antiemetics, and is involved in several neurological diseases. Cryo-electron microscopy of the 5-HT3R in complex with serotonin or setrons revealed that the protein has access to a wide conformational landscape. However, assigning known high-resolution structures to actual states contributing to the physiological response remains a challenge. In the present study, we used voltage-clamp fluorometry (VCF) to measure simultaneously, for 5-HT3R expressed at a cell membrane, conformational changes by fluorescence and channel opening by electrophysiology. Four positions identified by mutational screening report motions around and outside the serotonin-binding site through incorporation of cysteine-tethered rhodamine dyes with or without a nearby quenching tryptophan. VCF recordings show that the 5-HT3R has access to four families of conformations endowed with distinct fluorescence signatures: "resting-like" without ligand, "inhibited-like" with setrons, "pre-active-like" with partial agonists and "active-like" (open channel) with partial and strong agonists. Data are remarkably consistent with cryo-EM structures, the fluorescence partners matching respectively Apo, setron-bound, 5-HT bound-closed and 5-HT-bound-open conformations. Data show that strong agonists promote a concerted motion of all fluorescently labelled sensors during activation, while partial agonists, especially when loss-of-function mutations are engineered, stabilize both active and pre-active conformations. In conclusion, VCF, though the monitoring of electrophysiologically silent conformational changes, illuminates allosteric mechanisms contributing to signal transduction and their differential regulation by important classes of physiological and clinical effectors. Significance StatementHigh-resolution structures of serotonin-gated receptors (5-HT3AR) have evidenced a wide range of conformations that are challenging to annotate to physiologically relevant states. Voltage-clamp fluorometry allows to investigate the activation of 5-HT3AR by simultaneously following molecular motions and electrophysiological states at the plasma membrane. Here, we developed four fluorescent sensors reporting conformational changes at the serotonin binding site and at the extracellular domain and transmembrane domain interface. Investigation of a series of agonists, partial agonists and antagonists show that strong agonists promote a concerted motion of the whole protein during activation, while antagonists and partial agonists stabilize distinct closed-channel conformations. Data offer insights into allosteric mechanisms, unravelling the conformational dynamics of the receptors and helping to annotate high-resolution static structures.

molecular biology↗

bioRxiv2023

Short-chain mono-carboxylates as negative allosteric modulators of GLIC, and impact of a pre-β5 strand (Loop {Omega}) double mutation on crotonate, not butyrate effect.

The bacterial model GLIC remains one of the best known among pentameric ligand-gated ion channels (pLGICs), regarding their structure. GLIC is activated at low extracellular pH, but no agonist compound is known. Van Renterghem et al. (2023) showed that short-chain di-carboxylates potentiate GLIC activity, with strict dependence on two carboxylate binding pockets, previously characterized by crystallography (Sauguet et al., 2013, Fourati et al., 2015, 2020). An "in series" model was proposed, with compound binding at the inter-subunit pocket [homologous to the pLGICs orthotopic neutotransmitter binding site], and with involvement of the intra-subunit (or vestibular) pocket in coupling binding to gating. Here we characterize saturated, mono-carboxylates as negative modulators of GLIC, as previously shown for crotonate (Alqazzaz et al., 2016). Butyrate and crotonate have indistinguishable properties regarding negative modulation of WT GLIC. However, a double mutation in the pre-{beta}5 strand (Loop {Omega}) converts crotonate, as well as caffeate, but not butyrate, into positive modulators. We perform a mutational analysis of residue dependency in the two pockets, with the pre-{beta}5 strand either intact or mutated. We propose that positive modulation requires stringent conditions, with integrity of both pockets, whereas negative modulation is less labile. The vestibular pocket may be involved as an accessory binding site leading to negative, but not positive modulation. We propose that the pre-{beta}5 strand is involved in ligand-elicited modulation of GLIC gating, not in pHo-controlled gating. Possible involvement in Eukaryote pLGICs of regions corresponding to the vestibular pocket and the pre-{beta}5 strand/Loop {Omega} is discussed. Key points summaryO_LIUsing the bacterial proton-activated receptor-channel GLIC, we identify a locus in the pre-{beta}5 strand (Loop {Omega}) whose mutation inverses the effect of the mono-carboxylate crotonate from negative to positive modulation of the allosteric transitions, suggesting an involvement of the pre-{beta}5 strand in coupling the extracellular orthotopic receptor to pore gating in this pentameric ligand-gated ion channel. C_LIO_LIAs an extension to the previously proposed "in series" mechanism, we suggest that a orthotopic/orthosteric site - vestibular site - Loop {Omega} - {beta}5-{beta}6 "sandwich" - Pro-Loop/Cys-Loop series may be an essential component of orthotopic/orthosteric compound-elicited gating control in this pentameric ligand-gated ion channel, on top of which compounds targeting the vestibular site may provide modulation. C_LI

neuroscience↗

bioRxiv2023

Short-chain di-carboxylates as positive allosteric modulators of the pH-dependent pentameric ligand-gated ion channel GLIC: requirement of an intact vestibular pocket

GLIC is a prokaryotic orthologue of brain pentameric neurotransmitter receptors. Using whole-cell patch-clamp electrophysiology in a host cell line, we show that short-chain di-carboxylate compounds are positive modulators of pHo 5-evoked GLIC activity, with a rank order of action fumarate > succinate > malonate > glutarate. Potentiation by fumarate depends on intracellular pH, mainly as a result of a strong decrease of the pHo 5-evoked current when intracellular pH decreases. The modulating effect of fumarate also depends on extracellular pH, as fumarate is a weak inhibitor at pHo 6 and shows no agonist action at neutral pHo. A mutational analysis of residue-dependency for succinate and fumarate effects, based on two carboxylate-binding pockets previously identified by crystallography (Fourati et al. 2020), shows that positive modulation involves both the inter-subunit pocket, homologous to the neurotransmitter-binding orthotopic site, and the intra-subunit (also called vestibular) pocket. An almost similar pattern of mutational impact is observed for the effect of caffeate, a known negative modulator. We propose, for both di-carboxylate compounds and caffeate, a model where the inter-subunit pocket is the actual binding site, and the region corresponding to the vestibular pocket is required either for inter-subunit binding itself, or for binding-to-gating coupling during the allosteric transitions involved in pore gating modulation. Key points summaryO_LIUsing a bacterial orthologue of brain pentameric neurotransmitter receptors, we show that the orthotopic/orthosteric agonist site and the adjacent vestibular region are functionally inter-dependent in mediating compound-elicited modulation. We propose that the two sites in the extracellular domain are involved "in series", a mechanism which may have relevance to Eukaryote receptors. C_LIO_LIWe show that short-chain di-carboxylate compounds are positive modulators of GLIC. The most potent compound identified is fumarate, known to occupy the orthotopic/orthosteric site in previously published crystal structures. C_LIO_LIWe show that intracellular pH modulates GLIC allosteric transitions, as previously known for extracellular pH. C_LIO_LIWe report a caesium to sodium permeability ratio (PCs/PNa) of 0.54 for GLIC ion pore. C_LI

neuroscience↗

bioRxiv2023

Generation of nanobodies acting as silent and positive allosteric modulators of the α7 nicotinic acetylcholine receptor

The 7 nicotinic acetylcholine receptor (nAChR), a potential drug target for treating cognitive disorders, mediates communication between neuronal and non-neuronal cells. Although many competitive antagonists, agonists, and partial-agonists have been found and synthesized, they have not led to effective therapeutic treatments. In this context, small molecules acting as positive allosteric modulators binding outside the orthosteric, acetylcholine, site have attracted considerable interest. Two single-domain antibody fragments, C4 and E3, against the extracellular domain of the human 7-nAChR were generated through alpaca immunization with cells expressing a human 7-nAChR/mouse 5-HT3A chimera, and are herein described. They bind to the 7-nAChR but not to the other major nAChR subtypes, 4{beta}2 and 3{beta}4. E3 acts as a slowly associating positive allosteric modulator, strongly potentiating the acetylcholine-elicited currents, while not precluding the desensitization of the receptor. An E3-E3 bivalent construct shows similar potentiating properties but displays very slow dissociation kinetics conferring quasi-irreversible properties. Whereas, C4 does not alter the receptor function, but fully inhibits the E3-evoked potentiation, showing it is a silent allosteric modulator competing with E3 binding. Both nanobodies do not compete with -bungarotoxin, localizing at an allosteric extracellular binding site away from the orthosteric site. The functional differences of each nanobody, as well as the alteration of functional properties through nanobody modifications indicate the importance of this extracellular site. The nanobodies will be useful for pharmacological and structural investigations; moreover, they, along with the extracellular site, have a direct potential for clinical applications.

neuroscience↗

bioRxiv2023

An original potentiating mechanism revealed by the cryoEM structures of the human α7 nicotinic receptor in complex with nanobodies

The human 7 nicotinic receptor is a pentameric channel mediating cellular and neuronal communication. It has attracted considerable interest to design ligands for the treatment of neurological and psychiatric disorders. To develop a novel class of 7 ligands, we recently generated two nanobodies named E3 and C4 acting as positive and silent allosteric modulators respectively. Here, we solved the cryo-EM structures of the nanobody-receptor complexes. E3 and C4 bind to a common epitope involving two subunits at the apex of the receptor. They form by themselves a symmetric pentameric assembly that extends the extracellular domain. Unlike C4, the binding of E3 drives an active or desensitized conformation in the absence of orthosteric agonist, and mutational analysis shows a key contribution of a N-linked sugar moiety in mediating E3 potentiation. The nanobody E3, by remotely controlling the global allosteric conformation of the receptor, implements an original mechanism of regulation which opens new avenues for drug design.

neuroscience↗

bioRxiv2022

Illumination of a progressive allosteric mechanism mediating the glycine receptor activation

Pentameric ligand-gated ion channel mediate signal transduction at chemical synapses by transiting between resting and open states upon neurotransmitter binding. Here, we investigate the gating transition of the glycine receptor fluorescently labeled at the extracellular-transmembrane interface by voltage-clamp fluorimetry (VCF). Fluorescence reports a glycine-elicited conformational transition that precedes pore opening. Low concentrations of glycine, partial agonists or specific mixtures of glycine and strychnine trigger the full fluorescence signal while weakly activating the channel. Molecular dynamic simulations of a partial agonist bound-closed Cryo-EM structure show a highly dynamic personality: a marked structural flexibility at both the extracellular-transmembrane interface and the orthosteric site, generating docking properties that recapitulate VCF data. Data thus illuminate a progressive gating transition towards activation, displaying structural plasticity with novel implication concerning the mechanism of action of allosteric effectors.

biophysics↗

bioRxiv2020

Mutational analysis to explore long-range allosteric coupling and decoupling in a pentameric channel receptor

Pentameric ligand-gated ion channels (pLGICs) mediate chemical signaling through a succession of allosteric transitions that are yet not completely understood. On the prototypic bacterial channel GLIC, we explored the conformational landscape of the protein during pH-gating. To this aim, we introduced a series of allosteric mutations, and characterized the protein conformation over a broad pH range. We combined electrophysiological recordings, fluorescence quenching experiments monitoring key quaternary reorganizations, and simulations by normal mode analysis. Moderate loss-of-function mutations and the allosteric modulator propofol displace allosteric equilibria involved in pre-activation and pore opening processes, highlighting long-range allosteric coupling between distant regions of the protein. In contrast, total loss-of-function mutations stabilize the protein in unique intermediate conformations where motions are decoupled. Altogether, our data show that the protein can access a wide conformational landscape, raising the possibility of multiple conformational pathways during gating.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

The desensitization pathway of GABAA receptors, one subunit at a time

GABAA receptors mediate most inhibitory synaptic transmission in the brain of vertebrates. Following GABA binding and fast activation, these receptors undergo a slower desensitization, whose conformational pathway remains largely elusive. To explore the mechanism of desensitization, we used concatemeric 1{beta}2{gamma}2 GABAA receptors to selectively introduce gain-of-desensitization mutations one subunit at a time. A library of twenty-six mutant combinations was generated and their bi-exponential macroscopic desensitization rates measured. Introducing mutations at the different subunits shows a strongly asymmetric pattern with a key contribution of the {gamma}2 subunit, and combining mutations results in marked synergistic effects indicating a non-concerted mechanism. Kinetic modelling indeed suggests a pathway where subunits move independently, the desensitization of two subunits being required to occlude the pore. Our work thus hints towards a very diverse and labile conformational landscape during desensitization, with potential implications in physiology and pharmacology.

biophysics↗