Agonists and antagonists bind to an A-A interface in the heteromeric 5-HT3AB receptor

Abstract

The 5-HT3 receptor is a member of the Cys-loop family of transmitter receptors. It can function as a homopentamer (5-HT3A-only subunits) or as a heteropentamer. The 5-HT3AB receptor is the best characterized heteropentamer. This receptor differs from a homopentamer in its kinetics, voltage dependence, and single-channel conductance, but its pharmacology is similar. To understand the contribution of the 5-HT3B subunit to the binding site, we created homology models of 5-HT3AB receptors and docked 5-HT and granisetron into AB, BA, and BB interfaces. To test whether ligands bind in any or all of these interfaces, we mutated amino acids that are important for agonist and antagonist binding in the 5-HT3A subunit to their corresponding residues in the 5-HT3B subunit and vice versa. Changes in [3H]granisetron binding affinity (Kd) and 5-HT EC50 were determined using receptors expressed in HEK-293 cells and Xenopus oocytes, respectively. For all A-to-B mutant receptors, except T181N, antagonist binding was altered or eliminated. Functional studies revealed that either the receptors were nonfunctional or the EC50 values were increased. In B-to-A mutant receptors there were no changes in Kd, although EC50 values and Hill slopes, except for N170T mutant receptors, were similar to those for 5-HT3A receptors. Thus, the experimental data do not support a contribution of the 5-HT3B subunit to the binding pocket, and we conclude that both 5-HT and granisetron bind to an AA binding site in the heteromeric 5-HT3AB receptor.

Figure 1

(A) Location of the 5-HT3A subunit residues (stick representation) that were mutated to the corresponding 5-HT3B subunit residues. Two adjacent subunits (principal and complementary) show the positions of the six binding loops, A–F. Other residues of the putative ligand-binding site are shown in line representation. (B) ClustalW sequence alignment of the murine 5-HT3A (accession number: Q6J1J7) and 5-HT3B subunits (Q9JHJ5). 5-HT3A subunit residues that were mutated to 5-HT3B subunit residues in this study are highlighted as white text on black background. The six binding loops and the Cys loop are indicated by black lines above the text. Numbering of residues and structural features are taken from the AChBP protein crystal structure (13).

Figure 2

Docking of 5-HT (yellow, left panels) and granisetron (Grn, orange, right panels) into homomeric 5-HT₃A (14,19) and heteromeric 5-HT₃AB receptor homology models. The 5-HT3A subunit is shown in violet, and the 5-HT3B subunit is shown in teal. Residues within 4 Å of docked ligand are rendered in stick representation, color-coded according to the corresponding subunits, and numbered according to Fig. 1B. The + and − signs denote the principal and complementary face of the binding site, respectively. Proposed hydrogen bonds are shown as green dotted lines. The orientation of the docking models is the same as in Fig. 1A.

Figure 3

HEK cells labeled with a 5-HT3B-selective antisera reveal that both WT and mutant 5-HT3B receptor subunits reach the cell surface when coexpressed with WT 5-HT3A receptor subunits. Scale bar: 20 μm.

Figure 4

Examples of HEK cells labeled with 5-HT3A-selective antisera, which reveals that both WT and mutant 5-HT3A receptors subunits are expressed on the cell surface. Scale bar: 20 μm.

Figure 5

Example data for radioligand binding and functional studies of WT and mutant receptors. (A) K_d values were estimated using the 5-HT₃ receptor antagonist [³H]granisetron. The examples show binding curves for single experiments, fitted with a one-site binding equation. K_d values for a series of experiments were averaged for each mutant and are presented in Tables 2 and 5. (B) Concentration response curves in 5-HT₃ WT and mutant receptors measured using two-electrode voltage clamp and fitted with a four-parameter logistic equation. The calculated EC₅₀ values are shown in Tables 3 and 4. (C) Typical 5-HT responses from oocytes expressing WT and A-to-B mutant receptors.