051P Brighton
Winter Meeting December 2007 |
Disulphide trapping reveals the proximity and role in gating of the 5-HT3 receptor residues E80 and T307
Kerry Price, Sarah Lummis
Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
Members of the Cys-loop family of ligand-gated ion channels, which includes the 5-HT3 receptor, share many structural and functional characteristics. This study tests predictions from a homology model of the mouse 5-HT3 receptor (Price et al. 2005), based on the 4Å resolution nicotinic acetylcholine receptor structure (Unwin 2005), that a residue in loop 2 (E80) is close to, and could potentially interact with, a residue in the TM2-TM3 linker (T307).
Voltage clamp recordings were performed on Xenopus oocytes injected with ∼5ng mRNA produced by in vitro transcription of mutant mouse 5-HT3As receptor subunits in pGEMHE. For disulphide trapping experiments, receptors were initially tested with a maximal concentration of 5-HT (100 μM), then treated with copper phenanthroline (Cu:phen; 100 μM CuSO4: 400 μM 1,10-phenanthroline) for 2 min followed by a 10 min washout, then re-tested with 5-HT. Following recovery, 10 mM dithiothreitol (DTT) was applied for 3 min and the receptors were again tested with 5-HT after a 10 min washout.
Data are shown in Table 1. Small responses (< 200 nA) and rapid desensitization of E80C mutant receptors made data collection difficult, but robust responses were obtained from the single T307C and the double E80C + T307C mutants. Cu:phen dramatically reduced the magnitude of the response to 5-HT in E80C + T307C receptors, whilst having no effect on WT receptors. This suggests that Cu:phen can induce disulphide bond formation between these two introduced cysteines, which yields a receptor that cannot undergo normal gating movements in response to 5-HT. Normal function was not restored after application of DTT to reduce the disulphide; this may indicate that the receptor has been locked in a closed state which is inaccessible to DTT.
Table 1
|
EC50 (μM) |
pEC50 |
Post-Cu:phen response (% initial response) |
Post-DTT response (% initial response) |
| WT |
1.2 |
5.9 ± 0.02 (4) |
97 ± 7 (4) |
93 ± 5 (3) |
| T307C |
0.47 |
6.3 ± 0.05 (5) * |
68 ± 7 (8) * |
96 ± 12 (7) |
| E80C+T307C |
1.2 |
5.9 ± 0.03 (4) |
3 ± 1 (5) * |
10 ± 2 (6) * |
Data are mean ± standard error (n). *Statistically different from WT, P < 0.05, one-way ANOVA.
These data support our model showing the proximity of these loops and further suggest the importance of movement around residues E80 and T307C for receptor gating movements in the 5-HT3 receptor.
Price K et al. (2005) Proc. Br. Pharm. Soc., http://www.pA2online.org/abstracts/Vol3Issue2abst108P.pdf
Unwin N (2005) J. Mol. Biol., 346: 967-989
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