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Atomic force microscopy reveals the stoichiometry and subunit arrangement of 5-HT3 receptors
Nelson P Barrera1, Paul Herbert, Robert M Henderson
1Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
Abstract:
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.
Insights
Researchers used atomic force microscopy to reveal the 5-HT3 receptor
Area of Science:
- Neuroscience
- Structural Biology
- Biophysics
Background:
- The serotonin 3 (5-HT3) receptor is a crucial therapeutic target, particularly for antiemetic drugs in cancer therapy.
- This receptor, a cation-selective ligand-gated ion channel, exists as homomeric 5-HT3A or heteromeric 5-HT3A/B receptors.
- Heteromeric receptors exhibit higher single-channel conductance, more closely resembling native receptor properties.
Purpose of the Study:
- To elucidate the architectural organization and subunit arrangement of 5-HT3A and 5-HT3A/B receptors.
- To determine the stoichiometry and spatial configuration of subunits within the heteromeric 5-HT3A/B receptor complex.
Main Methods:
- Utilized atomic force microscopy (AFM) to visualize receptor structures.
- Engineered epitope tags onto A- and B-subunits for specific antibody binding.
- Imaged doubly liganded receptors to analyze subunit distribution and arrangement.
Main Results:
- Determined the subunit stoichiometry of the heteromeric 5-HT3A/B receptor to be 2A:3B.
- Identified a specific subunit arrangement: B-B-A-B-A around the receptor rosette.
- Observed distinct antibody angle distributions for A-subunits (single peak ~144°) and B-subunits (two peaks ~72°, ~144°).
Conclusions:
- The 2A:3B stoichiometry and B-B-A-B-A arrangement of the 5-HT3A/B receptor provide structural insights.
- This specific subunit arrangement likely explains differences in agonist Hill coefficients and single-channel conductances between receptor types.
- The findings advance our understanding of 5-HT3 receptor structure-function relationships and potential drug interactions.
