Discriminating between 5-HT₃A and 5-HT₃AB receptors

Abstract

The 5-HT3B subunit was first cloned in 1999, and co-expression with the 5-HT3A subunit results in heteromeric 5-HT₃AB receptors that are functionally distinct from homomeric 5-HT₃A receptors. The affinities of competitive ligands at the two receptor subtypes are usually similar, but those of non-competitive antagonists that bind in the pore often differ. A competitive ligand and allosteric modulator that distinguishes 5-HT₃A from 5-HT₃AB receptors has recently been described, and the number of non-competitive antagonists identified with this ability has increased in recent years. In this review, we discuss the differences between 5-HT₃A and 5-HT₃AB receptors and describe the possible sites of action of compounds that can distinguish between them.

Figure 1

Binding sites in 5-HT₃R. (A) The 5-HT₃R consists of five subunits that surround a central ion-conducting pore that is shown here from the side (left) and from the extracellular side (right) of the cell membrane. The orthosteric binding site (red) is located in the extracellular domain at the interface of two subunits (green and blue). The transmembrane domain consists of four α-helices (M1–M4) from each subunit, and the pore is formed by the convergence of five M2 α-helices (yellow); M1–M4 of the two facing subunits have been removed to view the pore more clearly. Competitive antagonists bind to the orthosteric site and the majority of non-competitive antagonists to the channel. Hydrophobic ligands may bind in inter-subunit cavities at the top of transmembrane domain α-helices. The orthosteric binding site is seen in more detail in Figure 2. (B) An alignment of amino acids that form the M2 α-helices (left) in a range of receptors, and their locations in the pore (right). Channel-lining residues mentioned in the text are highlighted as white on grey. The box shows the extent of the M2 α-helix as described by Hilf and Dutzler (2008). Accession numbers for the alignment are: human 5-HT3A P46098, mouse 5-HT3A Q6J1J7, human 5-HT3B O95264, mouse 5-HT3B Q9JHJ5, human GABA σ P24046, human GABA α1 P14867, GABA β2 P47870, GABA γ2 P18507, Glycine α1 P23415, Glycine β P48167, GluCl G5EBR3.

Figure 2

Human 5-HT3A and 5-HT3B subunits. (A) A protein sequence alignment highlighting the binding loops of the ECD and the α-helices of the transmembrane domain (M1–M4). The orange and blue colours show the residues that are shown in (B); a description of specific ligand–receptor interactions in the orthosteric binding site of the 5-HT₃R can be found in a review by Thompson et al. (2010a). The channel can also be seen in Figure 1. Accession numbers for the human sequences are 5-HT3A P46098, 5-HT3B O95264. (B) A homology model of the 5-HT₃A receptor extracellular domain, showing binding loops A–C of the principal (orange) face and D–F of the complementary (blue) face. Only two of the five subunits are shown for clarity.

Figure 3

Examples of electrophysiological () and radioligand binding () measurements at human 5-HT₃A and 5-HT₃AB receptors. (A) Concentration–response curves differ at human 5-HT₃A and 5-HT₃AB receptors. Higher concentrations of 5-HT are needed to elicit a current response at 5-HT₃AB receptors and the slope of the curves differs. Parameters derived from these curves are: 5-HT₃A, pEC₅₀ = 5.76 ± 0.03, EC₅₀ = 1.74 μM, n_H = 2.3, n = 6 and 5-HT₃AB, pEC₅₀ = 4.53 ± 0.04, EC₅₀ = 29.5 μM, n_H = 1.0, n = 6. (B) Saturation binding with the radioligand [³H]granisetron shows that like many other competitive ligands it has the same affinity at 5-HT₃A and 5-HT₃AB receptors. K_d values for these representative curves were 0.21 and 0.19 nM for 5-HT₃A and 5-HT₃AB receptors respectively. (C) Like many other non-competitive antagonists, the sensitivity of 5-HT₃R currents to PTX differs at the two receptor types. Parameters derived from these curves are: 5-HT₃A, pIC₅₀ = 5.02 ± 0.09, IC₅₀ = 9.55 μM, n_H = 0.7, n = 9 and 5-HT₃AB, pIC₅₀ = 4.26 ± 0.06, IC₅₀ = 55.0 μM, n_H = 0.7, n = 5. (D) VUF10166 is unusual as this competitive antagonist has differing affinities at 5-HT₃A and 5-HT₃AB receptors. K_d values for these representative curves were 0.08 and 12.6 nM for 5-HT₃A and 5-HT₃AB receptors respectively. (E) Similar to many other NCAs, the sensitivity of 5-HT₃R currents to DTZ also differs at 5-HT₃A and 5-HT₃AB receptors. Mutagenesis has shown that DTZ has a pore-binding site in the 5-HT₃A receptor that is responsible for its increased potency relative to 5-HT₃AB receptors. Parameters derived from these curves are: 5-HT₃A, pIC₅₀ = 4.68 ± 0.07, IC₅₀ = 20.9 μM, n_H = 0.8, n = 7 and 5-HT₃AB, pIC₅₀ = 3.53 ± 0.01, IC₅₀ = 295 μM, n_H = 0.8, n = 5. (F) In contrast to the electrophysiological measurements shown in panel (E), radioligand competition binding studies show that the binding affinity of DTZ is the same at 5-HT₃A and 5-HT₃AB receptors. This is consistent with the majority of other competitive antagonists that also have similar binding affinities at the two receptor types. K_i values for these representative curves were 180 μM for 5-HT₃A receptors and 169 μM for 5-HT₃AB receptors.