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5-HT3 Receptors*

  • Sarah C.R. Lummis
    Correspondence
    Wellcome Trust Senior Research Fellow in Basic Biomedical Science. To whom correspondence should be addressed
    Affiliations
    Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom
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  • Author Footnotes
    * This work was supported by the Wellcome Trust. This is the fifth article in the Thematic Minireview Series on Celebrating the Discovery of the Cysteine Loop Ligand-gated Ion Channel Superfamily.
Open AccessPublished:October 04, 2012DOI:https://doi.org/10.1074/jbc.R112.406496
      5-Hydroxytryptamine type 3 (5-HT3) receptors are cation-selective Cys loop receptors found in both the central and peripheral nervous systems. There are five 5-HT3 receptor subunits (A–E), and all functional receptors require at least one A subunit. Regions from noncontiguous parts of the subunit sequence contribute to the agonist-binding site, and the roles of a range of amino acid residues that form the binding pocket have been identified. Drugs that selectively antagonize 5-HT3 receptors (the “setrons”) are the current gold standard for treatment of chemotherapy-induced and postoperative nausea and vomiting and have potential for the treatment of a range of other conditions.

      Introduction

      5-Hydroxytryptamine (5-HT
      The abbreviations used are: 5-HT
      5-hydroxytryptamine
      nACh
      nicotinic acetylcholine
      PNS
      peripheral nervous system
      pS
      picosiemens
      ECD
      extracellular domain
      TMD
      transmembrane domain
      ICD
      intracellular domain
      AChBP
      ACh-binding protein
      IBS
      irritable bowel syndrome.
      ; serotonin) is one of, if not the most, versatile of all neurohormones or neurotransmitters. Its diverse range of functions are due to a large family of receptors: 5-HT1 to 5-HT7 (
      • Hoyer D.
      • Hannon J.P.
      • Martin G.R.
      Molecular, pharmacological, and functional diversity of 5-HT receptors.
      ). The 5-HT3 receptor is a Cys loop ligand-gated ion channel, most closely related to nicotinic acetylcholine (nACh) receptors, and it is structurally and functionally distinct from the other six classes of 5-HT receptor whose actions are mediated via G-proteins. The 5-HT3 receptor is a cation-selective ion channel capable of mediating fast excitatory neurotransmission in the CNS and peripheral nervous system (PNS) (
      • Sugita S.
      • Shen K.Z.
      • North R.A.
      5-Hydroxytryptamine is a fast excitatory transmitter at 5-HT3 receptors in rat amygdala.
      ). 5-HT3 receptors are located in many brain areas, with the highest levels in the brainstem, especially areas involved in the vomiting reflex such as the area postrema and nucleus tractus solitarius (
      • Miquel M.C.
      • Emerit M.B.
      • Nosjean A.
      • Simon A.
      • Rumajogee P.
      • Brisorgueil M.J.
      • Doucet E.
      • Hamon M.
      • Vergé D.
      Differential subcellular localization of the 5-HT3-As receptor subunit in the rat central nervous system.
      ,
      • Tecott L.H.
      • Maricq A.V.
      • Julius D.
      Nervous system distribution of the serotonin 5-HT3 receptor mRNA.
      ). The receptors are found both pre- and postsynaptically, and activation can modulate the release of a range of neurotransmitters, including dopamine, GABA, substance P, and acetylcholine (
      • Miquel M.C.
      • Emerit M.B.
      • Nosjean A.
      • Simon A.
      • Rumajogee P.
      • Brisorgueil M.J.
      • Doucet E.
      • Hamon M.
      • Vergé D.
      Differential subcellular localization of the 5-HT3-As receptor subunit in the rat central nervous system.
      ,
      • Blandina P.
      • Goldfarb J.
      • Craddock-Royal B.
      • Green J.P.
      Release of endogenous dopamine by stimulation of 5-hydroxytryptamine 3 receptors in rat striatum.
      ,
      • Thompson A.J.
      • Lummis S.C.
      5-HT3 receptors.
      ). 5-HT3 receptors also regulate gut motility, secretion, and peristalsis in the enteric nervous system and are involved in information transfer in the gastrointestinal tract (
      • Galligan J.J.
      Ligand-gated ion channels in the enteric nervous system.
      ).

      Background

      Serotonin was identified in the 1940s as a potent vasoconstrictor present in blood serum (
      • Rapport M.M.
      • Green A.A.
      • Page I.H.
      Purification of the substance which is responsible for the vasoconstrictor activity of serum.
      ). The proposal of a specific receptor for this compound was first raised in the literature in 1953, when Rocha e Silva et al. (
      • Rocha e Silva M.
      • Valle J.R.
      • Picarelli P.
      A pharmacological analysis of the mode of action of serotonin (5-hydroxytryptamine) upon the guinea pig ileum.
      ) noticed that 5-HT had actions on guinea pig ileum and that its effects could be blocked by cocaine at micromolar concentrations. At approximately the same time, Gaddum (
      • Gaddum J.H.
      Tryptamine receptors.
      ) proposed that 5-HT acted on specific receptors, of which there were two types: one in smooth muscle, which was inhibited by LSD (lysergic acid diethylamide), and another in the nervous system, which was not inhibited by LSD. However, the classic “discovery” of the 5-HT3 receptor is usually linked to the work of Gaddum and Picarelli in 1957 (
      • Gaddum J.H.
      • Picarelli Z.P.
      Two kinds of tryptamine receptor.
      ), who defined two classes of serotonin receptors in the ileum: M receptors, which were located primarily in the nervous system and inhibited by morphine, atropine, and cocaine, and D receptors, which were located mostly in muscle and blocked by dibenzyline. When the serotonin receptors were reclassified in 1986 (
      • Bradley P.B.
      • Engel G.
      • Feniuk W.
      • Fozard J.R.
      • Humphrey P.P.
      • Middlemiss D.N.
      • Mylecharane E.J.
      • Richardson B.P.
      • Saxena P.R.
      Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine.
      ,
      • Hoyer D.
      • Clarke D.E.
      • Fozard J.R.
      • Hartig P.R.
      • Martin G.R.
      • Mylecharane E.J.
      • Saxena P.R.
      • Humphrey P.P.
      International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin).
      ), the M receptor became the 5-HT3 receptor, and the D receptor the 5-HT2 receptor. These are just two of seven currently known 5-HT receptor families, most of which have a range of subtypes, in addition to splice variants and post-translationally modified receptors, creating one of the largest families of neurotransmitter receptors (
      • Hoyer D.
      • Clarke D.E.
      • Fozard J.R.
      • Hartig P.R.
      • Martin G.R.
      • Mylecharane E.J.
      • Saxena P.R.
      • Humphrey P.P.
      International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin).
      ).
      Despite being identified in 1957, it was not until the 1980s that the first selective 5-HT3 antagonists were developed, MDL 72222 or bemesetron (
      • Fozard J.R.
      MDL 72222: a potent and highly selective antagonist at neuronal 5-hydroxytryptamine receptors.
      ) and ICS 205-930 or tropisetron (
      • Richardson B.P.
      • Engel G.
      • Donatsch P.
      • Stadler P.A.
      Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs.
      ), and their antiemetic properties appreciated: MDL 72222 was found to be a potent antiemetic in cisplatin-treated ferrets (
      • Miner W.D.
      • Sanger G.J.
      Inhibition of cisplatin-induced vomiting by selective 5-hydroxytryptamine M-receptor antagonism.
      ,
      • Costall B.
      • Domeney A.M.
      • Naylor R.J.
      • Tattersall F.D.
      5-Hydroxytryptamine M-receptor antagonism to prevent cisplatin-induced emesis.
      ). Novel (second generation) antagonists were soon developed, including GR38032F (ondansetron) and BRL 43694 (granisetron). Use of these compounds, in addition to the older, nonselective, but still effective antagonists metoclopramide and cocaine, revealed a widespread distribution of 5-HT3 receptors in the PNS. The presence of 5-HT3-binding sites in the CNS was first established in 1987 using [3H]GR65630 (
      • Kilpatrick G.J.
      • Jones B.J.
      • Tyers M.B.
      Identification and distribution of 5-HT3 receptors in rat brain using radioligand binding.
      ), and single-channel studies published in 1989 provided unequivocal evidence that 5-HT3 receptors were indeed ligand-gated ion channels (
      • Derkach V.
      • Surprenant A.
      • North R.A.
      5-HT3 receptors are membrane ion channels.
      ). In 1991, the first 5-HT3 receptor subunit (5-HT3A) was cloned (
      • Maricq A.V.
      • Peterson A.S.
      • Brake A.J.
      • Myers R.M.
      • Julius D.
      Primary structure and functional expression of the 5-HT3 receptor, a serotonin-gated ion channel.
      ). The homology between this subunit and those from other Cys loop receptors clearly indicated 5-HT3 receptors were members of this family, but the 5-HT3A subunit was a little unusual in that it could readily form functional homomeric receptors. The biophysical properties of the expressed homomeric receptors differed, however, from those observed in some native preparations. For example, when expressed in HEK 293 cells, 5-HT3A receptors had a single-channel conductance of <1 picosiemen (pS), whereas channel activity in rabbit nodose ganglion revealed a single-channel conductance of 19 pS (
      • Gill C.H.
      • Peters J.A.
      • Lambert J.J.
      An electrophysiological investigation of the properties of a murine recombinant 5-HT3 receptor stably expressed in HEK 293 cells.
      ). This discrepancy was not explained until 1999, when a second subunit, the 5-HT3B receptor subunit, was identified (
      • Davies P.A.
      • Pistis M.
      • Hanna M.C.
      • Peters J.A.
      • Lambert J.J.
      • Hales T.G.
      • Kirkness E.F.
      The 5-HT3B subunit is a major determinant of serotonin-receptor function.
      ,
      • Dubin A.E.
      • Huvar R.
      • D'Andrea M.R.
      • Pyati J.
      • Zhu J.Y.
      • Joy K.C.
      • Wilson S.J.
      • Galindo J.E.
      • Glass C.A.
      • Luo L.
      • Jackson M.R.
      • Lovenberg T.W.
      • Erlander M.G.
      The pharmacological and functional characteristics of the serotonin 5-HT3A receptor are specifically modified by a 5-HT3B receptor subunit.
      ). Coexpression of this subunit with the A subunit resulted in properties that more closely represented those found in some native receptors. Since then, three other subunits (C–E) have been identified, considerably expanding the known complexity of the 5-HT3 receptor system (
      • Niesler B.
      • Frank B.
      • Kapeller J.
      • Rappold G.A.
      Cloning, physical mapping, and expression analysis of the human 5-HT3 serotonin receptor-like genes HTR3CHTR3DHTR3E.
      ). Over the last decade, much progress has been made in understanding the structure-function relationships of 5-HT3 receptors and their pathophysiological relevance (see Refs.
      • Thompson A.J.
      • Lummis S.C.
      5-HT3 receptors.
      and
      • Barnes N.M.
      • Hales T.G.
      • Lummis S.C.
      • Peters J.A.
      The 5-HT3 receptor–the relationship between structure and function.
      ,
      • Walstab J.
      • Rappold G.
      • Niesler B.
      5-HT3 receptors: role in disease and target of drugs.
      ,
      • Thompson A.J.
      • Lummis S.C.
      The 5-HT3 receptor as a therapeutic target.
      ,
      • Reeves D.C.
      • Lummis S.C.
      The molecular basis of the structure and function of the 5-HT3 receptor: a model ligand-gated ion channel (review).
      for reviews). There is nevertheless much left to be discovered, in particular in understanding the roles of the C, D, and E subunits, whose expression has only recently been confirmed (
      • Kapeller J.
      • Möller D.
      • Lasitschka F.
      • Autschbach F.
      • Hovius R.
      • Rappold G.
      • Brüss M.
      • Gershon M.D.
      • Niesler B.
      Serotonin receptor diversity in the human colon: expression of serotonin type 3 receptor subunits 5-HT3C, 5-HT3D, and 5-HT3E.
      ); such studies may allow the development of novel agents to treat disorders such as anxiety, schizophrenia, and Alzheimer disease, which were originally postulated to be targets of 5-HT3 receptor-specific compounds.

      Receptor Structure

      The functional 5-HT3 receptor, like other Cys loop receptors, is a pentameric assembly of five identical or non-identical subunits that surround, in a pseudo-symmetric manner, a water-filled ion channel (
      • Boess F.G.
      • Lummis S.C.
      • Martin I.L.
      Molecular properties of 5-hydroxytryptamine 3 receptor-type binding sites purified from NG108-15 cells.
      ,
      • Green T.
      • Stauffer K.A.
      • Lummis S.C.
      Expression of recombinant homo-oligomeric 5-hydroxytryptamine 3 receptors provides new insights into their maturation and structure.
      ). Each subunit has a large extracellular domain (ECD) that forms the ligand-binding site, a transmembrane domain (TMD) consisting of four membrane-spanning α-helices (M1–M4) that enable ions to cross the membrane, and an intracellular domain (ICD) formed by the large M3-M4 intracellular loop, which is responsible for receptor modulation, sorting, and trafficking, and which contains portals (openings) that influence ion conductance. The presence of portals has been deduced from nACh receptor data, but they are likely to exist, as these receptors are highly homologous (see Refs.
      • Thompson A.J.
      • Lummis S.C.
      5-HT3 receptors.
      ,
      • Maricq A.V.
      • Peterson A.S.
      • Brake A.J.
      • Myers R.M.
      • Julius D.
      Primary structure and functional expression of the 5-HT3 receptor, a serotonin-gated ion channel.
      ,
      • Reeves D.C.
      • Lummis S.C.
      The molecular basis of the structure and function of the 5-HT3 receptor: a model ligand-gated ion channel (review).
      , and
      • Thompson A.J.
      • Lester H.A.
      • Lummis S.C.
      The structural basis of function in Cys loop receptors.
      for reviews). The structural and functional similarity of these two receptors is such that chimeric receptors consisting of the ECD of the α7-nACh receptor and the TMD of the 5-HT3A receptor can be activated by ACh and have the channel properties of the 5-HT3A receptor (
      • Eiselé J.L.
      • Bertrand S.
      • Galzi J.L.
      • Devillers-Thiéry A.
      • Changeux J.P.
      • Bertrand D.
      Chimeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities.
      ). Thus, although there are no high resolution images of 5-HT3 receptors, the currently available cryo-electron microscope- and x-ray crystallography-derived structures are likely to be structurally representative, and those that have been used as templates for homology models include the nACh receptor, many ACh-binding proteins (AChBPs), and the bacterial homologs ELIC and GLIC (Erwinia and Gloeobacter ligand-gated ion channels) (e.g. Refs.
      • Thompson A.J.
      • Lester H.A.
      • Lummis S.C.
      The structural basis of function in Cys loop receptors.
      ,
      • Reeves D.C.
      • Sayed M.F.
      • Chau P.L.
      • Price K.L.
      • Lummis S.C.
      Prediction of 5-HT3 receptor agonist-binding residues using homology modeling.
      , and
      • Maksay G.
      • Bikádi Z.
      • Simonyi M.
      Binding interactions of antagonists with 5-hydroxytryptamine 3A receptor models.
      ). A model of one such subunit using the nACh receptor cryo-electron microscope structure as a template is shown in Fig. 1A; this reveals that the ECD (blue) is predominantly β-sheet and that the TMD (purple) is mostly α-helix, although most of the structure of the ICD, apart from a stretch of α-helix (yellow), is not currently known. Deletion studies revealed that this region is not essential for receptor expression, as the large intracellular loop of the mouse 5-HT3A receptor subunit can be replaced by the heptapeptide M3-M4 linker of GLIC without loss of function (
      • Jansen M.
      • Bali M.
      • Akabas M.H.
      Modular design of Cys loop ligand-gated ion channels: functional 5-HT3 and GABA ρ1 receptors lacking the large cytoplasmic M3M4 loop.
      ). Homology models of the 5-HT3 receptor ECD using the nACh receptor and AChBP as templates are broadly similar (Fig. 1B), although there are some subtle differences such as the orientation of the α-helix at the top of the receptor and the positions of some of the binding loops.
      Figure thumbnail gr1
      FIGURE 1Homology models of the 5-HT3 receptor. A, a single receptor subunit based on the nACh receptor structure (Protein Data Bank code 2BG9) showing the mostly β-sheet-containing ECD (blue), the four transmembrane α-helices (purple), and the α-helix that forms part of the ICD (orange). The structure of the remainder of this domain is not yet known. B, models of the 5-HT3 receptor ECD based on AChBP (blue; code 1UV6) and the nACh receptor (cyan; code 2BG9), with 5-HT docked into the intersubunit binding pocket.

      5-HT3 Receptor Subunits

      Five distinct 5-HT3 receptor subunits (A–E) have been identified so far (Fig. 2), which is relatively few for a Cys loop receptor, although the repertoire is increased by a number of different isoforms (
      • Niesler B.
      • Frank B.
      • Kapeller J.
      • Rappold G.A.
      Cloning, physical mapping, and expression analysis of the human 5-HT3 serotonin receptor-like genes HTR3CHTR3DHTR3E.
      ,
      • Holbrook J.D.
      • Gill C.H.
      • Zebda N.
      • Spencer J.P.
      • Leyland R.
      • Rance K.H.
      • Trinh H.
      • Balmer G.
      • Kelly F.M.
      • Yusaf S.P.
      • Courtenay N.
      • Luck J.
      • Rhodes A.
      • Modha S.
      • Moore S.E.
      • Sanger G.J.
      • Gunthorpe M.J.
      Characterization of 5-HT3C, 5-HT3D, and 5-HT3E receptor subunits: evolution, distribution, and function.
      ,
      • Niesler B.
      • Kapeller J.
      • Hammer C.
      • Rappold G.
      Serotonin type 3 receptor genes: HTR3ABCDE.
      ). There are, for example, a long and short form of the human 5-HT3A subunit that differ by 32 amino acids, three translational variants of the human 5-HT3B subunit, and five isoforms of the 5-HT3E subunit. The stoichiometry of heteromeric receptors is still not clear, although it has been established that only 5-HT3A subunits can form functional homomeric 5-HT3 receptors, and the presence of at least one 5-HT3A subunit appears to be obligatory in heteromeric receptors (
      • Niesler B.
      • Frank B.
      • Kapeller J.
      • Rappold G.A.
      Cloning, physical mapping, and expression analysis of the human 5-HT3 serotonin receptor-like genes HTR3CHTR3DHTR3E.
      ,
      • Holbrook J.D.
      • Gill C.H.
      • Zebda N.
      • Spencer J.P.
      • Leyland R.
      • Rance K.H.
      • Trinh H.
      • Balmer G.
      • Kelly F.M.
      • Yusaf S.P.
      • Courtenay N.
      • Luck J.
      • Rhodes A.
      • Modha S.
      • Moore S.E.
      • Sanger G.J.
      • Gunthorpe M.J.
      Characterization of 5-HT3C, 5-HT3D, and 5-HT3E receptor subunits: evolution, distribution, and function.
      ,
      • Niesler B.
      • Kapeller J.
      • Hammer C.
      • Rappold G.
      Serotonin type 3 receptor genes: HTR3ABCDE.
      ,
      • Niesler B.
      • Walstab J.
      • Combrink S.
      • Möller D.
      • Kapeller J.
      • Rietdorf J.
      • Bönisch H.
      • Göthert M.
      • Rappold G.
      • Brüss M.
      Characterization of the novel human serotonin receptor subunits 5-HT3C, 5-HT3D, and 5-HT3E.
      ).
      Figure thumbnail gr2
      FIGURE 25-HT3 receptor subunits. Shown is a Clustal alignment of representative human (h) 5-HT3 receptor A–E subunits, demonstrating the approximate locations of the binding loops on the principal (red) and complementary (cyan) faces, the Cys-Cys loop (green), and the transmembrane α-helices (black). Note the unusual sequence construction of the D subunit, which is missing a Cys-Cys loop and parts of loops D and E and which has a region of extra sequence in the M2-M3 loop. Accession numbers are as follows: NP_000860 (A subunit), NP_006019 (B subunit), NP_570126 (C subunit), NP_001157118 (D subunit), and NP_872385 (E subunit).
      Localization of the subunits reveals considerable overlap. Distribution of 5-HT3A receptor mRNA and protein is widespread and has been observed in many regions of the CNS (where it correlates well with radiolabeled antagonist binding studies (
      • Tecott L.H.
      • Maricq A.V.
      • Julius D.
      Nervous system distribution of the serotonin 5-HT3 receptor mRNA.
      ,
      • Kilpatrick G.J.
      • Jones B.J.
      • Tyers M.B.
      Identification and distribution of 5-HT3 receptors in rat brain using radioligand binding.
      )), in peripheral and sensory ganglia, and in a wide range of other tissues, including the gastrointestinal tract (
      • Holbrook J.D.
      • Gill C.H.
      • Zebda N.
      • Spencer J.P.
      • Leyland R.
      • Rance K.H.
      • Trinh H.
      • Balmer G.
      • Kelly F.M.
      • Yusaf S.P.
      • Courtenay N.
      • Luck J.
      • Rhodes A.
      • Modha S.
      • Moore S.E.
      • Sanger G.J.
      • Gunthorpe M.J.
      Characterization of 5-HT3C, 5-HT3D, and 5-HT3E receptor subunits: evolution, distribution, and function.
      ,
      • Morales M.
      • Battenberg E.
      • de Lecea L.
      • Sanna P.P.
      • Bloom F.E.
      Cellular and subcellular immunolocalization of the type 3 serotonin receptor in the rat central nervous system.
      ,
      • Morales M.
      • Battenberg E.
      • de Lecea L.
      • Bloom F.E.
      The type 3 serotonin receptor is expressed in a subpopulation of GABAergic neurons in the rat neocortex and hippocampus.
      ,
      • Laporte A.M.
      • Koscielniak T.
      • Ponchant M.
      • Vergé D.
      • Hamon M.
      • Gozlan H.
      Quantitative autoradiographic mapping of 5-HT3 receptors in the rat CNS using [125I]iodozacopride and [3H]zacopride as radioligands.
      ,
      • Morales M.
      • Battenberg E.
      • Bloom F.E.
      Distribution of neurons expressing immunoreactivity for the 5-HT3 receptor subtype in the rat brain and spinal cord.
      ). 5-HT3B subunit mRNA and protein were originally shown to be located in the spleen, colon, small intestine, and kidney, with some controversy as to their presence in the brain (
      • Davies P.A.
      • Pistis M.
      • Hanna M.C.
      • Peters J.A.
      • Lambert J.J.
      • Hales T.G.
      • Kirkness E.F.
      The 5-HT3B subunit is a major determinant of serotonin-receptor function.
      ,
      • Dubin A.E.
      • Huvar R.
      • D'Andrea M.R.
      • Pyati J.
      • Zhu J.Y.
      • Joy K.C.
      • Wilson S.J.
      • Galindo J.E.
      • Glass C.A.
      • Luo L.
      • Jackson M.R.
      • Lovenberg T.W.
      • Erlander M.G.
      The pharmacological and functional characteristics of the serotonin 5-HT3A receptor are specifically modified by a 5-HT3B receptor subunit.
      ,
      • Monk S.A.
      • Desai K.
      • Brady C.A.
      • Williams J.M.
      • Lin L.
      • Princivalle A.
      • Hope A.G.
      • Barnes N.M.
      Generation of a selective 5-HT3B subunit-recognizing polyclonal antibody: identification of immunoreactive cells in rat hippocampus.
      ,
      • Reeves D.C.
      • Lummis S.C.
      Detection of human and rodent 5-HT3B receptor subunits by anti-peptide polyclonal antibodies.
      ,
      • Morales M.
      • Wang S.D.
      Differential composition of 5-hydroxytryptamine 3 receptors synthesized in the rat CNS and peripheral nervous system.
      ). Later studies showing that there are several 5-HT3B receptor isoforms provided an answer to this conundrum: different tissue preferences of the different subunits. The longer B subunit variant is broadly expressed in many tissues, including the kidney, liver, brain, and gastrointestinal tract, whereas the shorter variant has a brain-specific expression pattern (
      • Holbrook J.D.
      • Gill C.H.
      • Zebda N.
      • Spencer J.P.
      • Leyland R.
      • Rance K.H.
      • Trinh H.
      • Balmer G.
      • Kelly F.M.
      • Yusaf S.P.
      • Courtenay N.
      • Luck J.
      • Rhodes A.
      • Modha S.
      • Moore S.E.
      • Sanger G.J.
      • Gunthorpe M.J.
      Characterization of 5-HT3C, 5-HT3D, and 5-HT3E receptor subunits: evolution, distribution, and function.
      ,
      • Tzvetkov M.V.
      • Meineke C.
      • Oetjen E.
      • Hirsch-Ernst K.
      • Brockmöller J.
      Tissue-specific alternative promoters of the serotonin receptor gene HTR3B in human brain and intestine.
      ). 5-HT3 receptor C–E subunits were first identified in humans, and genes for these proteins have now been shown to exist in a range of species, although not in rodents (
      • Niesler B.
      • Frank B.
      • Kapeller J.
      • Rappold G.A.
      Cloning, physical mapping, and expression analysis of the human 5-HT3 serotonin receptor-like genes HTR3CHTR3DHTR3E.
      ,
      • Holbrook J.D.
      • Gill C.H.
      • Zebda N.
      • Spencer J.P.
      • Leyland R.
      • Rance K.H.
      • Trinh H.
      • Balmer G.
      • Kelly F.M.
      • Yusaf S.P.
      • Courtenay N.
      • Luck J.
      • Rhodes A.
      • Modha S.
      • Moore S.E.
      • Sanger G.J.
      • Gunthorpe M.J.
      Characterization of 5-HT3C, 5-HT3D, and 5-HT3E receptor subunits: evolution, distribution, and function.
      ,
      • Karnovsky A.M.
      • Gotow L.F.
      • McKinley D.D.
      • Piechan J.L.
      • Ruble C.L.
      • Mills C.J.
      • Schellin K.A.
      • Slightom J.L.
      • Fitzgerald L.R.
      • Benjamin C.W.
      • Roberds S.L.
      A cluster of novel serotonin receptor 3-like genes on human chromosome 3.
      ). A recent study suggested that they all have a relatively widespread distribution, although initial studies suggested that the D and E subunits had a very restricted expression in the gastrointestinal tract. Studies examining protein levels have lagged behind the genetic work, but expression of the C–E subunits at the protein level in the gastrointestinal tract has recently been demonstrated (
      • Kapeller J.
      • Möller D.
      • Lasitschka F.
      • Autschbach F.
      • Hovius R.
      • Rappold G.
      • Brüss M.
      • Gershon M.D.
      • Niesler B.
      Serotonin receptor diversity in the human colon: expression of serotonin type 3 receptor subunits 5-HT3C, 5-HT3D, and 5-HT3E.
      ).

      5-HT3 Receptor Binding Pocket

      The agonist-binding site lies at the interface of two adjacent subunits in the extracellular N-terminal domain and is formed by three loops (A–C) from one (the principal) subunit and three β-strands (referred to as loops D–F) from the adjacent or complementary subunit, as in other Cys loop receptors. Only a few residues within each loop face into the binding pocket, with other residues having roles in maintaining the structure of the pocket and/or contributing to the conformational changes that result in channel opening (
      • Thompson A.J.
      • Lummis S.C.
      5-HT3 receptors.
      ). Evidence from AChBP structures suggests that the binding pocket contracts around agonists, which may initiate the conformational change that ultimately leads to channel opening, whereas antagonists tend to have little effect or may cause binding site expansion (
      • Thompson A.J.
      • Lester H.A.
      • Lummis S.C.
      The structural basis of function in Cys loop receptors.
      ).
      Key residues that contribute to the 5-HT3 receptor ligand-binding site include one or more from each of the six binding loops (see Refs.
      • Thompson A.J.
      • Lummis S.C.
      5-HT3 receptors.
      ,
      • Barnes N.M.
      • Hales T.G.
      • Lummis S.C.
      • Peters J.A.
      The 5-HT3 receptor–the relationship between structure and function.
      , and
      • Thompson A.J.
      • Padgett C.L.
      • Lummis S.C.
      Mutagenesis and molecular modeling reveal the importance of the 5-HT3 receptor F-loop.
      for comprehensive reviews). In loop A, attention has focused on the sequence 128Asn-Glu-Phe130, as substitutions here have large effects on receptor function (
      • Boess F.G.
      • Steward L.J.
      • Steele J.A.
      • Liu D.
      • Reid J.
      • Glencorse T.A.
      • Martin I.L.
      Analysis of the ligand-binding site of the 5-HT3 receptor using site-directed mutagenesis: importance of glutamate 106.
      ,
      • Steward L.J.
      • Boess F.G.
      • Steele J.A.
      • Liu D.
      • Wong N.
      • Martin I.L.
      Importance of phenylalanine 107 in agonist recognition by the 5-hydroxytryptamine 3A receptor.
      ,
      • Sullivan N.L.
      • Thompson A.J.
      • Price K.L.
      • Lummis S.C.
      Defining the roles of Asn-128, Glu-129, and Phe-130 in loop A of the 5-HT3 receptor.
      ,
      • Yan D.
      • Schulte M.K.
      • Bloom K.E.
      • White M.M.
      Structural features of the ligand-binding domain of the serotonin 5-HT3 receptor.
      ). Recent data indicate that only Glu-129 faces into the binding pocket, where it forms a hydrogen bond with the hydroxyl of 5-HT (
      • Price K.L.
      • Bower K.S.
      • Thompson A.J.
      • Lester H.A.
      • Dougherty D.A.
      • Lummis S.C.
      A hydrogen bond in loop A is critical for the binding and function of the 5-HT3 receptor.
      ). Mutations in loop B show that many residues here are important for receptor function, and these data, when combined with modeling data, suggest that loop B is an obligate rigid structure (
      • Spier A.D.
      • Lummis S.C.
      The role of tryptophan residues in the 5-hydroxytryptamine 3 receptor ligand-binding domain.
      ,
      • Thompson A.J.
      • Lochner M.
      • Lummis S.C.
      Loop B is a major structural component of the 5-HT3 receptor.
      ,
      • Venkataraman P.
      • Joshi P.
      • Venkatachalan S.P.
      • Muthalagi M.
      • Parihar H.S.
      • Kirschbaum K.S.
      • Schulte M.K.
      Functional group interactions of a 5-HT3R antagonist.
      ). One residue plays an especially critical role, Trp-183, which forms a cation-π interaction with the primary amine of 5-HT (
      • Spier A.D.
      • Lummis S.C.
      The role of tryptophan residues in the 5-hydroxytryptamine 3 receptor ligand-binding domain.
      ,
      • Beene D.L.
      • Brandt G.S.
      • Zhong W.
      • Zacharias N.M.
      • Lester H.A.
      • Dougherty D.A.
      Cation-π interactions in ligand recognition by serotonergic (5-HT3A) and nicotinic acetylcholine receptors: the anomalous binding properties of nicotine.
      ). Loop C shows the largest species variability and is important in determining the species specificity of various drugs (
      • Suryanarayanan A.
      • Joshi P.R.
      • Bikádi Z.
      • Mani M.
      • Kulkarni T.R.
      • Gaines C.
      • Schulte M.K.
      The loop C region of the murine 5-HT3A receptor contributes to the differential actions of 5-hydroxytryptamine and m-chlorophenylbiguanide.
      ). However, point mutations throughout the loop C region did not identify any residues that were essential for binding of the agonist m-chlorophenylbiguanide or the antagonist (+)-tubocurarine, suggesting that multiple regions of the binding site are important (
      • Hope A.G.
      • Belelli D.
      • Mair I.D.
      • Lambert J.J.
      • Peters J.A.
      Molecular determinants of (+)-tubocurarine binding at recombinant 5-hydroxytryptamine 3A receptor subunits.
      ,
      • Mochizuki S.
      • Miyake A.
      • Furuichi K.
      Identification of a domain affecting agonist potency of meta-chlorophenylbiguanide in 5-HT3 receptors.
      ). One residue that is critical for both agonist and antagonist binding is Tyr-234, which forms part of the aromatic box found in all Cys loop receptors (
      • Lester H.A.
      • Dibas M.I.
      • Dahan D.S.
      • Leite J.F.
      • Dougherty D.A.
      Cys loop receptors: new twists and turns.
      ). Loop D also contributes an aromatic residue (Trp-90) to the binding pocket, and double-mutant cycle analysis at Trp-90 and Arg-92 has indicated that the aromatic rings of the competitive antagonist granisetron are located close to Trp-90 and that the azabicyclic rings lie close to Arg-92 (
      • Yan D.
      • White M.M.
      Spatial orientation of the antagonist granisetron in the ligand-binding site of the 5-HT3 receptor.
      ). Loop E residues Tyr-141, Tyr-143, Gly-148, Glu-149, Val-150, Gln-151, Asn-152, Tyr-153, and Lys-154 may all be important for granisetron binding and perhaps function, although it is not clear if some of these effects are due to alterations in the binding site structure (
      • Yan D.
      • White M.M.
      Spatial orientation of the antagonist granisetron in the ligand-binding site of the 5-HT3 receptor.
      ,
      • Price K.L.
      • Lummis S.C.
      The role of tyrosine residues in the extracellular domain of the 5-hydroxytryptamine 3 receptor.
      ,
      • Venkataraman P.
      • Venkatachalan S.P.
      • Joshi P.R.
      • Muthalagi M.
      • Schulte M.K.
      Identification of critical residues in loop E in the 5-HT3AsR binding site.
      ). As yet, the structure of loop F is not well defined, although a study of granisetron binding has implicated Trp-195, Asp-204, and Ser-206 as potentially important residues for ligand binding; alternatively, these residues may influence conformational changes in or close to the binding pocket (
      • Thompson A.J.
      • Padgett C.L.
      • Lummis S.C.
      Mutagenesis and molecular modeling reveal the importance of the 5-HT3 receptor F-loop.
      ).
      There are also binding sites for a range of other ligands and modulators (Fig. 3). Of these, the pore-binding sites are currently the best characterized, e.g. picrotoxin and the ginkgolides block the 5-HT3 receptor channel by interacting with the 6′ residue (
      • Thompson A.J.
      • Lester H.A.
      • Lummis S.C.
      The structural basis of function in Cys loop receptors.
      ,
      • Das P.
      • Dillon G.H.
      Molecular determinants of picrotoxin inhibition of 5-hydroxytryptamine type 3 receptors.
      ); there also may be specific binding pockets for many other compounds, including ions, steroids, alcohols, anesthetics, and a range of small molecules (
      • Thompson A.J.
      • Lummis S.C.
      5-HT3 receptors.
      ,
      • Barnes N.M.
      • Hales T.G.
      • Lummis S.C.
      • Peters J.A.
      The 5-HT3 receptor–the relationship between structure and function.
      ,
      • Davies P.A.
      Allosteric modulation of the 5-HT3 receptor.
      ,
      • Trattnig S.M.
      • Harpsøe K.
      • Thygesen S.B.
      • Rahr L.M.
      • Ahring P.K.
      • Balle T.
      • Jensen A.A.
      Discovery of a novel allosteric modulator of 5-HT3 receptors: inhibition and potentiation of Cys loop receptor signaling through a conserved transmembrane intersubunit site.
      ).
      Figure thumbnail gr3
      FIGURE 35-HT3 receptor binding sites. Shown is a model of the 5-HT3 receptor (with two subunits removed for clarity) showing granisetron (green) and picrotoxin (red) docked into their known binding sites in the ECD and in the pore, respectively. Other possible binding sites (pink), whose locations have not yet been confirmed, are an allosteric site in the ECD, an interhelical site in the TMD, and a lipid transmembrane site at the membrane-receptor boundary.

      Receptor Function

      Homomeric 5-HT3A receptors mediate rapidly activating and desensitizing inward currents, which are carried primarily by Na+ and K+ ions (
      • Derkach V.
      • Surprenant A.
      • North R.A.
      5-HT3 receptors are membrane ion channels.
      ). The receptors are also permeable to Ca2+ and other small organic cations (
      • Maricq A.V.
      • Peterson A.S.
      • Brake A.J.
      • Myers R.M.
      • Julius D.
      Primary structure and functional expression of the 5-HT3 receptor, a serotonin-gated ion channel.
      ,
      • Yang J.
      Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells.
      ). As in other Cys loop receptors, the residues that line the ion-accessible inner face of the M2-generated pore are predominantly non-polar, and it is M2 residues that primarily control ion flux and size selection through the channel (
      • Panicker S.
      • Cruz H.
      • Arrabit C.
      • Slesinger P.A.
      Evidence for a centrally located gate in the pore of a serotonin-gated ion channel.
      ,
      • Reeves D.C.
      • Goren E.N.
      • Akabas M.H.
      • Lummis S.C.
      Structural and electrostatic properties of the 5-HT3 receptor pore revealed by substituted cysteine accessibility mutagenesis.
      ,
      • McKinnon N.K.
      • Reeves D.C.
      • Akabas M.H.
      5-HT3 receptor ion size selectivity is a property of the transmembrane channel, not the cytoplasmic vestibule portals.
      ). Charge selectively is mediated predominantly via the Glu residue in the M1-M2 loop, at the so-called −1′ position, and when combined with the introduction of a positively charged residue at the 20′ position or insertion of a Pro at the −2′ position and a V9′T mutation, the channel conducts predominantly anions (
      • Gunthorpe M.J.
      • Lummis S.C.
      Conversion of the ion selectivity of the 5-HT3A receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily.
      ,
      • Thompson A.J.
      • Lummis S.C.
      A single ring of charged amino acids at one end of the pore can control ion selectivity in the 5-HT3 receptor.
      ). Residues in the α-helical stretch of the ICD and in the ECD also play roles in determining single-channel conductance and relative permeability to Ca2+ (
      • Livesey M.R.
      • Cooper M.A.
      • Deeb T.Z.
      • Carland J.E.
      • Kozuska J.
      • Hales T.G.
      • Lambert J.J.
      • Peters J.A.
      Structural determinants of Ca2+ permeability and conduction in the human 5-hydroxytryptamine type 3A receptor.
      ,
      • Livesey M.R.
      • Cooper M.A.
      • Lambert J.J.
      • Peters J.A.
      Rings of charge within the extracellular vestibule influence ion permeation of the 5-HT3A receptor.
      ).
      5-HT3B receptor subunits do not form functional homomeric receptors but can coexpress with A subunits to yield heteromeric 5-HT3AB receptors that differ from 5-HT3A receptors in their EC50, Hill slope, desensitization kinetics, calcium permeability, shape of current-voltage relationship, and, most noticeably, single-channel conductance, which is much larger: ∼16 pS in 5-HT3AB receptors compared with <1 pS in 5-HT3A receptors (
      • Davies P.A.
      • Pistis M.
      • Hanna M.C.
      • Peters J.A.
      • Lambert J.J.
      • Hales T.G.
      • Kirkness E.F.
      The 5-HT3B subunit is a major determinant of serotonin-receptor function.
      ,
      • Dubin A.E.
      • Huvar R.
      • D'Andrea M.R.
      • Pyati J.
      • Zhu J.Y.
      • Joy K.C.
      • Wilson S.J.
      • Galindo J.E.
      • Glass C.A.
      • Luo L.
      • Jackson M.R.
      • Lovenberg T.W.
      • Erlander M.G.
      The pharmacological and functional characteristics of the serotonin 5-HT3A receptor are specifically modified by a 5-HT3B receptor subunit.
      ). Despite these biophysical differences, the pharmacology of the orthosteric sites of 5-HT3A and 5-HT3AB receptors is almost identical, and to date only one compound has been identified that can distinguish between them (
      • Thompson A.J.
      • Verheij M.H.
      • de Esch I.J.
      • Lummis S.C.
      VUF10166, a novel compound with differing activities at 5-HT3A and 5-HT3AB receptors.
      ). This is consistent with the action of agonists and competitive antagonists being at an AA interface, as has been described for both human and mouse receptors (
      • Lochner M.
      • Lummis S.C.
      Agonists and antagonists bind to an A-A interface in the heteromeric 5-HT3AB receptor.
      ,
      • Thompson A.J.
      • Price K.L.
      • Lummis S.C.
      Cysteine modification reveals which subunits form the ligand-binding site in human heteromeric 5-HT3AB receptors.
      ), but conflicting with data suggesting a BABBA arrangement determined using atomic force microscopy (
      • Barrera N.P.
      • Herbert P.
      • Henderson R.M.
      • Martin I.L.
      • Edwardson J.M.
      Atomic force microscopy reveals the stoichiometry and subunit arrangement of 5-HT3 receptors.
      ).
      There have been only two studies to date examining the functional effects of roles of the C–E subunits (
      • Holbrook J.D.
      • Gill C.H.
      • Zebda N.
      • Spencer J.P.
      • Leyland R.
      • Rance K.H.
      • Trinh H.
      • Balmer G.
      • Kelly F.M.
      • Yusaf S.P.
      • Courtenay N.
      • Luck J.
      • Rhodes A.
      • Modha S.
      • Moore S.E.
      • Sanger G.J.
      • Gunthorpe M.J.
      Characterization of 5-HT3C, 5-HT3D, and 5-HT3E receptor subunits: evolution, distribution, and function.
      ,
      • Niesler B.
      • Walstab J.
      • Combrink S.
      • Möller D.
      • Kapeller J.
      • Rietdorf J.
      • Bönisch H.
      • Göthert M.
      • Rappold G.
      • Brüss M.
      Characterization of the novel human serotonin receptor subunits 5-HT3C, 5-HT3D, and 5-HT3E.
      ). None of these subunits form functional homomers but do result in functional receptors when coexpressed with the A subunit, although there is no reported difference in radioligand binding, current-voltage relationships, or kinetics of whole cell currents of the presumed heteromers compared with the homomer. The physiological roles of these subunits are therefore still unknown. The role of the 5-HT3D subunit is particularly tantalizing; there are two variants, one of which is missing a significant proportion of the ECD, whereas the other is lacking a Cys-Cys loop, both critical features of the vast majority of functionally characterized Cys loop receptor subunits. Perhaps these subunits can modify receptor function when in a very specific stoichiometry or are involved in receptor expression and trafficking. More studies are needed on these potentially interesting subunits.

      Therapeutic Use and Potential

      There are currently a range of 5-HT3 antagonists available for clinical use in Europe, including tropisetron (Navaban®), ondansetron (Zofran®, Emetron®), granisetron (Kytril®), dolasetron (Anzemet®), and palonosetron (Aloxi®). These drugs have revolutionized the treatment of nausea and vomiting in cancer patients receiving chemotherapy or radiation therapy, which is their largest therapeutic use (
      • Aapro M.S.
      5-HT3 receptor antagonists. An overview of their present status and future potential in cancer therapy-induced emesis.
      ). The efficiency of these drugs may depend on the particular variants of 5-HT3 receptors expressed by the patient; , for example, one isoform of the 5-HT3B receptor has a promoter deletion that is associated with reduced efficacy of tropisetron and ondansetron (
      • Niesler B.
      • Weiss B.
      • Fischer C.
      • Nöthen M.M.
      • Propping P.
      • Bondy B.
      • Rietschel M.
      • Maier W.
      • Albus M.
      • Franzek E.
      • Rappold G.A.
      Serotonin receptor gene HTR3A variants in schizophrenic and bipolar affective patients.
      ,
      • Niesler B.
      • Flohr T.
      • Nöthen M.M.
      • Fischer C.
      • Rietschel M.
      • Franzek E.
      • Albus M.
      • Propping P.
      • Rappold G.A.
      Association between the 5′ UTR variant C178T of the serotonin receptor gene HTR3A and bipolar affective disorder.
      ,
      • Niesler B.
      5-HT3 receptors: potential of individual isoforms for personalized therapy.
      ).
      Irritable bowel syndrome (IBS) is a common gastrointestinal disorder affecting 10–15% of adults and is another major therapeutic area for 5-HT3 receptor-selective compounds, perhaps not surprisingly as these receptors have roles in gastrointestinal motility, sensation, and secretion. Alosetron (Lotronex®, Lotronox®), a 5-HT3 receptor antagonist, has been approved for the treatment of IBS, but there have been problems with constipation and, more rarely, ischemic colitis, and it is now less frequently used (primarily in female patients suffering from IBS with diarrhea). A range of other 5-HT3-selective compounds, including some partial agonists, may prove more successful and are currently being explored (
      • Walstab J.
      • Rappold G.
      • Niesler B.
      5-HT3 receptors: role in disease and target of drugs.
      ,
      • Machu T.K.
      Therapeutics of 5-HT3 receptor antagonists: current uses and future directions.
      ).
      An important consideration is that the 5-HT3 receptor-related actions of all drugs now on the market have been determined using homomeric 5-HT3A receptors. This may not prove to be the most useful testing protocol given that other subunits may play important roles. Emerging studies suggest that alterations in a number of 5-HT3 receptor subunits contribute to a range of disorders. Thus, mutations in the A, B, D, and E subunits have been associated with bipolar disorder, depression, anxiety, IBS, and anorexia (see Ref.
      • Niesler B.
      5-HT3 receptors: potential of individual isoforms for personalized therapy.
      for a recent review). A greater understanding of the roles of C–E subunit-containing heteromeric receptors may therefore allow a wide range of other diseases to be treated with 5-HT3 receptor-selective drugs, potentially including addiction, pruritis, emesis, fibromyalgia, migraine, chronic heart pain, bulimia, and neurological phenomena such as anxiety, psychosis, nociception, and cognitive function (
      • Walstab J.
      • Rappold G.
      • Niesler B.
      5-HT3 receptors: role in disease and target of drugs.
      ,
      • Niesler B.
      5-HT3 receptors: potential of individual isoforms for personalized therapy.
      ,
      • Machu T.K.
      Therapeutics of 5-HT3 receptor antagonists: current uses and future directions.
      ).

      Conclusions

      The 5-HT3 receptor is a widely expressed, cation-selective member of the Cys loop receptor family. A range of studies, in particular heterologous expression and molecular modeling, have revealed many molecular details of its distribution, structure, function, and pharmacology. Nevertheless, information on receptor stoichiometry and the roles of these receptors in the CNS and PNS is still limited, suggesting there is much potential for therapeutic intervention in areas beyond those for which 5-HT3 receptor-specific drugs are proving highly successful.

      Acknowledgments

      I thank J. Ashby, K. Price, and A. Thompson for assistance with figures and D. Weston for critical reading of the manuscript.

      Author Profile

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