Effects of N-Terminal-Helix and M3-M4-Loop Deletions on Channel Activity in a Chimeric Acetylcholine-Glutamate Cys-Loop Receptor

Abstract

Eukaryotic Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that respond to neurotransmitters such as acetylcholine, serotonin, γ-aminobutyric acid, glycine, glutamate or histamine by opening (and closing) of an intrinsic transmembrane channel. Each subunit has a long extracellular N-terminal segment, four transmembrane helices (M1 to M4) and a long intracellular M3-M4 connecting segment (“M3-M4 loop”). Recently, the X-ray crystal structure of a prokaryotic pLGIC from the cyanobacterium Gloeobacter violaceus (termed “GLIC”) was determined at atomic resolution. This prokaryotic receptor does not respond to any of the aforementioned neurotransmitters, but it is activated by protons. In addition, compared to eukaryotic Cys-loop receptors, GLIC lacks a short N-terminal helical segment in its ligand-binding domain, and has a much shorter M3-M4 loop. To determine whether (or not) these segments are crucial for the function of a eukaryotic acetylcholine-glutamate Cys-loop chimeric receptor (α7-GluClβR), we deleted those segments of the chimera that are missing in GLIC. Ligand-binding assays performed on transfected living cells indicate that chimeras lacking most of the M3-M4 loop can readily bind 3H-α-bungarotoxin (a competitive antagonist) and nicotine (an agonist). These deletion chimeras were visualized on the cell surface by confocal microscopy using rhodaminylated α-bungarotoxin and specific antibodies. In addition, chimeras lacking the M3-M4 loop display ACh-induced currents with unchanged EC50, Hill coefficient and ionic selectivity. In contrast, chimeras lacking the N-terminal helical segment do not bind 3H-α-bungarotoxin. However, these N-terminus-truncated receptors migrate as non-degraded proteins in SDS PAGE and are readily visualized on the surface of transfected cells with specific anti-HA tag antibodies. Electrophysiological experiments are currently performed to determine whether (or not) acetylcholine, nicotine or protons activate the N-terminus truncated chimeras. Supported by the Wolfson Family Foundation and the Israel Science Foundation.