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 Cys-loop (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.

Figure 1

Alignment of loops A and B from the mouse and human 5-HT₃A subunits, the Torpedo nACh α1 subunit, the human nACh α7 subunit, and the Lymnaea AChBP. Residues contributing significant interactions through their side chains are highlighted in gray. The numbering is that of the mouse 5-HT₃A subunit.

Figure 2

Schematic of the collection of interactions being probed here. Dashed red lines are hydrogen bonds. Purple dashed line is a cation−π interaction.

Figure 3

(A) Structures of unnatural amino acids considered here. Unless specified, a, b, c, and d are H. (B) The α-hydroxy acid strategy for probing backbone hydrogen bonding interactions. (C) Structures of agonists considered here.

Figure 4

Examples of responses for 5-HT on receptors in which a nonsense mutation (TAG) at L184 is suppressed by THG73 tRNA bearing either leucine (wild-type recovery) or α-hydroxy leucine expressed in Xenopus oocytes and their corresponding concentration–response curves. Circles correspond to L184TAG + THG73-Leu; squares correspond to L184TAG + THG73-Lah. Each data point represents the mean ± SEM (n = 7–23).

Figure 5

Results of select mutagenesis studies.

Figure 6

Triple mutant cycle analysis. ΔΔG values (kcal/mol) are given on each face of the cube. ΔΔΔG for the diagonal from lower left front to upper right back is 1.5 kcal/mol. The structural image is a simple docking of 5-HT into a previously described homology model of the receptor and is for illustrative purposes only. See text for a full description and analysis.

Figure 7

Structures and electrostatic potential surfaces for agonists considered here. Structures and surfaces were generated at the HF 3-21G* level. Electrostatic potentials are plotted with blue most positive and red least positive over a range from 0 to +750 kJ/mol. For the individual molecules, the electrostatic potentials span the following ranges: 5-HT, 13–733; 5-FT, 47–745; mCPBG, 116–636; mCPG, 152–698.