Modulation of the Erwinia ligand-gated ion channel (ELIC) and the 5-HT3 receptor via a common vestibule site

Abstract

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site.

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

“…The first, an exterior site just below the orthosteric pocket, structurally resolved on GABA A [28, 29] and a nAChR [30] and the second, an exterior binding site directly overlapping and slightly above that of α-Btx, found with the 5-HT 3 receptor [20]. A third site on the complementary subunit to the right of and slightly above the orthosteric pocket, visualized on ELIC [22] and GABA A [31] respectively, is likely occluded in the α7-nAChR due to a unique glycosylation site. This site in the prokaryotic ELIC was found to have PAM effects.…”

“…In addition, nanobodies have a number of advantages over small molecules, notably: they usually have a high affinity, typically in the nanomolar range, as well as a high specificity conferred by the large surface of the nanobody-antigen interaction. As an example: within the pentameric ligand-gated ion channel family, nanobodies acting as PAMs and NAMs were reported on the serotonin type 3 (5-HT 3 ) receptor [20], the GABA A receptor [21], and the bacterial ELIC [22]. The generation and functional characterization of two nanobodies specifically targeting the α7-nAChR is presented herein.…”

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

“…In the pLGIC family, nanobodies were previously generated and used mainly to assist structural determination. Most bind at the ortho- or pseudo-ortho-steric site in the 5HT 3 R 20 , GABA A R 21,22 and prokaryotic homolog ELIC 23 , except for a single NAM nanobody binding in the vestibule of ELIC 23 . It is noteworthy that E3 and C4 were generated through immunization of alpacas with cells expressing the fully glycosylated α7-nAChR ECD, while previous nanobodies involved immunization with purified proteins partly depleted from N-linked glycosylation by enzymatic cleavage (5HT 3 R) or production in GntI-cells (GABA A R).…”

Lateral fenestrations in the extracellular domain of the glycine receptor contribute to the main chloride permeation pathway

“…Second, amino acid substitutions at the position corresponding to K104 in GlyR-1 with residues that annihilate (23) or revert the charge (21,22) strongly affect the unitary conductance in both anionic and cationic pLGICs [i.e., -aminobutyric acid type A receptor (GABA A R) K105, 5-HT 3 R D132, and muscle nAChR D97] without altering the charge selectivity (39). Third, lateral fenestrations have been already detected in GABA A R (40,41), 5HT 3 R (42), STeLIC (endosymbiont of Tevnia jerichonana pentameric ligand gated ion channel) (43), and ELIC (Erwinia chrysanthemi pentameric ligand gated ion channel) (44), and these observations are now extended to a larger set of pLGICs (table S8). Fourth, the existence of lateral fenestrations for ion conductance appears to overcome the problem of glycans in recent cryo-EM structures of GlyR 2 (45) and GABAR 112 (46) or 132 (47), where sugar moieties located at the apex of the receptor clearly hinder and possibly occlude the apical pathway.…”