Functional Screening of G Protein–Coupled Receptors by Measuring Intracellular Calcium with a Fluorescence Microplate Reader

Abstract

Ligand binding studies reveal information about affinity to G protein—coupled receptors (GPCRs) rather than functional properties. Increase in intracellular Ca2+appears to represent a universal second messenger signal for a majority of recombinant GPCRs. Here, we exploit Ca2+signaling as a fast and sensitive functional screening method for a number of GPCRs coupled to different G proteins. Ca2+fluorescence measurements are performed using Oregon Green 488 BAPTA-1/AM and a microplate reader equipped with an injector. Buffer alone or test compounds dissolved in buffer are injected into a cell suspension, and fluorescence intensity is recorded for 30 s. Each of the GPCRs tested—Gq-coupled P2Y2, Gs-coupled dopamine D1 and D5, Gi-coupled dopamine D2L, and Gq/11-coupled muscarinic acetylcholine M1—yielded a significant rise in intracellular free [Ca2+] on agonist stimulation. Agonist stimulation was dose dependent, as shown for ATP or UTP stimulation of P2Y2receptors (EC50= 1 μM), SKF38393 stimulation of hD1 and hD5 (EC50= 18.1 nM and 2.7 nM), and quinpirole at hD2L (EC50= 6.5 nM). SCH23390 (at hD1 and hD5) and spiperone, haloperidol, and clozapine (at hD2L) competitively antagonized the Ca2+response. Furthermore, the Ca2+assay served to screen suramin analogs for antagonistic activity at P2Y2receptors. Screening at dopamine receptors revealed LE300, a new lead for a dopamine receptor antagonist. Advantages of the assay include fast and simple 96- or 384-well plate format (high-throughput screening), use of a visible light-excitable fluorescent dye, applicability to a majority of GPCRs, and simultaneous analysis of distinct Ca2+fluxes.

α-1 adrenergic receptor agonists modulate ductal secretion of BDL rats via Ca2+- and PKC-dependent stimulation of cAMP

“…Calcium fluorescence measurements in cholangiocytes were performed using fluo-3 AM (Molecular Probes, Eugene, OR) and a Fluoroskan Ascent FL (ThermoLabsystems, Helsinki, Finland) microplate reader equipped with three injectors. 11,27 Cholangiocytes (4 ϫ 10 4 per well) were loaded for 1 hour at room temperature with 5 mol/L of fluo-3 AM in Tyrode's salt solution (137 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl 2 , 0.2 mmol/L NaH 2 PO 4 , 12 mmol/L NaHCO 3 , and 5.5 mmol/L glucose) with 0.1% Pluronic F-127 (Molecular Probes). After washes with Tyrode's salt solution, the loaded cells were added to a 96-well black microplate.…”

Quantitative comparison of functional screening by measuring intracellular Ca2+ with radioligand binding at recombinant human dopamine receptors

“…Recently, a functional assay based on the change in intracellular Ca 2+ upon agonist stimulation was described for differently coupled GPCRs (G s , G, Gq). 11 In this report, it was shown that compounds described in the literature as agonists at D1 and/or D2 receptors are indeed capable of inducing a Ca 2+ signal ( Table 3). The specificity of this signal was proved by antagonist injection, which did not yield a Ca 2+ response (Figure 1 , Figure 2), and by injection of agonists into control cells lacking dopamine receptor expression (HEK293 wildtype cells, data not shown).…”

“…These data suggest that apparent functional and radioligand binding K values are significantly correlated but that an increase in radioligand binding yields a smaller increase in functional effects (measured as Ca D2 receptors (B), respectively. K i of LE300 was estimated as previously described [11]. Apparent functional K i values were obtained by measuring the inhibition of agonist (100nM SKF38393 at D1, 30nM quinpirole at D2 receptors) induced increase in intracellular Ca 2+ after preincubation with the respective test compound.…”

“…12 The functional Ca 2+ assay allows screening at various G s , G, Gq, and Go coupled receptors, among them the recombinantly expressed human dopamine receptors hD1 and hD2L. 11 Further, the assay can be performed in a 384 well plate format, thus allowing a medium-throughput screening. The Ca 2+ assay distinguishes between agonists and antagonists, and yields information about the potency of an agonist (EC50: effective concentration 50%) or an antagonist (IC 50 : inhibitory concentration 50%, or apparent K value: apparent inhibition constant calculated according to the Cheng-Prusoff equation).…”

Regulation of neuromodulator receptor efficacy—implications for whole-neuron and synaptic plasticity

“…GIRK channels) (20), I h and other K+ channels (21), high-voltage gated calcium channels (L-type, P/Q/R-type and N-type calcium channels) (22; 23), and also sodium channels (24; 25). D1 receptors furthermore have the effect of increasing NMDA transmission (26; 27), and may have complex effects on local calcium levels (28).…”