Site-Directed Spin Labeling Reveals Pentameric Ligand-Gated Ion Channel Gating Motions

Cosma D Dellisanti,James M Raspanti,Valerie A Grant,Susan M Hanson,Borna Ghosh,Kenneth A Satyshur,Candice S Klug,Cynthia Czajkowski

doi:10.1016/j.bpj.2011.11.2284

Cosma D Dellisanti, James M Raspanti + Show 6 more

Open Access

https://doi.org/10.1016/j.bpj.2011.11.2284

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Abstract

Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, the flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, 9) to the transmembrane channel domain (M2-M3 loop) are essential for coupling ligand binding to channel gating. Comparing the recent crystal structures of two bacterial pLGIC homologues, ELIC and GLIC, suggests channel opening is associated with rearrangements in these loops but the specifics of these motions remain unknown. Here, using site-directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy, we measured proton-induced movements of loop 2, loop 9, and M2-M3 loop in functional GLIC channels reconstituted into liposomes. The loops undergo significant proton-dependent motion, with loop 2 and loop 9 moving inward toward the channel lumen, and the M2-M3 loop moving at least 5 angstrom outward away from the channel lumen. The movements are consistent with the gating motions predicted by comparing the ELIC and GLIC structures, and suggest the ELIC structure represents the resting state of pLGICs.

Highlights

The movements are consistent with the gating motions predicted by comparing the ELIC and GLIC structures, and suggest the ELIC structure represents the resting state of Pentameric ligand-gated ion channels (pLGICs)
To determine how many of the five agonist binding sites are required to be occupied by the agonist for maximal open channel stability we applied an electrical fingerprinting strategy in the homomeric receptor model, a7-5HT3A
We find that receptors can be activated by occupancy of only one agonist binding site but open channel lifetime is brief, and occupancy of three non-consecutive sites is required for maximal open channel lifetime

Summary

Introduction

Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. The movements are consistent with the gating motions predicted by comparing the ELIC and GLIC structures, and suggest the ELIC structure represents the resting state of pLGICs. 2123-MiniSymp The Ring of Glutamates in the Charge-Selectivity Filter Region of the Nicotinic Receptor Forms a System of Unanticipated Complexity Gisela D. 2124-MiniSymp Contribution of Agonist Binding Sites and Coupling Regions to Activation and Desensitization of Homomeric Cys-Loop Receptors Cecilia B.

Results

Conclusion