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Cell lines expressing recombinant aequorin and a G-protein coupled receptor for functional screening

he detection of antagonists, CHO-5HT2B- Gα16-aequorin cells were incubated with various concentrations of the compounds to be tested. An agonist (α-methyl-5-HT) at a single concentration was then injected on the mixture of cells + antagonist and the emitted light was recorded and integrated for 30 seconds. Non-surmountable antagonists (Methiothepin, Methysergide) as well as surmountable antagonists (Mesulergine, Mianserin, Ketanserin) prevented, in a dosedependent manner the activation of the 5- HT2B receptor by α-methyl-5-HT (Figure 6B). Similar results were obtained with other antagonists (Ritanserin, rauwolscine, Yohimbine, Spiperone, SB206553 not shown).

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Custom cloning of the G-protein coupled receptor and establishment of a stable cell line, expressing the receptor and apoaequorin.

Selection of the most appropriate calciumcoupling system, if necessary.

Pharmacological characterization of the receptor, development and characterization of the functional assay in a 96-well plate.

A control cell line expressing apoaequorin only is provided (on request) to validate the specificity of the functional response.

References:

Button, D. and Brownstein, M. (1993) Aequorin-expressing mammalian cell lines used to report Ca2+ mobilization. Cell Calcium 14, 663-671.

Milligan, G., Marshall, F., and Rees, S. (1996) Gα16 as a universal G protein adapter: implications for agonist screening strategies. TIPS 17, 235-237.

Mottheakis, L.C. and Ohler, L.D. (2000) Seeing the light: calcium imaging in cells for drug discovery. Drug Discovery Today: HTS supplement 1, (1) 18- 19.

Rink, T.J. (1990) Receptor-mediat
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