Investigations into the Binding Affinities of Different Human 5-HT₄ Receptor Splice Variants

Abstract

This study examined whether the drug-receptor-binding sites of 5 selected human 5-HT₄ receptor splice variants [h5-HT_4(a), h5-HT_4(b), h5-HT_4(c), h5-HT_4(d) and h5-HT_4(g)] display preferential affinities towards agonists. The agonists selected on the basis of chemical diversity and clinical relevance were: 5-HT₄ benzamides, renzapride, zacopride and prucalopride; the benzimidazolones, DAU 6236 and BIMU 1; the aromatic ketone, RS67333, and the indole carbazimidamide tegaserod. The rank order of affinities ranging across the splice variants was: tegaserod (pK_i: 7.38–7.91) ≧ Y-36912 (pK_i: 7.03–7.85) = BIMU 1 (pK_i: 6.92–7.78) ≧ DAU 6236 (pK_i: 6.79–7.99) ≧ 5-HT (pK_i: 5.82–7.29) ≧ 5-MeOT (pK_i: 5.64–6.83) ≧ renzapride (pK_i: 4.85–5.56). We obtained affinity values for the 5-HT_4(b), (d) and (g) variants for RS67333 (pK_i: 7:48–8.29), prucalopride (pK_i: 6.86–7.37) and zacopride (pK_i: 5.88–7.0). These results indicate that the ligands interact with the same conserved site in each splice variant. Some splice variants have a higher affinity for certain agonists and the direction of selectivity followed a common trend of lowest affinity at the (d) variant. However, this trend was not evident in functional experiments. Our findings suggest that it may be possible to design splice variant selective ligands, which may be of relevance for experimental drugs but may be difficult to develop clinically.

Luminal 5‐HT4 receptors—A successful target for prokinetic actions

“…This is a significant consideration for design of future pharmacological agents. 5‐HT 4 R agonists, including prucalopride, show preferential affinity for particular isoforms (Table ). This is ideal when designing experimental drugs targeting tissues predominantly expressing a particular splice variant, but whether agents are readily absorbed systemically becomes a key consideration when many 5‐HT 4 R isoforms are commonly expressed in other tissues.…”

PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease

“…PPNAD tissues overexpressed HTR4 mRNAs versus normal adrenals ( Figure 3A). It has been demonstrated that alternative splicing of HTR4 transcripts has the potential to generate several receptor isoforms (a, b, d, g, and i) that differ in the structure of their C-terminal tail and their pharmacological properties (31). Reverse transcriptase PCR (RT-PCR) analysis showed that the expression pattern of 5-HT 4 R isoforms is distinct in PPNAD tissues from normal adrenals (Figure Table 3).…”

Assessment of the pharmacological properties of 5-methoxyindole derivatives at 5-HT4 receptors

“…Membranes were isolated from COS 7 cells transiently transfected with the human recombinant 5‐HT 4(b) receptor splice variant as previously described [17] . Membranes were obtained from at least three separate transfections for all the ligands tested in binding assays.…”

“…Membranes were obtained from at least three separate transfections for all the ligands tested in binding assays. Radioligand binding assays using [ 3 H]GR‐113808 with or without the competing unlabelled ligand were performed as previously described [17] …”

“…Binding data was fitted by least squares analysis using the one site fit K i model using the equations: logEC50 = log(10 logKi × (1 + radioligand/K d )) and Y = Bottom + (Top‐Bottom)/(1 + 10 (X‐LogEC50) ) where EC50 is the concentration at 50% of the maximum response, the radioligand is 0.25 n m [ 3 H]GR‐113808 and K d is the equilibrium dissociation constant that was previously determined to be 0.087 n m in our hands [17] . Concentration–effect curves were analysed using a sigmoidal dose–response (variable slope) function.…”