013P University of Oxford
BPS 75th Anniversary Meeting December 2007 |
Human 5-HT3 Receptor B-subunit Transcriptional Variants Alter Channel Function.
Andrew Thompson, Sarah Lummis
Cambridge University, Cambridge, United Kingdom
5-HT3 receptors (5-HT3R) are members of the Cys-loop family of ligand-gated ion channels. To date 5 subunits (A - E) have been functionally characterised (Nielser et al, 2007). Only A can form functional homomeric Rs, but the other subunits can combine with A to form heteromeric complexes. Recently, 2 novel variants of the B subunit (Brain-1; Br1 & Brain-2; Br2) were identified and found to be abundantly expressed in human brain (Tzvetkov et al, 2007). Here we describe the properties of these variants.
Human 5-HT3A (accession number: P46098), 5-HT3B (AF080582), Br1 and Br2 (constructed using PCR) subunit cDNAs were cloned into pGEMHE or pcDNA3.1 for oocyte or HEK293 cell expression respectively. Xenopus oocytes were injected with 5 ng cRNA (1:3, A:B for heteromeric 5-HT3R) and currents recorded 2 - 5 days later. Binding affinities for 5-HT3R-selective ligands were estimated using standard procedures from transfected HEK293 cells (Thompson et al, 2007).
[3H]Granisetron binding could only be detected when A alone or A+B subunits were expressed. Kd and Ki values for a range of ligands were similar for A-only and heteromeric Rs, but 5-HT EC50 values were significantly different (Fig 1, Table 1). Immunofluorescence of HEK 293 cells supported heteromeric cell surface expression, and also revealed significant B, but not A, subunit localisation in the nucleus.
Our data show that the 5-HT3B receptor subunit variants are expressed as heteromeric Rs at the cell surface. They cause distinct changes to EC50 and nH values, but do not alter the binding site pharmacology, indicating the different 5-HT3B receptor subunits differentially effect receptor gating.
Fig1. Kd and Ki of 5-HT3R ligands. Values mean ± SEM. n < 3.
Table 1. Parameters derived from Fig1.
| Subunit Types | pEC50 (μM) Mean ± SEM | nH Mean ± SEM | n |
| A | 5.76 ± 0.02 | 2.71 ± 0.30 | 6 |
| AB | 4.55 ± 0.04* | 1.01 ± 0.10* | 7 |
| ABr1 | 5.09 ± 0.06* | 0.93 ± 0.15* | 8 |
| ABr2 | 5.48 ± 0.04* | 1.72 ± 0.22* | 6 |
* Sig dif (P < 0.001) to wild type using ANOVA with Dunnett’s Post-Test.
Niesler, B. et al.(2007) Mol Pharmacol, 72, 8-17.
Tzvetkov, M.V et al (2007) Gene, 386, 52-62.
Thompson A. J. et al (2007) Br J Pharmacol, 151, 666-677. |
