Phylogenetic analyses of 5-hydroxytryptamine 3 (5-HT3) receptors in Metazoa

Abstract

The 5-hydroxytrptamine 3 (5-HT3) receptor is a member of the 'Cys-loop' family and the only pentameric ligand gated ion channel among the serotonin receptors. 5-HT3 receptors play an important role in controlling growth, development, and behaviour in animals. Several 5-HT3 receptor antagonists are used to treat diseases (e.g., irritable bowel syndrome, nausea and emesis). Humans express five different subunits (A-E) enabling a variety of heteromeric receptors to form but all contain 5HT3A subunits. However, the information available about the 5-HT3 receptor subunit occurrence among the metazoan lineages is minimal. In the present article we searched for 5-HT3 receptor subunit homologs from different phyla in Metazoa. We identified more than 1000 5-HT3 receptor subunits in Metazoa in different phyla and undertook simultaneous phylogenetic analysis of 526 5HT3A, 358 5HT3B, 239 5HT3C, 70 5HT3D, and 173 5HT3E sequences. 5-HT3 receptor subunits were present in species belonging to 11 phyla: Annelida, Arthropoda, Chordata, Cnidaria, Echinodermata, Mollusca, Nematoda, Orthonectida, Platyhelminthes, Rotifera and Tardigrada. All subunits were most often identified in Chordata phylum which was strongly represented in searches. Using multiple sequence alignment, we investigated variations in the ligand binding region of the 5HT3A subunit protein sequences in the metazoan lineage. Several critical amino acid residues important for ligand binding (common structural features) are commonly present in species from Nematoda and Platyhelminth gut parasites through to Chordata. Collectively, this better understanding of the 5-HT3 receptor evolutionary patterns raises possibilities of future pharmacological challenges facing Metazoa including effects on parasitic and other species in ecosystems that contain 5-HT3 receptor ligands.

Fig 1

Venn diagram summarising the number of 5-HT₃ receptor A, B, C, D and E subunit sequences identified in different phyla from the animal kingdom. A detailed breakdown of subunit distribution is listed in S2 Table. The figure contains the numbers of B, C, D and E subunits for those species that also contain subunit A, thus our dataset by definition contained zero (0) sequences with only B, C, D, or E subunits.

Fig 2. Phylogenetic tree of 5HT3A homolog proteins from animal phyla.

The colours of the species in the tree correspond to the colours of the phyla in the figure legend: Chordata (green with the human sequence highlighted with amber background), Nematoda (dark red), Arthropoda (orange), Platyhelminthes (dark purple), Mollusca (cyan), Rotifera (yellow), Tardigrada (maroon), Hemichordata (purple), Orthonectida (pale green), Echinodermata (teal), Annelida (blue) and Cnidaria (grey). This analysis involved 494 amino acid sequences. There was a total of 292 alignment positions in the final dataset. Evolutionary analyses were conducted in MEGA X and tree editing was performed in iTOL. Tree scale represents the number of differences between sequences.

Fig 3

Homology models of heteromeric 5-HT₃AE receptors (a) Extracellular view showing 5-HT₃AE receptor heteromer formed with three A (maroon) subunits and two E (green) subunits with AAEAE stoichiometry (based on the AABAB stoichiometry [114]) where A+A- interface (ligand binding region) is indicated. (b) The A+A- extracellular interface of two adjacent subunits (principal subunit (A+) and complementary subunit (A-)) highlighting the six loops that converge to form the ligand binding site. Only two of the five subunits have been shown for ease of viewing. c) The loops in the principal subunit (loop A (cyan), B [115], C (pink)) and the complementary subunit (loops D (red), E (yellow), F (blue)) and the critical amino acid residues participating in the binding site are labelled. Human 5HT3A and 5HT3E subunit tertiary structures modelled using information from mouse 5HT₃A receptor structures with PDB 6np0.1 [74] and PDB 6w1m.1 respectively by Swiss modelling software [94] and further edited in by UCSF Chimera software [116]. Residue numbers of the human 5HT3A (AAP35868.1) subunit (Fig 4) are used as the comparator in the following sections. Each 5HT3A subunit contains an extracellular N terminal helix (M1-P21 residues) signal peptide that helps in receptor translocation to the plasma membrane [117, 118]. The remainder of the extracellular region contains multiple beta strands with the orthosteric ligand binding site formed by β1 – β10 strands and six loops (T87-K239) followed by the signature Cys-loop (C163-C177) [97]. This sequence then leads into the four sequential transmembrane (TM) domains: TM1 (V252-L272), TM2 (R279-D299), TM3 (C318-K338) and TM4 (K457-W478). The intracellular loop (ICL) between TM3-TM4 (L341-L455) is involved in channel gating activities [40, 119, 120]. Resistance to inhibitors of cholinesterase 3 (RIC-3) a chaperone protein binds to the RIC-3 binding region present in between TM3 and TM4 domains to help in receptor translocation to cell membranes [–123].

Fig 4. Amino acid sequence of the human 5HT3A subunit (AAP35868.1).

The Cys-loop is marked in salmon, the extracellular loops are labelled in blue and transmembrane (TM) domains are depicted in purple, RIC-3 binding region (RIC-3 BR) depicted in pink. Functionally important amino acids as described in Table 2 are highlighted in grey.

Fig 5. Consensus amino acid logos of 5HT3A subunit regions derived from 449 different animal species.

Human 5HT3A subunit tertiary structure is modelled using information from PDB 6np0.1 using Swiss modelling [94] software and edited in UCSF Chimera software [116]. Sequence logos of loops A to F, TM domains and Cys-loop represented according to Table 2 and Figs 4 and S6 and S7.