A Coupled Array of Noncovalent Interactions Impacts the Function of the 5-HT₃A Serotonin Receptor in an Agonist-Specific Way

Abstract

The serotonin type 3A (5-HT 3 A) receptor is a Cysloop (pentameric) neurotransmitter-gated ion channel found in the central and peripheral nervous systems and implicated in numerous diseases. In previous studies with the endogenous agonist serotonin, we identified two interactions critical for receptor function: a cation−π interaction with W183 in loop B (TrpB) and a hydrogen bond to E129 in loop A. Here we employ mutant cycle analyses utilizing conventional and unnatural amino acid mutagenesis to demonstrate that a third residue, D124 of loop A, forms two functionally important hydrogen bonds to the backbone of loop B. We also show that these three interactions, the cation−π interaction, the backbone hydrogen bonds, and the E129 hydrogen bond, are tightly coupled to each other, suggesting they function as a single unit. We also identify key functional differences between serotonin and the competitive partial agonist m-chlorophenyl biguanide (mCPBG) at these residues. mCPBG displays no cation−π at TrpB and extreme sensitivity to the positioning of E129, on which it is reliant for initiation of channel gating. KEYWORDS: Serotonin, mCPBG, Cys-loop, binding site, gating, unnatural amino acid mutagenesis ■ INTRODUCTIONThe serotonin type 3 receptor (5-HT 3 ) is a Cys-loop (pentameric) neurotransmitter-gated ion channel whose activation elicits fast excitatory responses.1,2 The 5-HT 3 receptor is expressed in both the central and peripheral nervous systems and is known to be involved in multiple psychiatric and neurological disorders.3 Five distinct subunits of the 5-HT 3 receptor have been identified, 4 but only the A subunit forms functional homomeric receptors. These subunits, consisting of a large N-terminal extracellular domain, four transmembrane helices (M1−4), and a large intracellular loop (M3−M4), arrange into a symmetrical pentamer around a central pore.As with other pentameric receptors, the ligand binding domain is located at the interface between the extracellular domains of two adjacent subunits. More specifically, the binding site is composed of six loops from both the principle (A−C) and complementary (D−F) faces. Agonist binding leads to an opening of the channel gate at the M2 helix, over 60 Å distant.In previous studies of the 5-HT 3 A receptor, we identified critical roles for two residues. W183 on loop B (termed TrpB) makes a cation−π interaction to serotonin, 5 playing the same role as the aligning residue in most nicotinic acetylcholine receptors (nAChR).6−8 E129 on loop A of the 5-HT 3 receptor makes a hydrogen bond that is critical to receptor function. 9 We proposed that the hydrogen bond occurs with the OH of the agonist, serotonin. The aligning residue of nAChRs is Y93 (TyrA) (Figure 1).In the present work, we evaluate the role of a second residue on loop A that is very highly conserved across the entire Cysloop family of neurotransmitter-gated ion channels, D124 of the 5-HT 3 A receptor. In the nAChR family, the aligning residue, D89, makes a number of hydrogen bonds to loop B, ...

A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors

“…Loop A residue N101 couples to T6'S, supporting a previous proposition that this residue is involved in receptor gating (29). The mutation E102Q couples to T6'S, in line with a previouslypublished result (23). We observe that the T152K mutation affects ligand binding and also couples to the T6'S; thus we propose that this residue is involved in both binding and gating.…”

“…Previous work in the 5-HT 3A R on loop A residues N101 and E102 showed that mutations to these residues that introduce a net positive charge (N101K and E102Q) generate very large losses of function (23,29). This parallels our observation on the T152K mutation, which is the most disruptive of the single mutations in the triad swap.…”

“…The data from the single, double, and triple mutants enables a triple mutant cycle analysis, which can be represented as a cube ( Figure 3). The difference between each opposite face gives a ∆∆∆G°, which quantifies the interaction energy among all three residues as a unit (23). We find in each case that ∆∆∆G° = 0.95 kcal•mol -1 .…”

“…For these studies we typically used more conservative mutations than were used in the "triad swapping" experiments (Table 3). For residues E102 and E209, we employ mutations to glutamine and/or aspartate; the E102D mutation was not examined as it has been observed previously that this mutation has little effect on the response of receptors to 5-HT (23). We employ a more drastic mutation at N101, N101K.…”

An Unaltered Orthosteric Site and a Network of Long-Range Allosteric Interactions for PNU-120596 in α7 Nicotinic Acetylcholine Receptors

“…In this analysis, the first perturbation is mutation of the protein (with conventional or non-canonical amino acids); and the second perturbation is not a mutation, but the addition of PNU-120596. Non-additivity of the two perturbations indicates that the protein mutation differentially impacts receptor function depending on whether PNU-120596 is or is not present, suggesting that the residue under study plays an important role in allosteric modulation (Daeffler, et al, 2012; Gleitsman, et al, 2009; Miles, et al, 2012). The analysis parallels that of the common double mutant cycle analysis, including the notion of an interaction energy, designated as ΔΔG.…”