Identification of a pre-active conformation of a pentameric channel receptor.

Anaïs Menny,Philipp AM Schmidpeter,Pierre-Jean Corringer,Crina M Nimigean,Delphine Joseph,Zaineb Fourati,Stuart J Edelstein,Solène N Lefebvre,Marc Delarue,Emmanuelle Drège

doi:10.7554/elife.23955

Abstract

Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical signaling through global allosteric transitions. Despite the existence of several high-resolution structures of pLGICs, their dynamical properties remain elusive. Using the proton-gated channel GLIC, we engineered multiple fluorescent reporters, each incorporating a bimane and a tryptophan/tyrosine, whose close distance causes fluorescence quenching. We show that proton application causes a global compaction of the extracellular subunit interface, coupled to an outward motion of the M2-M3 loop near the channel gate. These movements are highly similar in lipid vesicles and detergent micelles. These reorganizations are essentially completed within 2 ms and occur without channel opening at low proton concentration, indicating that they report a pre-active intermediate state in the transition pathway toward activation. This provides a template to investigate the gating of eukaryotic neurotransmitter receptors, for which intermediate states also participate in activation.

Highlights

Pentameric ligand-gated ion channels mediate fast chemical signaling between cells
Following the medium resolution structure of the Torpedo marmorata nAChR by electron microscopy in lipids (Miyazawa et al, 2003), the first X-ray structures of full-length Pentameric ligand-gated ion channels (pLGICs) were obtained on the bacterial homologs Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC) (Hilf and Dutzler, 2008) and Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) (Bocquet et al, 2009; Hilf and Dutzler, 2009; Sauguet et al, 2014), followed by structures of eukaryotic members: the GluClaR (Hibbs and Gouaux, 2011; Althoff et al, 2014), the b3GABAAR (Miller et al, 2014), the 5-HT3R (Hassaine et al, 2014), the a1GlyR (Du et al, 2015) by electron microscopy, the a3GlyR
We focused our analysis on regions known to contribute critically to allosteric transitions of pLGICs and that are solvent-accessible, starting from the most membrane-distal region of the extracellular domain (ECD)

Summary

Introduction

Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical signaling between cells. Prominent members of the family include nicotinic acetylcholine (nAChRs), serotonin-type-3 (5-HT3Rs), glycine (GlyRs) and g-aminobutyric acid-A (GABAARs) receptors that are widely expressed in the nervous system Their mutation can cause congenital myasthenia, epilepsy, hyperekplexia and possibly autistic and schizophrenic syndromes (Steinlein, 2012). Following the medium resolution structure of the Torpedo marmorata nAChR by electron microscopy in lipids (Miyazawa et al, 2003), the first X-ray structures of full-length pLGICs were obtained on the bacterial homologs Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC) (Hilf and Dutzler, 2008) and Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) (Bocquet et al, 2009; Hilf and Dutzler, 2009; Sauguet et al, 2014), followed by structures of eukaryotic members: the GluClaR (Hibbs and Gouaux, 2011; Althoff et al, 2014), the b3GABAAR (Miller et al, 2014), the 5-HT3R (Hassaine et al, 2014), the a1GlyR (Du et al, 2015) by electron microscopy, the a3GlyR

Methods

Results

Conclusion