Short-chain mono-carboxylates as negative modulators of allosteric transitions in Gloeobacter violaceus ligand-gated ion channel, and impact of a pre-ß5 strand (Loop Ω) double mutation on crotonate, not butyrate effect.

Using the bacterial proton-activated pentameric receptor-channel Gloeobacter violaceus ligand-gated ion channel (GLIC): (1) We characterize saturated, mono-carboxylates as negative modulators of GLIC (as previously shown for crotonate; Alqazzaz et al., Biochemistry, 2016, 55, 5947). Butyrate and crotonate have indistinguishable properties regarding negative modulation of wt GLIC. (2) We identify a locus in the pre-ß5 strand (Loop Ω) 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-ß5 strand in coupling the extracellular orthotopic receptor to pore gating. (3) As an extension to the previously proposed "in series" mechanism, we suggest that a orthotopic/orthosteric site-vestibular site-Loop Ω-ß5-ß6 "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.

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

The α7 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 α7-nAChR were generated through alpaca immunization with cells expressing a human α7-nAChR/mouse 5-HT3A chimera, and are herein described. They bind to the α7-nAChR but not to the other major nAChR subtypes, α4ß2 and α3ß4. 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 α-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.

Fumarate as positive modulator of allosteric transitions in the pentameric ligand-gated ion channel GLIC: requirement of an intact vestibular pocket.

Gloeobacter violaceus ligand-gated ion channel (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 dicarboxylate 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 dicarboxylate 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: Using a bacterial orthologue of brain pentameric neurotransmitter receptors, we show that the orthotopic/orthosteric agonist site and the adjacent vestibular region are functionally interdependent 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 for eukaryote receptors. We show that short-chain dicarboxylate compounds are positive modulators of the Gloeobacter violaceus ligand-gated ion channel (GLIC). The most potent compound identified is fumarate, known to occupy the orthotopic/orthosteric site in previously published crystal structures. We show that intracellular pH modulates GLIC allosteric transitions, as previously known for extracellular pH. We report a caesium to sodium permeability ratio (PCs /PNa ) of 0.54 for GLIC ion pore.

Crystal structures of a pentameric ion channel gated by alkaline pH show a widely open pore and identify a cavity for modulation.

Pentameric ligand-gated ion channels (pLGICs) constitute a widespread class of ion channels, present in archaea, bacteria, and eukaryotes. Upon binding of their agonists in the extracellular domain, the transmembrane pore opens, allowing ions to go through, via a gating mechanism that can be modulated by a number of drugs. Even though high-resolution structural information on pLGICs has increased in a spectacular way in recent years, both in bacterial and in eukaryotic systems, the structure of the open channel conformation of some intensively studied receptors whose structures are known in a nonactive (closed) form, such as Erwinia chrysanthemi pLGIC (ELIC), is still lacking. Here we describe a gammaproteobacterial pLGIC from an endo-symbiont of Tevnia jerichonana (sTeLIC), whose sequence is closely related to the pLGIC from ELIC with 28% identity. We provide an X-ray crystallographic structure at 2.3 Å in an active conformation, where the pore is found to be more open than any current conformation found for pLGICs. In addition, two charged restriction rings are present in the vestibule. Functional characterization shows sTeLIC to be a cationic channel activated at alkaline pH. It is inhibited by divalent cations, but not by quaternary ammonium ions, such as tetramethylammonium. Additionally, we found that sTeLIC is allosterically potentiated by aromatic amino acids Phe and Trp, as well as their derivatives, such as 4-bromo-cinnamate, whose cocrystal structure reveals a vestibular binding site equivalent to, but more deeply buried than, the one already described for benzodiazepines in ELIC.

Full mutational mapping of titratable residues helps to identify proton-sensors involved in the control of channel gating in the Gloeobacter violaceus pentameric ligand-gated ion channel.

The Gloeobacter violaceus ligand-gated ion channel (GLIC) has been extensively studied by X-ray crystallography and other biophysical techniques. This provided key insights into the general gating mechanism of pentameric ligand-gated ion channel (pLGIC) signal transduction. However, the GLIC is activated by lowering the pH and the location of its putative proton activation site(s) still remain(s) unknown. To this end, every Asp, Glu, and His residue was mutated individually or in combination and investigated by electrophysiology. In addition to the mutational analysis, key mutations were structurally resolved to address whether particular residues contribute to proton sensing, or alternatively to GLIC-gating, independently of the side chain protonation. The data show that multiple residues located below the orthosteric site, notably E26, D32, E35, and D122 in the lower part of the extracellular domain (ECD), along with E222, H235, E243, and H277 in the transmembrane domain (TMD), alter GLIC activation. D122 and H235 were found to also alter GLIC expression. E35 is identified as a key proton-sensing residue, whereby neutralization of its side chain carboxylate stabilizes the active state. Thus, proton activation occurs allosterically to the orthosteric site, at the level of multiple loci with a key contribution of the coupling interface between the ECD and TMD.

Emerging Molecular Mechanisms of Signal Transduction in Pentameric Ligand-Gated Ion Channels.

Nicotinic acetylcholine, serotonin type 3, γ-amminobutyric acid type A, and glycine receptors are major players of human neuronal communication. They belong to the family of pentameric ligand-gated ion channels, sharing a highly conserved modular 3D structure. Recently, high-resolution structures of both open- and closed-pore conformations have been solved for a bacterial, an invertebrate, and a vertebrate receptor in this family. These data suggest that a common gating mechanism occurs, coupling neurotransmitter binding to pore opening, but they also pinpoint significant differences among subtypes. In this Review, we summarize the structural and functional data in light of these gating models and speculate about their mechanistic consequences on ion permeation, pathological mutations, as well as functional regulation by orthosteric and allosteric effectors.

Crystal structures of a pentameric ligand-gated ion channel provide a mechanism for activation.

Pentameric ligand-gated ion channels mediate fast chemical transmission of nerve signals. The structure of a bacterial proton-gated homolog has been established in its open and locally closed conformations at acidic pH. Here we report its crystal structure at neutral pH, thereby providing the X-ray structures of the two end-points of the gating mechanism in the same pentameric ligand-gated ion channel. The large structural variability in the neutral pH structure observed in the four copies of the pentamer present in the asymmetric unit has been used to analyze the intrinsic fluctuations in this state, which are found to prefigure the transition to the open state. In the extracellular domain (ECD), a marked quaternary change is observed, involving both a twist and a blooming motion, and the pore in the transmembrane domain (TMD) is closed by an upper bend of helix M2 (as in locally closed form) and a kink of helix M1, both helices no longer interacting across adjacent subunits. On the tertiary level, detachment of inner and outer ß sheets in the ECD reshapes two essential cavities at the ECD-ECD and ECD-TMD interfaces. The first one is the ligand-binding cavity; the other is close to a known divalent cation binding site in other pentameric ligand-gated ion channels. In addition, a different crystal form reveals that the locally closed and open conformations coexist as discrete ones at acidic pH. These structural results, together with site-directed mutagenesis, physiological recordings, and coarse-grained modeling, have been integrated to propose a model of the gating transition pathway.

The effect of crebanine on memory and cognition impairment via the alpha-7 nicotinic acetylcholine receptor.

AIMS: The aims of the present study were to investigate the effect of crebanine on memory and cognition impairment in mice and to elucidate the underlying molecular mechanisms. MAIN METHODS: The memory-enhancing effects of crebanine were assessed with a water maze test using scopolamine-induced amnesic mice. The molecular mechanism was explored in silico by docking crebanine against acetylcholine binding proteins (AChBPs) and in vitro with a radioligand competition assay using (±)-[(3)H]-epibatidine. The pharmacological behavior was assessed by observing changes to the functional activity of α7-nAChRs expressed in Xenopus oocytes and by fluorescent assays on recombinant ligand gated ion channel (LGIC) receptors expressed in mammalian cells. KEY FINDINGS: The administration of crebanine significantly improved the cognitive deficits induced by scopolamine, as measured by the water maze test. The docking results demonstrated that crebanine bound to the active binding site of the AChBP template with a good docking energy. Crebanine significantly inhibited the binding of (±)-[(3)H]-epibatidine to AChBPs with K(i) values of 179 nM and 538 nM for Ls and Ac, respectively. Further functional assays performed using two separate protocols indicated that crebanine is an antagonist of the α7-nAChR with an IC(50) of 19.1µM. SIGNIFICANCE: The observed actions of crebanine against amnesia and its effect on α7-nAChRs will be beneficial for target-based drug design; crebanine or its scaffold can be used as the starting point to develop a drug for Alzheimer's disease. The cognition-enhancing effects of crebanine and the underlying mechanism based on α7-nAChRs are consistent with its traditional use. These findings demonstrate the potential utility of crebanine in the development of neurodegenerative therapy.

Synthesis of selective agonists for the α7 nicotinic acetylcholine receptor with in situ click-chemistry on acetylcholine-binding protein templates.

The acetylcholine-binding proteins (AChBPs), which serve as structural surrogates for the extracellular domain of nicotinic acetylcholine receptors (nAChRs), were used as reaction templates for in situ click-chemistry reactions to generate a congeneric series of triazoles from azide and alkyne building blocks. The catalysis of in situ azide-alkyne cycloaddition reactions at a dynamic subunit interface facilitated the synthesis of potentially selective compounds for nAChRs. We investigated compound sets generated in situ with soluble AChBP templates through pharmacological characterization with α7 and α4ß2 nAChRs and 5-hydroxytryptamine type 3A receptors. Analysis of activity differences between the triazole 1,5-syn- and 1,4-anti-isomers showed a preference for the 1,4-anti-triazole regioisomers among nAChRs. To improve nAChR subtype selectivity, the highest-potency building block for α7 nAChRs, i.e., 3α-azido-N-methylammonium tropane, was used for additional in situ reactions with a mutated Aplysia californica AChBP that was made to resemble the ligand-binding domain of the α7 nAChR. Fourteen of 50 possible triazole products were identified, and their corresponding tertiary analogs were synthesized. Pharmacological assays revealed that the mutated binding protein template provided enhanced selectivity of ligands through in situ reactions. Discrete trends in pharmacological profiles were evident, with most compounds emerging as α7 nAChR agonists and α4ß2 nAChR antagonists. Triazoles bearing quaternary tropanes and aromatic groups were most potent for α7 nAChRs. Pharmacological characterization of the in situ reaction products established that click-chemistry synthesis with surrogate receptor templates offered novel extensions of fragment-based drug design that were applicable to multisubunit ion channels.

Generation of candidate ligands for nicotinic acetylcholine receptors via in situ click chemistry with a soluble acetylcholine binding protein template.

Nicotinic acetylcholine receptors (nAChRs), which are responsible for mediating key physiological functions, are ubiquitous in the central and peripheral nervous systems. As members of the Cys loop ligand-gated ion channel family, neuronal nAChRs are pentameric, composed of various permutations of α (α2 to α10) and ß (ß2 to ß4) subunits forming functional heteromeric or homomeric receptors. Diversity in nAChR subunit composition complicates the development of selective ligands for specific subtypes, since the five binding sites reside at the subunit interfaces. The acetylcholine binding protein (AChBP), a soluble extracellular domain homologue secreted by mollusks, serves as a general structural surrogate for the nAChRs. In this work, homomeric AChBPs from Lymnaea and Aplysia snails were used as in situ templates for the generation of novel and potent ligands that selectively bind to these proteins. The cycloaddition reaction between building-block azides and alkynes to form stable 1,2,3-triazoles was used to generate the leads. The extent of triazole formation on the AChBP template correlated with the affinity of the triazole product for the nicotinic ligand binding site. Instead of the in situ protein-templated azide-alkyne cycloaddition reaction occurring at a localized, sequestered enzyme active center as previously shown, we demonstrate that the in situ reaction can take place at the subunit interfaces of an oligomeric protein and can thus be used as a tool for identifying novel candidate nAChR ligands. The crystal structure of one of the in situ-formed triazole-AChBP complexes shows binding poses and molecular determinants of interactions predicted from structures of known agonists and antagonists. Hence, the click chemistry approach with an in situ template of a receptor provides a novel synthetic avenue for generating candidate agonists and antagonists for ligand-gated ion channels.

Creating an α7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: crystallographic and ligand selectivity analyses.

Determining the structure of the ligand-binding domain of the nicotinic acetylcholine receptor (nAChR) has been a long standing goal in the design of selective drugs useful in implicated diseases for this prevalent receptor family. Acetylcholine-binding proteins have proven to be valuable surrogates with structural similarity and sequence identity to the extracellular domain of the nicotinic receptor, yet these soluble proteins have their unique features and do not serve as exact replicates of the nAChRs of interest. Here we systematically modify the sequence of these proteins toward the homomeric human α7 nAChR. These chimeric proteins exhibit a shift in affinities to reflect α7 binding characteristics yet maintain expression levels and stability conducive for crystallization. We also present a pentameric humanoid nAChR extracellular domain with the structural determination of the α7 nAChR glycosylation site.

Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors.

We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing a Ca(2+) permeable LGIC and a genetically encoded Förster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human α7 and human α4ß2 nicotinic acetylcholine receptors, mouse 5-HT(3A) serotonin receptors and a chimera of human α7/mouse 5-HT(3A) receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.