title: Minimal structural rearrangement of the cytoplasmic pore during activation of the 5-HT3A receptor.
abstract: Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT(3A)) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd2+) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2'C and, to a lesser extent, G-2'C, showed high affinity inhibition with Cd2+ when applied extracellularly in the open state. Cd2+ inhibition in S2'C was attenuated if applied in the presence of an open-channel inhibitor and showed voltage-dependent recovery, indicating a direct effect of Cd2+ in the pore. When applied intracellularly, Cd2+ appeared to bind S2'C receptors in the closed state. The ability of cysteine side chains at the 2' and -2' positions to coordinate Cd2+ in both the native open and closed states of the channel suggests that the cytoplasmic selectivity filter of 5-HT(3A) receptors maintains a narrow pore during channel gating.
authors.affiliation: Peptide Biology Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Sandip
authors.initials: S
authors.lastname: Panicker
authors.forename: Hans
authors.initials: H
authors.lastname: Cruz
authors.forename: Christine
authors.initials: C
authors.lastname: Arrabit
authors.forename: Ka Fai
authors.initials: KF
authors.lastname: Suen
authors.forename: Paul A
authors.initials: PA
authors.lastname: Slesinger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000737:chemistry; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D006801:Humans; D007477:Ions; D008024:Ligands; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000378:metabolism; D058831:Serotonin 5-HT3 Receptor Antagonists; D013997:Time Factors; D014162:Transfection; D014981:Xenopus
chemical_list: D018076:DNA, Complementary; D007477:Ions; D008024:Ligands; D058831:Serotonin 5-HT3 Receptor Antagonists; D002104:Cadmium; D012701:Serotonin; D003545:Cysteine