title: Molecular dissection of Cl--selective Cys-loop receptor points to components that are dispensable or essential for channel activity.
abstract: Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that bind neurotransmitters to open an intrinsic transmembrane ion channel pore. The recent crystal structure of a prokaryotic pLGIC from the cyanobacterium Gloeobacter violaceus (GLIC) revealed that it naturally lacks an N-terminal extracellular α helix and an intracellular domain that are typical of eukaryotic pLGICs. GLIC does not respond to neurotransmitters acting at eukaryotic pLGICs but is activated by protons. To determine whether the structural differences account for functional differences, we used a eukaryotic chimeric acetylcholine-glutamate pLGIC that was modified to carry deletions corresponding to the sequences missing in the prokaryotic homolog GLIC. Deletions made in the N-terminal extracellular α helix did not prevent the expression of receptor subunits and the appearance of receptor assemblies on the cell surface but abolished the capability of the receptor to bind α-bungarotoxin (a competitive antagonist) and to respond to the neurotransmitter. Other truncated chimeric receptors that lacked the intracellular domain did bind ligands; displayed robust acetylcholine-elicited responses; and shared with the full-length chimeric receptor similar anionic selectivity, effective open pore diameter, and unitary conductance. We suggest that the integrity of the N-terminal α helix is crucial for ligand accommodation because it stabilizes the intersubunit interfaces adjacent to the neurotransmitter-binding pocket(s). We also conclude that the intracellular domain of the chimeric acetylcholine-glutamate receptor does not modulate the ion channel conductance and is not involved in positioning of the pore-lining helices in the conformation necessary for coordinating a Cl- ion within the intracellular vestibule of the ion channel pore.
authors: Laboratory of Ion Channels, Bar-Ilan University, Ramat Gan 52900, Israel; Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 52900, Israel.
authors: Dekel D
authors: DD
authors: Bar-Lev
authors: Laboratory of Ion Channels, Bar-Ilan University, Ramat Gan 52900, Israel; Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 52900, Israel.
authors: Nurit
authors: N
authors: Degani-Katzav
authors: Scientific Equipment Unit, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel.
authors: Alexander
authors: A
authors: Perelman
authors: Laboratory of Ion Channels, Bar-Ilan University, Ramat Gan 52900, Israel; Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 52900, Israel. Electronic address: yoav.paas@biu.ac.il.
authors: Yoav
authors: Y
authors: Paas
mesh_terms: D000109:Acetylcholine / Q000737:chemistry; D000595:Amino Acid Sequence; D002038:Bungarotoxins / Q000737:chemistry; D002713:Chlorine / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D004594:Electrophysiology; D006801:Humans; D007477:Ions / Q000737:chemistry; D008024:Ligands; D018613:Microscopy, Confocal / Q000379:methods; D008969:Molecular Sequence Data; D018377:Neurotransmitter Agents / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011522:Protons; D017470:Receptors, Glutamate / Q000737:chemistry; D011978:Receptors, Nicotinic / Q000737:chemistry
chemical_list: D002038:Bungarotoxins; D007477:Ions; D008024:Ligands; D018377:Neurotransmitter Agents; D011522:Protons; D017470:Receptors, Glutamate; D011978:Receptors, Nicotinic; D002713:Chlorine; D003545:Cysteine; D000109:Acetylcholine