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

Marijke Brams,Kerry L Price,Cedric Govaerts,Radovan Spurny,Genevieve L Evans,Chris Ulens,Sarah Cr Lummis,Jan Steyaert,Kumiko Kambara,Daniel Bertrand,Ryan E Hibbs,Els Pardon,Anant Gharpure

doi:10.7554/elife.51511

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.

Highlights

In 1965, Monod, Wyman and Changeux postulated the model of allosteric modulation in proteins (Monod et al, 1965)
Using the prokaryote ion channel Erwinia ligand-gated ion channel (ELIC) as a model system, we investigated whether nanobodies could be selected with allosteric modulator activity on ligand-gated ion channels
While none of the nanobodies had any functional effect on ELIC when applied alone, we found that co-application with the agonist GABA evoked a response that broadly falls into three categories

Summary

Introduction

In 1965, Monod, Wyman and Changeux postulated the model of allosteric modulation in proteins (Monod et al, 1965). Changeux subsequently devoted much of his scientific career to the study of allosteric proteins, with specific attention paid to the nicotinic acetylcholine receptor (nAChR) This protein is a ligandgated ion channel (LGIC) and in effect has no substrate, but the principle of allosteric modulation is similar in that binding of acetylcholine (ACh) shifts the thermodynamic equilibrium from a closed channel state to an open channel state through binding to a site ~50 Aaway from the channel. Using cysteine-scanning mutagenesis and electrophysiological recordings, we show the vestibule site can be targeted for modulation of the human 5-HT3A receptor as a proof of principle relevant to other eukaryotic receptors

Results and discussion

Conclusion

Materials and methods