Characterization of a typical ligand-gated ion channel using the flexstation K.L. Price and S.C.R. Lummis, Dept of Biochemistry, Tennis Court Road, Cambridge, CB2 1AG, UK

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The 5-HT₃ receptor is a member of the Cys-loop family of ligand-gated ion channels (LGIC), which includes the nicotinic acetylcholine, GABA_A and glycine receptors (Reeves et al., 2002). Activation of these receptors by agonist results in a change in membrane potential in the postsynaptic cell due to an influx of ions. The receptors are cation or anion selective, and some of those that are cation selective also are permeable to Ca²⁺ ions. One such protein is the 5-HT₃ receptor which has been characterised in vivo using both electrophysiological (membrane potential) measurements and Ca²⁺ imaging using Fura 2 loaded cells. This protein therefore provides an excellent material for examining the use of the FLEXstation to characterise these proteins using both membrane potential and Ca²⁺ imaging dyes (Hargreaves et al., 1994; Baxter et al. 2002). Here, we present data obtained from characterising the recombinant 5-HT₃ receptor stably expressed in HEK 293 cells and show that both types of dyes yield usable data when analysed using a FLEXstation.

Mouse 5-HT_3A subunit cDNA cloned from N1E-115 cells (Hargreaves et al. 1994) in the vector pcDNA₃ (Invitrogen) was transfected into HEK293 cells using calcium phosphate precipitation (Chen and Okayama, 1988). A stable cell line with high levels of [³H]granisetron (a 5-HT₃ receptor antagonist) binding was selected using geneticin. FLEXstation assays were performed on 96 well plates 1-3 days after passage using either the FLEXstation Calcium Plus Assay Kit or the Membrane Potential Assay Kit (Molecular Devices) according to recommendations, with the following modifications. Cells were washed twice with 1x Reagent Buffer Component B (Molecular Devices) then 100µl of this buffer plus 100µl Loading Buffer was incubated with cells at room temperature. Responses were only detected when cells were washed prior to dye loading.

Robust responses were observed with both the membrane potential and Ca²⁺ imaging dyes, although responses were larger from the former. Analysis of these data yielded pEC₅₀ values of 5.96 ± 0.04µM (EC₅₀=1.1µM; n=6) and 5.62 ± 0.06µM (EC₅₀=2.4µM; n=3) respectively. Further experiments with the membrane potential dye yielded pEC₅₀s of 5.51 ± 0.05µM (EC₅₀=3.1µM; n=6) and 6.1 ± 0.04µM (EC₅₀=0.8mM; n=6) for the 5-HT₃ receptor agonists 2-Me-5-HT and mCPBG (m-chlorophenylbiguanide). These agree well with previously published results (e.g. Hargreaves et al., 1994). The responses were inhibited by the 5-HT₃ receptor antagonists d-tubocurarine, MDL72222, ondansetron and diltiazem at mM concentrations. The results suggest that the FLEXstation is a suitable tool to characterise ligand-gated ion channel responses.

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