Abstract

Crystal structures of Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated prokaryotic homologue of pentameric ligand-gated ion channel (LGIC) from G. violaceus, have provided high-resolution models of the channel architecture and its role in selective ion conduction and drug binding. However, it is still unclear which functional states of the LGIC gating scheme these crystal structures represent. Much of this uncertainty arises from a lack of thorough understanding of the functional properties of these prokaryotic channels. To elucidate the molecular events that constitute gating, we have carried out an extensive characterization of GLIC function and dynamics in reconstituted proteoliposomes by patch clamp measurements and EPR spectroscopy. We find that GLIC channels show rapid activation upon jumps to acidic pH followed by a time-dependent loss of conductance because of desensitization. GLIC desensitization is strongly coupled to activation and is modulated by voltage, permeant ions, pore-blocking drugs, and membrane cholesterol. Many of these properties are parallel to functions observed in members of eukaryotic LGIC. Conformational changes in loop C, measured by site-directed spin labeling and EPR spectroscopy, reveal immobilization during desensitization analogous to changes in LGIC and acetylcholine binding protein. Together, our studies suggest conservation of mechanistic aspects of desensitization among LGICs of prokaryotic and eukaryotic origin.

Highlights

  • Gloeobacter violaceus homologue (GLIC), a prokaryotic homologue of pentameric ligand-gated ion channel (LGIC), is activated by protons, and crystal structures suggest a putative open conformation

  • We investigate the structural correlates of the underlying conformational transition by site-directed spin labeling and EPR spectroscopy of residues comprising of loop C

  • The native Cys (Cys-26) in the extracellular domain (ECD) of GLIC was labeled with fluoresceinmaleimide and tetramethylrhodamine-maleimide [35,36,37] and reconstituted in E. coli polar lipids, 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine:1-palmitoyl-2-oleoyl-sn-glycero-3phospho-(1Ј-rac-glycerol) (3:1), or asolectin (Fig. 1A)

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Summary

Background

GLIC, a prokaryotic homologue of pentameric ligand-gated ion channel (LGIC), is activated by protons, and crystal structures suggest a putative open conformation. GLIC desensitization is strongly coupled to activation and is modulated by voltage, permeant ions, pore-blocking drugs, and membrane cholesterol Many of these properties are parallel to functions observed in members of eukaryotic LGIC. We investigate the structural correlates of the underlying conformational transition by site-directed spin labeling and EPR spectroscopy of residues comprising of loop C (connecting strands ␤9 and ␤10 in the ECD) These findings provide insights into the functioning of GLIC and thereby aid in pairing up structural data with functional measurements

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