Biology subjects

Nemecz, A.

Publications and source records attributed to Nemecz, A..

3 recordsLinked to original sources

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↗