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 approximately 144 degrees , whereas the distribution for antibodies against the B-subunit had two peaks at approximately 72 degrees and 144 degrees . 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.
Barrera
Nelson P
NP
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
Herbert
Paul
P
Henderson
Robert M
RM
Martin
Ian L
IL
Edwardson
J Michael
JM
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000737:chemistry; D002460:Cell Line; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D018625:Microscopy, Atomic Force; D046912:Multiprotein Complexes; D015202:Protein Engineering; D021122:Protein Subunits; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D046912:Multiprotein Complexes; D021122:Protein Subunits; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins