title: Modular design of Cys-loop ligand-gated ion channels: functional 5-HT3 and GABA rho1 receptors lacking the large cytoplasmic M3M4 loop.
abstract: Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the alpha-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-HT(3A)) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-HT(3A)-glvM3M4 receptors were comparable to 5-HT(3A) wild type. The channels remained cation-selective but the 5-HT(3A)-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-HT(3) and acetylcholine receptors, significantly attenuated 5-HT-induced currents with wild-type 5-HT(3A) but not 5-HT(3A)-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-rho1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains.
authors.affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA.
authors.forename: Michaela
authors.initials: M
authors.lastname: Jansen
authors.forename: Moez
authors.initials: M
authors.lastname: Bali
authors.forename: Myles H
authors.initials: MH
authors.lastname: Akabas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D000458:Cyanobacteria / Q000235:genetics; D004110:Diltiazem / Q000494:pharmacology; D005260:Female; D058788:GABA-B Receptor Antagonists; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008012:Lidocaine / Q000031:analogs & derivatives / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D017294:Ondansetron / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D010852:Picrotoxin / Q000494:pharmacology; D018080:Receptors, GABA-B / Q000235:genetics / Q000502:physiology*; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000494:pharmacology; D058831:Serotonin 5-HT3 Receptor Antagonists; D012965:Sodium Chloride / Q000494:pharmacology; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D004110:Diltiazem; D058788:GABA-B Receptor Antagonists; D047908:Intracellular Signaling Peptides and Proteins; D008012:Lidocaine; D017294:Ondansetron; D010852:Picrotoxin; D018080:Receptors, GABA-B; D044406:Receptors, Serotonin, 5-HT3; D012701:Serotonin; D058831:Serotonin 5-HT3 Receptor Antagonists; D012965:Sodium Chloride; D005680:gamma-Aminobutyric Acid; C496701:RIC3 protein, human; C006072:QX-222