Biology subjects

Medjebeur, K.

Publications and source records attributed to Medjebeur, K..

4 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↗

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↗

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↗