Atomic force microscopy reveals the stoichiometry and subunit arrangement of 5-HT₃ receptors

The 5-HT₃ receptor is a cation-selective ligand-gated ion channel of the Cys-loop superfamily. The receptor is an important therapeutic target, with receptor antagonists being widely used as antiemetics in cancer therapy. The two known receptor subunits, A and B, form homomeric 5-HT _3A receptors and heteromeric 5-HT_3A/B receptors. The heteromeric receptor has the higher single-channel conductance and more closely mimics the properties of the native receptor. We have used atomic force microscopy to study the architecture of 5-HT_3A and 5-HT _3A/B receptors. We engineered different epitope tags onto the A- and B-subunits and imaged receptors that were doubly liganded by anti-epitope antibodies. We found that, for the 5-HT_3A/B receptor, the distribution of angles between antibodies against the A-subunit had a single peak at ≈144°, whereas the distribution for antibodies against the B-subunit had two peaks at ≈72° and 144°. Our results indicate that the subunit stoichiometry is 2A:3B and that the subunit arrangement around the receptor rosette is B-B-A-B-A. This arrangement may account for the difference between the agonist Hill coefficients and the single-channel conductances for the two types of receptor.