title: Importance of the C-terminus of the human 5-HT3A receptor subunit.
abstract: Amongst the family members of Cys-loop LGICs, the atypical ability of the 5-HT3A subunit to form functional homomeric receptors allowed a direct investigation of the role of the C-terminus. Deletion of the three C-terminal amino acids (DeltaGln453-DeltaTyr454-DeltaAla455) from the h5-HT3A subunit prevented formation of a specific radioligand binding site as well as expression within the cell membrane. Removal of merely the C-terminal residue (DeltaAla455) reduced specific radioligand binding (to 4+/-1% relative to the wild-type; cells grown at 37 degrees C) and also cell membrane expression; these reductions were less evident when the DeltaAla455 expressing cells were grown at 27 degrees C (specific radioligand binding levels 27+/-5% relative to wild-type also grown at 27 degrees C). Mutation of the h5-HT3A C-terminal amino acid, alanine, for either glycine (Ala455Gly), valine (Ala455Val) or leucine (Ala455Leu) reduced specific radioligand binding levels by 24+/-23%, 32+/-12% and 88+/-1%, respectively; the latter mutant also displaying reduced membrane expression. In contrast, mutation to alanine of the two amino acids preceding the C-terminal alanine (Gln453Ala and Tyr454Ala) had no detrimental effects on specific radioligand binding or cell membrane expression levels. The present study demonstrates an important role for the C-terminus in the formation of the functional h5-HT3A receptor. The partial restoration of 5-HT3 receptor binding and cell membrane expression when cells expressing C-terminal mutant 5-HT3A subunits were grown at a lower temperature (27 degrees C) suggests that the C-terminus stabilises the 5-HT3 receptor allowing subunit folding and subsequent maturation.
authors.affiliation: Cellular and Molecular Neuropharmacology Research Group, School of Experimental and Clinical Medicine, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Amy S
authors.initials: AS
authors.lastname: Butler
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Lindesay
authors.forename: Terri J
authors.initials: TJ
authors.lastname: Dover
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Kennedy
authors.forename: Valerie B
authors.initials: VB
authors.lastname: Patchell
authors.forename: Barry A
authors.initials: BA
authors.lastname: Levine
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Hope
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Barnes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D017829:Granisetron / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D018408:Patch-Clamp Techniques; D011869:Radioligand Assay; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012702:Serotonin Antagonists / Q000494:pharmacology
chemical_list: D017829:Granisetron; D044406:Receptors, Serotonin, 5-HT3; D012702:Serotonin Antagonists