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

Abstract

Cys-loop receptors are pentameric ligand-gated ion channels that facilitate communication within the nervous system. Upon neurotransmitter binding, these receptors undergo an allosteric activation mechanism connecting the binding event to the membrane-spanning channel pore, which expands to conduct ions. Some of the earliest steps in this activation mechanism are carried out by residues proximal to the binding site, the relative positioning of which may reflect functional differences among members of the Cys-loop family of receptors. Herein, we investigated key side-chain interactions near the binding site via mutagenesis and two-electrode voltage-clamp electrophysiology in serotonin-gated 5-HT_3A receptors (5-HT_3ARs) and nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. We found that a triad of residues aligning to Thr-152, Glu-209, and Lys-211 in the 5-HT_3AR can be exchanged between the homomeric 5-HT_3AR and the muscle-type nAChR α-subunit with small functional consequences. Via triple mutant cycle analysis, we demonstrated that this triad forms an interdependent network in the muscle-type nAChR. Furthermore, nAChR-type mutations of the 5-HT_3AR affect the affinity of nicotine, a competitive antagonist of 5-HT_3ARs, in a cooperative manner. Using mutant cycle analyses between the 5-HT_3A triad, loop A residues Asn-101 and Glu-102, β9 residue Lys-197, and the channel gate at Thr-257, we observed that residues in this region are energetically linked to the channel gate and are particularly sensitive to mutations that introduce a net positive charge. This study expands our understanding of the differences and similarities in the activation mechanisms of Cys-loop receptors.

Keywords: Cys-loop receptor; electrophysiology; gating; ligand-binding protein; mutagenesis; nicotinic acetylcholine receptors (nAChR); serotonin; structure-function.

Figure 1.

Region of interest in this study. A, view of two adjacent receptor subunits from the 5-HT_3AR crystal structure (PDB code 4PIR). The inset shows residues mutated in this study highlighted in yellow and binding-site residues of the aromatic box shown in green for context; red = oxygen, blue = nitrogen. B, sequence alignment of subunits contributing to the primary binding face of human-type 5-HT_3ARs, nAChRs, and GABA_ARs. Cells are colored according to side-chain chemistry. Unfilled cells indicate non-conservation with regard to the triad in that receptor subtype. TMD, transmembrane domain; ICD, intracellular domain. *, Residue numbers corresponding to the 5-HT_3AR crystal structure (PDB code 4PIR). See “Experimental procedures” for sequence accession numbers.

Figure 2.

Mutating residues Thr-152, Glu-E209, and Lys-211 in 5-HT_3ARs to their equivalents in nAChRs has non-additive effects on receptor function. A, dose-response curves (±S.E.) of wildtype and mutant receptors to 5-HT. Variants are ordered from top to bottom in order of increasing EC₅₀. B, scatterplots illustrating losses of function (±S.D.) of mutants on a logarithmic scale. The double mutants and triple mutant display considerable deviations from additivity.

Figure 3.

Triple mutant cycle in the muscle-type nAChR. Each face of the cube represents a double mutant cycle. Double mutant cycles from wildtype are meaningfully coupled, however, any pair of mutations is uncoupled in the background of the other mutation of the triad. This analysis yields ΔΔΔG = 0.95 kcal mol⁻¹ for the three mutations. EC₅₀ values (μm) are provided in parentheses.

Figure 4.

Dose-response curves of 5-HT_3AR variants in the presence of varying concentrations of nicotine. As a competitive antagonist, nicotine shifts the dose-response curve of 5-HT_3ARs to higher EC₅₀ values. Some variants are more sensitive to competitive inhibition by nicotine than others. Error bars represent S.E.

Figure 5.

Schild analysis of inhibition of 5-HT_3ARs by nicotine. A–F, Schild plots comparing inhibition of the wildtype 5-HT_3AR to the mutants T152K, E209D, K211T, T152K/K211T, E209D/K211T, and T152K/E209D/K211T, respectively. Meaningful changes in nicotine K_d are observed, and the mutations have non-additive effects. Error bars are shown as S.E., and are often smaller than the data points.

Figure 6.

Greatest-magnitude coupling energies observed between pairs of mutations at residues in 5-HT_3ARs. Full data can be found in Tables 1 and 3.

Figure 7.

In 5-HT_3ARs, mutations that remove the cationic amine on Lys-211 functionally couple to nearby mutations that introduce positive charge. A, mutant cycles with N101K. B, mutant cycles with E102Q. C, mutant cycles with E209Q. D, mutant cycles with T152K. Full pharmacological data can be found in Table 3. Dose-response curves provided in Fig. S8. Error bars represent S.D.