Defining the roles of Asn-128, Glu-129 and Phe-130 in loop A of the 5-HT₃receptor

Abstract

The ligand binding pocket of Cys-loop receptors consists of a number of binding loops termed A-F. Here we examine the 5-HT 3 receptor loop A residues Asn-128, Glu-129 and Phe-130 using modelling, mutagenesis, radioligand binding and functional studies on HEK 293 cells. Replacement of Asn-128 results in receptors that have wild type [ 3 H]granisetron binding characteristics but large changes (ranging from a five-fold decrease to a 1500-fold increase) in the 5-HT EC 50 when compared to wild type receptors. Phe-130 mutant receptors show both increases and decreases in K d and EC 50 values, depending on the amino acid substituted. The most critical of these residues appears to be Glu-129; its replacement with a range of other amino acids results in non-binding and non-functional receptors. Lack of binding and function in some, but not all, of these receptors is due to poor membrane expression. These data suggest that Glu-129 is important primarily for receptor expression, although it may also play a role in ligand binding; Phe-130 is important for both ligand binding and receptor function, and Asn-128 plays a larger role in receptor function than ligand binding. In light of these results, we have created two new homology models of the 5-HT 3 receptor, with alternative positions of loop A. In our preferred model Glu-129 and Phe-130 contribute to the binding site, while the location of Asn-128 immediately behind the binding pocket could contribute to the conformation changes that result in receptor gating. This study provides a new model of the 5-HT 3 receptor binding pocket, and also highlights the importance of experimental data to support modelling studies.

The extracellular subunit interface of the 5-HT3receptors: a computational alanine scanning mutagenesis study

“…Y148 has been the object of mutation studies ( Thompson et al, 2005;Venkataraman et al, 2002;Yan & White, 2005): it has been proposed to play a role in both receptor function and ligand binding. Interestingly, all the residues which in our model interact with Y148 (Y138 and W85, W178 and L179) underwent experimental mutations (see Table 1): the results for W178 were discussed previously and support its primary role in the interface stabilization (Beene et al, 2002;Reeves et al, 2003;Spier & Lummis, 2000;Thompson et al, 2005Thompson et al, , 2008; W85 was shown to be important for ligand binding Spier & Lummis, 2000;Sullivan et al, 2006;Thompson et al, 2005;Yan & White, 2005), as it will be discussed in the details further on; the results of experimental alanine scanning mutagenesis studies on Y138 demonstrated its involvement in receptor structure and function Thompson et al, 2005;Venkataraman et al, 2002) and evidences from experimental studies on mutants of L179 seem to support its involvement in channel opening (Thompson et al, 2005. As a whole, these seem to have both structural and functional roles.…”

“…H180 is supposed to be involved in receptor binding and function: its mutations cause changes in agonist binding but not in antagonist binding and the H180-mutated receptor is not functional ; similarly, also L179 is supposed to be involved in subunit rotation and conformational modification occurring during opening of the channel . In addition, W178 is also adjacent to two other null spot residues, E124 and F125 are also considered to be relevant for receptor function (Boess et al, 1997;Price et al, 2008;Sullivan et al, 2006;Thompson et al, 2005Thompson et al, , 2008. As a whole, combining the information from CASM and experimental mutagenesis, the interface pattern W116-H180-L179-W178-E124-F125 seems to define a pathway (or part of a more extended pathway) of residues involved in protein conformational transitions upon ligand binding, triggering receptor activation.…”

“…As far as the complementary subunit interface is concerned, according to experimental evidences, Y148 and W85 are directly involved in ligand binding and/ or receptor function, while the unique hot spot residue Y83 and the warm spot Y68 are not involved in receptor function (Beene et al, 2004;Spier & Lummis, 2000;Sullivan et al, 2006;Thompson et al, 2005;Venkataraman et al, 2002;Yan & White, 2005). Two other nearby aromatic polar residues, Y136 and Y138, have been mutated experimentally to alanine and are both supposed to play a role in receptor function Thompson et al, 2005;Venkataraman et al, 2002).…”

“…W85 is sandwiched between P150 on the complementary subunit (Figure 4 (e)) and the hot spot residue W178 on the principal subunit, with which it also forms a π-π stacking that is largely preserved throughout the whole simulation (Figure 4(a)). W85 residue has also been the subject of mutagenesis studies Spier & Lummis, 2000;Sullivan et al, 2006;Thompson et al, 2005;Yan & White, 2005) and it has been shown to be important for ligand binding, however no evidence for its being involved in receptor function or structure stabilization has been found so far.…”

The binding orientations of structurally-related ligands can differ; A cautionary note

“…In loop A, N128 also points towards the binding site, but consistent with the absence of specific interactions with the equivalent residue in 2YME (Y91), the affinity of granisetron was not altered. In previous work, a similar absence of effects on granisetron has also been reported for range of other substitutions at this location (Price et al., 2008, Sullivan et al., 2006). However, the effects of N128C we measured on the affinity of tropisetron (7-fold) and previous reports of changes to the EC 50 of 5-HT, m CPBG and the binding affinity of VUF10166 suggests that effects may depend upon the ligand studied (Price et al., 2008, Sullivan et al., 2006, Thompson et al., 2014).…”