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Aequorin functional assay for characterization of G-protein-coupled receptors: Implementation with cryopreserved transiently transfected cells
Analytical Biochemistry 400 (2010) 184–189
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Aequorin functional assay for characterization of G-protein-coupled receptors: Implementation with cryopreserved transiently transfected cells Bruce Jones a,*, Beverly Holskin a, Sheryl Meyer a, Thao Ung a, Vincent Dupriez b, Sandra Y. Flores b, Emmanuel Burgeon b, Mark Ator a, Emir Duzic a a b
Cephalon, Inc., Worldwide Discovery Research, 145 Brandywine Parkway, West Chester, PA 19380, USA PerkinElmer Life and Analytical Sciences, 8 Imperiastraat, BE-1930 Zaventem, Belgium
a r t i c l e
i n f o
Article history: Received 27 October 2009 Received in revised form 6 January 2010 Accepted 21 January 2010 Available online 28 January 2010 Keywords: Aequorin Transient transfection Muscarinic G-protein-coupled receptor Cryopreserved cells
a b s t r a c t Assay technologies that measure intracellular Ca2+ release are among the predominant methods for evaluation of GPCR function. These measurements have historically been performed using cell-permeable fluorescent dyes, although the use of the recombinant photoprotein aequorin (AEQ) as a Ca2+ sensor has gained popularity with recent advances in instrumentation. The requirement of the AEQ system for cells expressing both the photoprotein and the GPCR target of interest has necessitated the laborintensive development of cell lines stably expressing both proteins. With the goal of streamlining this process, transient transfections were used to either (1) introduce AEQ into cells stably expressing the GPCR of interest or (2) introduce the GPCR into cells stably expressing the AEQ protein, employing the human muscarinic M1 receptor as a model system. Robust results were obtained from cryopreserved cells prepared by both strategies, yielding agonist and antagonist pharmacology in good agreement with literature values. Good reproducibility was observed between multiple transient transfection events. These results indicate that transient transfection is a viable and efficient method for production of cellular reagents for use in AEQ assays. Ó 2010 Elsevier Inc. All rights reserved.
The diversity of processes that are regulated by guanine nucleotide binding proteins (G-proteins) via stimulation of G-proteincoupled receptors (GPCRs)1 defines the critical importance of this class of receptors. Binding events leading to conformational changes are, in part, translated through the heterotrimeric G-proteins, resulting in specific cellular responses [1]. Multiple techniques are available to detect these responses, although functional cell-based approaches [2] are commonly employed since activation of the cellular pathways involves some level of amplification. These functional approaches utilize the coupling of the GPCR and an associated G-protein to affect cellular responses through several second messengers. Measurement of changes in the concentration of the intracellular second messenger Ca2+ is frequently employed as a technique for assay of these targets. Classically, measurement of cytosolic Ca2+ levels utilizes low molecular weight fluorescent probes whose spectral response or intensity of response changes in the presence of Ca2+. On chelation of Ca2+, these dyes emit a fluorescent response that is directly proportional to the concentration of free calcium [3–7]. * Corresponding author. Fax: +1 (610) 738 6305. E-mail address: [email protected] (B. Jones). 1 Abbreviations used: Ach, acetylcholine; AEQ, aequorin; apoAEQ, apoaequorin; FBS, fetal bovine serum; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; GPCRs, G-protein-coupled receptors; hM1R, human M1 muscarinic receptor; IRLU, integrated relative luminescent units; Oxo, oxotremorine; pen/strep, penicillin/ streptomycin. 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.01.028
In recent years, recombinant photoproteins have been employed as an alternative functional technique for measurement of intracellular Ca2+. These calcium-sensitive photoproteins emit luminescent signals in the presence of free Ca2+ with virtually no background signal. Furthermore, by utilizing recombinant approaches photoproteins can be transiently or stably expressed in a range of cellular phenotypes and directed to specific subcellular compartments, thus providing a substantial advantage over dyes to monitor Ca2+ levels [8–10]. Aequorin (AEQ) is a calcium-sensitive photoprotein that was first isolated from the umbrella of the jellyfish Aequorea victoria [11]. The active protein (aequorin) is formed from apoaequorin (apoAEQ) and its prosthetic group, coelenterazine, which covalently binds to the protein in the presence of molecular oxygen [12]. Calcium binding to the holoprotein AEQ induces a conformational shift that results in the oxidation of coelenterazine, yielding coelenteramide. The relaxation of the excited state of coelenteramide bound to apoAEQ results in the flash emission of blue light at 470 nm [11]. AEQ has a low affinity for Ca2+ [13] and large dynamic range, making it an ideal sensor for biological Ca2+ dynamics [14,15]. While the use of AEQ as a Ca2+ probe to study GPCR pharmacology has several clear advantages, in practice the necessity for cell line development has been a drawback to adoption of this technique. In order to use AEQ as a Ca2+ probe, the cell system must
Aequorin, GPCRs and cryopreserved transiently transfected cells / B. Jones et al. / Anal. Biochem. 400 (2010) 184–189
express both the apoAEQ protein and the target GPCR. In general, this has been accomplished by the establishment of stable cell lines, although multiple reports exist with successful signal generation using transient transfections of plasmids or viral vectors coding for apoAEQ [16–18]. Stable cell line development normally requires 6 to 9 months; however, the use of transient transfections to either transfect the apoAEQ protein into receptor-bearing cells or transfect a receptor of interest into cells stably expressing apoAEQ would greatly reduce the time necessary for reagent development. Additionally, using transient transfections would also allow for greater flexibility when working with multiple receptor systems. Herein we describe the use of readily available reagents to transiently transfect human M1 muscarinic receptor (hM1R)-bearing cells with a plasmid coding for the apoAEQ protein and the transfection of hM1R into cells stably expressing the apoAEQ protein. Both methods successfully produced AEQ-responsive cells exhibiting agonist and antagonist pharmacology consistent with muscarinic M1 receptor activation. Materials and methods CHO-K1 cells stably expressing mitochondrially targeted apoAEQ (CHO-A23), the AEQ plasmid (Cat. No. ES-002-AC) which expresses the mitochondrially targeted apoAEQ plasmid under the control of a CMV promoter, CHO-K1 cells stably expressing hM1R (Cat. No. ES-210-C), and the bicistronic expression plasmid (pEFIN5-M1) containing the coding sequence for human muscarinic M1 (GenBank CAA68560.1) receptor were obtained from Perkin Elmer Life Sciences (Waltham, MA). Ham’s F-12 media was obtained from Lonza (Walkersville, MD), fetal bovine serum (FBS) from Thermo Scientific Hyclone (Logan, UT), penicillin/streptomycin (pen/strep), G418 and HEPES from MediaTech (Manassas, VA), Zeocin from Invitrogen (Carlsbad, CA), and carbenicillin from MP Biomedical (Solon, OH). Opti-MEM was purchased from Invitrogen and DMSO is a product of Sigma–Aldrich (St. Louis, MO). Digitonin and the muscarinic ligands acetylcholine, oxotremorine-M, 4diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and atropine were purchased from Sigma–Aldrich. Coelenterazine-h was purchased from Promega (Madison, WI). BSA (fraction V) was purchased from EMD Chemicals (Gibbstown, NJ). Cell culture techniques Strategy 1: Transient transfection of hM1R into CHO-A23 cells stably expressing AEQ CHO-A23 cells were cultured in Ham’s F12 media containing 10% FBS, 1 pen/strep, Zeocin (250 lg/ml) and scaled up to a one-chamber Corning (Lowell, MA) CellSTACK. The pEFIN5-M1 plasmid was transformed into chemically competent DH5a Escherichia coli (Invitrogen) and cryopreserved. For plasmid preparations, a freshly plated colony was scaled up to inoculate 500 ml LB broth with 100 lg/ml carbenicillin and grown at 37 °C with shaking at 300 rpm for 18 h and harvested. The frozen cell pellet was used to prepare endotoxin-free DNA using the EndoFree Mega kit (Qiagen; Valencia, CA) following the manufacturer’s instructions. Transient transfection protocols used Lipofectamine™ 2000 reagent (Invitrogen) as follows. CHO-A23 cells were plated at 8 106 cells/150 mm plate in Ham’s F12 media containing 10% FBS and incubated overnight. The following day hM1R DNA/Lipofectamine™ 2000 complexes were formed at 1:1.2 DNA:Lipofectamine™ 2000. On Day 3, the complexes were removed, fresh complete media were added, and on Day 4 the transfections were harvested as described below.
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Strategy 2: Transient transfection of AEQ into CHO-K1 cells stably expressing hM1R CHO-hM1R cells were cultured in Ham’s F12 media containing 10% FBS, 1 pen/strep, G418 (400 lg/ml) scaled up to a one-chamber Corning CellSTACK. Cells were plated in Ham’s F12 media containing 10% FBS and incubated overnight. The AEQ plasmid was cultured in LB broth containing 35 lg/ml zeocin and endotoxin-free AEQ DNA was used in the transient transfection protocol described above. Harvest Forty-eight hours post transfection, the media were removed, the plates were washed with PBS, and then PBS containing 5 mM EDTA was added to remove cells. The cells were counted and each variation was pooled at 2 107 cells/ml using 10% FBS/90% Ham’s F-12 media. An equal volume of 80% FBS/20% DMSO was added to the cell suspension just prior to freezing in Cryo 1 °C freezing containers (Nalgene) at 80 °C. The following day the vials were placed in vapor phase above liquid nitrogen for storage until used for assay. Aequorin assays Luminescence measures were performed on a FDSS6000 (Hamamatsu; Bridgewater, NJ) reader which is a fully integrated system containing internal liquid handling capacity. Assay buffer consisted of DMEM/Ham’s F-12 50/50 mix, no phenol red containing L-glutamine (MediaTech), 15 mM HEPES, and 0.1% BSA. The functional luminescence-based assay was performed as follows. Frozen cells were removed from liquid nitrogen storage and rapidly thawed in a 37 °C water bath for 2 min. The cells were loaded with coelenterazine-h by washing and adding 15 ml of assay buffer to 1 ml of cells and centrifuged at 276g for 3 min. The pellet was resuspended at 1.7 106 cells/ml of assay buffer containing 5 lM coelenterazine-h. Aliquots of 15 ml of cells were placed into 15-ml conical bottom tubes, completely filling the tube with assay buffer, and incubated overnight for 18 h at 22 °C in the dark with gentle rotation (Labnet H5100; Edison, NJ, 10 rpm) in order to maintain the cells in suspension. The cells were then removed from the rotator and diluted to the final cell concentration with assay buffer at 22 °C. During the assay, cells were maintained in suspension by gentle stirring with a magnetic stir bar incorporated into a cell reservoir as part of the FDSS6000 liquid handling features. For the measurement of agonist-stimulated Ca2+ release, compounds were plated into 384-well plates and introduced into the FDSS6000 reader. After a 10 s basal determination, cells were added to the compound plate at 5000 cells per well. For agonist responses, the light emission was recorded for 90 s and the results reported as integrated relative luminescent units (IRLU; area under the response curve as function of time). For determination of antagonist activity, cells and compound were allowed to incubate for 3 min prior to the addition of an EC80 concentration of agonist. All cell and agonist additions were carried out using the internal liquid handling capacity of the FDSS6000. Digitonin Digitonin, which produces the maximal luminescent signal, was used as a reference response and included for each determination of agonist concentration response curves. The reference response was determined in the presence of 100 lM digitonin with light emission recorded for 90 s and reported as IRLUs. Data analysis Each of the two transfection strategies was repeated on three separate occasions. To evaluate each of these transfections, five
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separate determinations of agonist concentration–response curves as well as concentration–response curves of the inhibition of the EC80 response of agonist were completed. Data reported for each of the three separate transfections were the average for each of the five separate determinations of each individual transfection. All concentration response data were analyzed using GraphPad Prism 5 and values determined using a four-parameter logistic equation for the curve fitting. Agonist activity was reported as percentage of digitonin response obtained with 100 lM digitonin. EC80 was calculated for each concentration–response curve and the average of the five values was determined. This average was used as the EC80 value when determining inhibition by antagonists. To determine if the individual transfection events were similar from event to event, each agonist and antagonist profile was evaluated using a one-way analysis of variance with an a priori alpha level of 0.05. Similar analyses were used for the EC50 and IC50 values as well as the maximum response. Results The pharmacology of each transient transfection condition was characterized by agonist profiles using acetylcholine (ACh) and oxotremorine (Oxo), along with antagonist profiles in which the EC80 responses of ACh and Oxo were antagonized by 4-DAMP and atropine.
separate transfections (Fig. 1) with the relative average EC50 value of the agonists ACh and Oxo determined as 31 ± 8 and 35 ± 9 nM, respectively (Table 1). The difference in magnitude of the EC50 values for the three transfection events was 2.3-fold for ACh and 2.5fold for Oxo (Table 1). Some variability was noted from transfection event to transfection event (Fig. 1). The maximum response for ACh ranged from 10210 to 18147 IRLU, representing a 1.8-fold difference, with an average of 14060 IRLU. For Oxo, the range was 9952 IRLU to 17843 IRLU, representing a 1.8-fold difference, with an average of 13912 IRLU (Table 1). No significant differences were found in the agonist potency or maximum response for either the ACh- or Oxo-stimulated response among the three transfection events. The antagonist activity of 4-DAMP for the inhibition of the EC80 response of ACh was consistent in the three separate transfections (average IC50 value of 1.2 ± 0.2 nM; Table 2), which was slightly more potent than the antagonist activity against atropine (7.2 ± 0.8 nM; Table 2). The data obtained for inhibition of the EC80 response of Oxo were similar to those of ACh, with average IC50 values of 2.0 ± 0.6 and 8.3 ± 3 nM for 4-DAMP and atropine,
Table 1 Strategy 1: Values for the potency and maximum response for acetylcholine- and oxotremorine-stimulated responses in cells stably expressing AEQ and transiently transfected with hM1R. Acetylcholine
Strategy 1: Transient transfection of hM1R into CHO-A23 cells stably expressing AEQ Following transfection of the hM1R plasmid into cells stably expressing AEQ, both ACh and Oxo produced concentration-dependent increases in Ca2+ that were reproducible among the three
TT No. 1 TT No. 2 TT No. 3 Average
Oxotremorine
EC50 (nM)
Max (IRLU)
EC50 (nM)
Max (IRLU)
18 ± 3 33 ± 5 41 ± 11 31 ± 8
18147 ± 2912 10210 ± 3064 13822 ± 3591 14060 ± 2994
20 ± 4 34 ± 6 50 ± 17 35 ± 9
17843 ± 3089 9952 ± 3147 13941 ± 3638 13912 ± 2278
Values are mean ± SEM of five independent experiments.
Fig. 1. Strategy 1: Agonist responses for cells stably expressing apoAEQ protein and transiently transfected with hM1R. Data represent three independent transfections (TT No. 1, TT No. 2, and TT No. 3); with each curve the average of five separate experiments. ACh- (A) and Oxo- (C) stimulated responses are presented as percentage of digitonin response. Both agonists produced concentration-dependent increases in Ca2+ with average EC50 values of 31 ± 12 nM for ACh (A) and 35 ± 15 nM for Oxo (C). As a comparison the actual IRLUs are included for ACh- (B) and Oxo- (D) stimulated responses to illustrate potential variability in the maximal brightness. The maximal response for ACh (B) ranged from 10120 to 18147 IRLU whereas the maximal response for Oxo (D) ranged from 9952 to 17843. There were no significant differences in the maximal brightness among the three separate transient transfections.
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Aequorin, GPCRs and cryopreserved transiently transfected cells / B. Jones et al. / Anal. Biochem. 400 (2010) 184–189 Table 2 Strategy 1: IC50 values for the inhibition of agonist responses in cells stably expressing AEQ and transiently transfected with hM1R. Acetylcholine 4-DAMP (nM) TT No. 1 TT No. 2 TT No. 3 Average
1.5 ± 0.2 1.1 ± 0.1 1.0 ± 0.2 1.2 ± 0.2
Oxotremorine Atropine (nM) 8.7 ± 2 6.1 ± 1 6.8 ± 2 7.2 ± 0.8
4-DAMP (nM) 3.1 ± 0.6 1.5 ± 0.4 1.4 ± 0.3 2.0 ± 0.6
Table 3 Strategy 2: Values for the potency and maximum response for acetylcholine- and oxotremorine-stimulated responses in cells stably expressing hM1R and transiently transfected with AEQ. Acetylcholine
Atropine (nM) 11 ± 3 8.2 ± 2 5.8 ± 1 8.3 ± 3
Values are mean ± SEM of five independent experiments.
TT No. 4 TT No. 5 TT No. 6 Average
Oxotremorine
EC50 (nM)
Max (IRLU)
EC50 (nM)
Max (IRLU)
44 ± 12 36 ± 2 41 ± 2 40 ± 2
27272 ± 5107 46556 ± 6626 36451 ± 4792 36760 ± 5568
32 ± 3 43 ± 2 47 ± 4 41 ± 4
23336 ± 6395 47150 ± 7747 36433 ± 5964 35640 ± 6885
Values are mean ± SEM of five independent experiments.
respectively (Table 2). No significant differences were found in the antagonist potency for inhibition of either the ACh- or Oxo-stimulated response among the three transfection events.
Strategy 2: Transient transfection of AEQ into cells stably expressing M1R The transfection of the AEQ plasmid into cells stably expressing hM1R resulted in concentration-dependent increases in Ca2+ with both ACh and Oxo that were reproducible among the three separate transfections (Fig. 2). The agonists were equipotent with the relative average EC50 value for the three transfections of 40 ± 2 nM for ACh and 41 ± 4 nM for Oxo (Table 3). The differences in magnitude of the EC50 values for the three separate transfection events of 1.2- and 1.5-fold for ACh and Oxo, respectively, were not significant (Table 3). Again, the maximum responses did show some nonsignificant variability from transfection event to transfection event (Fig. 2). The range of responses for ACh was 27272 to 46556 IRLU (1.7-fold difference) with an average of 36760 IRLU. Similarly, the range of responses for Oxo was 23336 to 47150 IRLU
(2.0-fold difference) with an average of 35640 IRLU (Table 3). The inhibition of the EC80 response of ACh by 4-DAMP showed an average IC50 value of 3.4 ± 1 nM for the three transfections and was equipotent to atropine, which produced an average IC50 value of 4.4 ± 2 nM (Table 4). The IC50 values for the inhibition of the Oxo EC80 response were 4.7 ± 2 and 7.5 ± 3 nM for 4-DAMP and atropine, respectively (Table 4). No significant differences were found in the antagonist potency for inhibition of either the ACh- or the Oxo-stimulated response. Digitonin For the transfection of hM1R into AEQ-expressing cells (Strategy 1), the agonist-stimulated response was approximately 45% of the digitonin response, whereas the agonist response for the transfection of AEQ into the hM1R-bearing cells (Strategy 2) was approximately 60% of the digitonin response (Table 5). The maximum brightness, as determined by the digitonin response, for transfection Strategy 2 was approximately twice that of Strategy 1 (Table 5).
Fig. 2. Strategy 2: Agonist responses for cells stably expressing hM1R and transiently transfected with apoAEQ. Data represent three independent transfections (TT No. 4, TT No. 5, and TT No. 6); with each curve the average of five separate experiments. ACh- (A) and Oxo- (C) stimulated responses are presented as percentage of digitonin response. Both agonists produced concentration-dependent increases in Ca2+ with average EC50 values of 40 ± 4 nM for ACh (A) and 41 ± 8 nM for Oxo (C). As a comparison the actual IRLUs are included for ACh- (B) and Oxo- (D) stimulated responses to illustrate potential variability in the maximal brightness. The maximal response for ACh (B) ranged from 27272 to 46556 IRLU, whereas the maximal response for Oxo (D) ranged from 23336 to 47150. There were no significant differences in the maximal brightness among the three separate transient transfections.
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Table 4 Strategy 2: IC50 values for the inhibition of acetylcholine and oxotremorine responses in cells stably expressing hM1R and transiently transfected with AEQ. Acetylcholine
TT No. 4 TT No. 5 TT No. 6 Average
Oxotremorine
4-DAMP (nM)
Atropine (nM)
4-DAMP (nM)
Atropine (nM)
5.4 ± 2 2.4 ± 0.8 2.4 ± 0.5 4.3 ± 1
8.5 ± 3 2.7 ± 0.4 2.1 ± 0.4 4.4 ± 2
8.8 ± 3 2.8 ± 0.7 2.6 ± 0.9 4.7 ± 2
13 ± 3 6.3 ± 2 3.3 ± 1 7.5 ± 3
Values are mean ± SEM of five independent experiments.
Table 5 Maximal stimulated response for digitonin, acetylcholine, and oxotremorine.
Digitonin ACh Oxo
Strategy 1 (hM1R into stable AEQ cells)
Strategy 2 (AEQ into hM1Rbearing cells)
27437 ± 4629 12204 ± 1482 12090 ± 2064
52488 ± 10022 34125 ± 6714 31142 ± 5824
Values are mean ± SEM (IRLU) of the three individual transfections within each transfection strategy.
Discussion The role of Ca2+ as a second messenger is well understood. Likewise, the use of transient transfection technology is commonplace for the study of receptor function as well as calcium flux. The results presented above describe the successful use of cryopreserved transiently transfected cells and the more recently developed AEQ technology platform for the measurement of GPCR-mediated increases in intracellular Ca2+ [19–21]. The use of AEQ as a biosensor for the measurement of intracellular Ca2+ release has been well established [3,11,22–26]. With the cloning of apoAEQ cDNA [27,28], certain advantages of AEQ technology compared to the Ca2+-sensitive fluorescent dyes have emerged. Most important is the ability to measure intracellular Ca2+ in specific subcellular locations without background signal, toxicity, or potential to alter the Ca2+ dynamics. Likewise there are methodological advantages provided by use of AEQ that allow an increase of throughput with a decrease in reagent cost in comparison to the fluorescent dyes [21]. While the AEQ technology has some substantial advantages over fluorescent dye assays, it does require the use of cells expressing both the target of interest and the apoAEQ protein. The commercial availability of both the apoAEQ plasmid and the parental cells expressing apoAEQ provides an opportunity for the rapid development of reagents using transient transfections. Since both the plasmid and the cells are available, a strategy of either (1) transfecting the target of interest into cells expressing the apoAEQ or (2) transfecting the apoAEQ plasmid into cells expressing the target of interest could be employed. The present work demonstrates successful application of both strategies. The muscarinic M1 receptor is coupled to a pertussis toxininsensitive G protein of the Gaq family. Utilizing this system to examine the utility of combining transient transfections and the AEQ technology platform as a means for developing reagents has the advantages of direct Gaq coupling along with a well-characterized molecular pharmacology literature. The present study examined two strategies: the first (Strategy 1) involved transiently transfecting the hM1R into CHO-K1 cells stably expressing the apoAEQ protein, while the second (Strategy 2) utilized transient transfection of the AEQ plasmid into CHO-K1 cells stably expressing hM1R. The pharmacology was characterized by the use of the endogenous agonist ACh and the synthetic alkaloid agonist Oxo along with the antagonists 4-DAMP and atropine. For both trans-
fection conditions, ACh and Oxo provided concentration-dependent increases in calcium release. The EC50 values for both ACh and Oxo were similar in both transfection strategies (31 ± 8 and 35 ± 9 nM, respectively, for Strategy 1, and 40 ± 2 and 41 ± 4 nM, respectively, for Strategy 2). These values are well within the range of previously reported values for functional assay systems [6,29– 35] and compare well to the values obtained using other luminometers and plate formats on a double stable CHO-AEQ-M1 cell line (EC50 = 11.6 ± 4 nM for ACh and 7.2 ± 2 nM for Oxo; PerkinElmer, data not shown). The antagonists 4-DAMP and atropine were used to inhibit the EC80 response of both ACh and Oxo. For Strategy 1 (hM1R into stable AEQ cells), both 4-DAMP and atropine produced concentration-dependent inhibition of the ACh- and Oxo-induced Ca2+ flux consistent with previously reported literature values [6,35–39]. The 4-DAMP inhibition was slightly more potent than atropine on the ACh-mediated response (IC50 = 1.2 ± 0.2 and 7.2 ± 0.8 nM, respectively) as well as the Oxo-mediated response (IC50 = 2.0 ± 0.6 and 8.3 ± 3 nM, respectively). For Strategy 2 (AEQ plasmid into hM1R stables), both 4-DAMP and atropine again produced concentration-dependent inhibition of the Ca2+ flux consistent with literature values. In this case there was little difference in the inhibition values obtained with either antagonist for inhibition of the ACh or Oxo induced Ca2+ flux. There was no significant difference in the results obtained between the two different transfection strategies, supporting the suggestion that employing either strategy would yield appropriate pharmacological systems. As with most functional assays, the agonist-stimulated maximal response can vary from experiment to experiment as is the case herein. Modern reagents have increased the reproducibility of the transient transfection, but this technique still has inherent variability and is most probably the major source of observed experiment-to-experiment variations. As a consequence, the use of appropriate internal controls such as reference agonists and digitonin is recommended. For this study digitonin was included as the internal control. Digitonin is a glycoside obtained from Digitalis purporea that effectively water-solubilizes lipids, thus permeabilizing membranes. In doing so, digitonin allows influx of Ca2+ from the buffer, exposing all the available aequorin to Ca2+. This in turn causes the maximum possible luminescence (or maximal brightness) achievable by the cells to occur. For both strategies, the average maximal response produced by agonist stimulation was less than the maximal brightness obtainable with digitonin, indicating that agonist stimulation of hM1R did not lead to full consumption of the calcium tracer in the cells. Transient transfection of AEQ into the hM1R-bearing cells (Strategy 2) produced a maximal digitonin induced brightness that was nearly twofold higher than that obtained from cells prepared by the transfection of hM1R into the AEQ-expressing cells (Strategy 1). Likewise the average maximal agonist-stimulated response was greater in Strategy 2 compared to Strategy 1. There was nearly a 3fold increase in the agonist-stimulated response for Strategy 2 compared to Strategy 1. This is consistent with a greater expression of apoAEQ protein in the transient transfection compared to stables. The slightly lower ratio of agonist to digitonin response in Strategy 1 could be explained by the persistence of cells stably expressing apoAEQ that were not transfected by hM1R in the cell population, as usually occurs with the currently available transient transfection techniques. In contrast, Strategy 2 produces a cell population in which only the transfected cells are able to generate a luminescent signal. The ratio of agonist to digitonin response is therefore higher, since the nontransfected cells do not contribute to the digitonin signal. It is also noteworthy that all of these data were generated with cryopreserved ‘‘assay-ready” cells. This is becoming the norm for delivering stable cell lines for functional cell-based assays, but reports with cryopreserved transiently transfected cells are still
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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Aequorin functional assay for characterization of G-protein-coupled receptors: Implementation with cryopreserved transiently transfected cells Bruce Jones a,*, Beverly Holskin a, Sheryl Meyer a, Thao Ung a, Vincent Dupriez b, Sandra Y. Flores b, Emmanuel Burgeon b, Mark Ator a, Emir Duzic a a b
Cephalon, Inc., Worldwide Discovery Research, 145 Brandywine Parkway, West Chester, PA 19380, USA PerkinElmer Life and Analytical Sciences, 8 Imperiastraat, BE-1930 Zaventem, Belgium
a r t i c l e
i n f o
Article history: Received 27 October 2009 Received in revised form 6 January 2010 Accepted 21 January 2010 Available online 28 January 2010 Keywords: Aequorin Transient transfection Muscarinic G-protein-coupled receptor Cryopreserved cells
a b s t r a c t Assay technologies that measure intracellular Ca2+ release are among the predominant methods for evaluation of GPCR function. These measurements have historically been performed using cell-permeable fluorescent dyes, although the use of the recombinant photoprotein aequorin (AEQ) as a Ca2+ sensor has gained popularity with recent advances in instrumentation. The requirement of the AEQ system for cells expressing both the photoprotein and the GPCR target of interest has necessitated the laborintensive development of cell lines stably expressing both proteins. With the goal of streamlining this process, transient transfections were used to either (1) introduce AEQ into cells stably expressing the GPCR of interest or (2) introduce the GPCR into cells stably expressing the AEQ protein, employing the human muscarinic M1 receptor as a model system. Robust results were obtained from cryopreserved cells prepared by both strategies, yielding agonist and antagonist pharmacology in good agreement with literature values. Good reproducibility was observed between multiple transient transfection events. These results indicate that transient transfection is a viable and efficient method for production of cellular reagents for use in AEQ assays. Ó 2010 Elsevier Inc. All rights reserved.
The diversity of processes that are regulated by guanine nucleotide binding proteins (G-proteins) via stimulation of G-proteincoupled receptors (GPCRs)1 defines the critical importance of this class of receptors. Binding events leading to conformational changes are, in part, translated through the heterotrimeric G-proteins, resulting in specific cellular responses [1]. Multiple techniques are available to detect these responses, although functional cell-based approaches [2] are commonly employed since activation of the cellular pathways involves some level of amplification. These functional approaches utilize the coupling of the GPCR and an associated G-protein to affect cellular responses through several second messengers. Measurement of changes in the concentration of the intracellular second messenger Ca2+ is frequently employed as a technique for assay of these targets. Classically, measurement of cytosolic Ca2+ levels utilizes low molecular weight fluorescent probes whose spectral response or intensity of response changes in the presence of Ca2+. On chelation of Ca2+, these dyes emit a fluorescent response that is directly proportional to the concentration of free calcium [3–7]. * Corresponding author. Fax: +1 (610) 738 6305. E-mail address: [email protected] (B. Jones). 1 Abbreviations used: Ach, acetylcholine; AEQ, aequorin; apoAEQ, apoaequorin; FBS, fetal bovine serum; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; GPCRs, G-protein-coupled receptors; hM1R, human M1 muscarinic receptor; IRLU, integrated relative luminescent units; Oxo, oxotremorine; pen/strep, penicillin/ streptomycin. 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.01.028
In recent years, recombinant photoproteins have been employed as an alternative functional technique for measurement of intracellular Ca2+. These calcium-sensitive photoproteins emit luminescent signals in the presence of free Ca2+ with virtually no background signal. Furthermore, by utilizing recombinant approaches photoproteins can be transiently or stably expressed in a range of cellular phenotypes and directed to specific subcellular compartments, thus providing a substantial advantage over dyes to monitor Ca2+ levels [8–10]. Aequorin (AEQ) is a calcium-sensitive photoprotein that was first isolated from the umbrella of the jellyfish Aequorea victoria [11]. The active protein (aequorin) is formed from apoaequorin (apoAEQ) and its prosthetic group, coelenterazine, which covalently binds to the protein in the presence of molecular oxygen [12]. Calcium binding to the holoprotein AEQ induces a conformational shift that results in the oxidation of coelenterazine, yielding coelenteramide. The relaxation of the excited state of coelenteramide bound to apoAEQ results in the flash emission of blue light at 470 nm [11]. AEQ has a low affinity for Ca2+ [13] and large dynamic range, making it an ideal sensor for biological Ca2+ dynamics [14,15]. While the use of AEQ as a Ca2+ probe to study GPCR pharmacology has several clear advantages, in practice the necessity for cell line development has been a drawback to adoption of this technique. In order to use AEQ as a Ca2+ probe, the cell system must
Aequorin, GPCRs and cryopreserved transiently transfected cells / B. Jones et al. / Anal. Biochem. 400 (2010) 184–189
express both the apoAEQ protein and the target GPCR. In general, this has been accomplished by the establishment of stable cell lines, although multiple reports exist with successful signal generation using transient transfections of plasmids or viral vectors coding for apoAEQ [16–18]. Stable cell line development normally requires 6 to 9 months; however, the use of transient transfections to either transfect the apoAEQ protein into receptor-bearing cells or transfect a receptor of interest into cells stably expressing apoAEQ would greatly reduce the time necessary for reagent development. Additionally, using transient transfections would also allow for greater flexibility when working with multiple receptor systems. Herein we describe the use of readily available reagents to transiently transfect human M1 muscarinic receptor (hM1R)-bearing cells with a plasmid coding for the apoAEQ protein and the transfection of hM1R into cells stably expressing the apoAEQ protein. Both methods successfully produced AEQ-responsive cells exhibiting agonist and antagonist pharmacology consistent with muscarinic M1 receptor activation. Materials and methods CHO-K1 cells stably expressing mitochondrially targeted apoAEQ (CHO-A23), the AEQ plasmid (Cat. No. ES-002-AC) which expresses the mitochondrially targeted apoAEQ plasmid under the control of a CMV promoter, CHO-K1 cells stably expressing hM1R (Cat. No. ES-210-C), and the bicistronic expression plasmid (pEFIN5-M1) containing the coding sequence for human muscarinic M1 (GenBank CAA68560.1) receptor were obtained from Perkin Elmer Life Sciences (Waltham, MA). Ham’s F-12 media was obtained from Lonza (Walkersville, MD), fetal bovine serum (FBS) from Thermo Scientific Hyclone (Logan, UT), penicillin/streptomycin (pen/strep), G418 and HEPES from MediaTech (Manassas, VA), Zeocin from Invitrogen (Carlsbad, CA), and carbenicillin from MP Biomedical (Solon, OH). Opti-MEM was purchased from Invitrogen and DMSO is a product of Sigma–Aldrich (St. Louis, MO). Digitonin and the muscarinic ligands acetylcholine, oxotremorine-M, 4diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and atropine were purchased from Sigma–Aldrich. Coelenterazine-h was purchased from Promega (Madison, WI). BSA (fraction V) was purchased from EMD Chemicals (Gibbstown, NJ). Cell culture techniques Strategy 1: Transient transfection of hM1R into CHO-A23 cells stably expressing AEQ CHO-A23 cells were cultured in Ham’s F12 media containing 10% FBS, 1 pen/strep, Zeocin (250 lg/ml) and scaled up to a one-chamber Corning (Lowell, MA) CellSTACK. The pEFIN5-M1 plasmid was transformed into chemically competent DH5a Escherichia coli (Invitrogen) and cryopreserved. For plasmid preparations, a freshly plated colony was scaled up to inoculate 500 ml LB broth with 100 lg/ml carbenicillin and grown at 37 °C with shaking at 300 rpm for 18 h and harvested. The frozen cell pellet was used to prepare endotoxin-free DNA using the EndoFree Mega kit (Qiagen; Valencia, CA) following the manufacturer’s instructions. Transient transfection protocols used Lipofectamine™ 2000 reagent (Invitrogen) as follows. CHO-A23 cells were plated at 8 106 cells/150 mm plate in Ham’s F12 media containing 10% FBS and incubated overnight. The following day hM1R DNA/Lipofectamine™ 2000 complexes were formed at 1:1.2 DNA:Lipofectamine™ 2000. On Day 3, the complexes were removed, fresh complete media were added, and on Day 4 the transfections were harvested as described below.
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Strategy 2: Transient transfection of AEQ into CHO-K1 cells stably expressing hM1R CHO-hM1R cells were cultured in Ham’s F12 media containing 10% FBS, 1 pen/strep, G418 (400 lg/ml) scaled up to a one-chamber Corning CellSTACK. Cells were plated in Ham’s F12 media containing 10% FBS and incubated overnight. The AEQ plasmid was cultured in LB broth containing 35 lg/ml zeocin and endotoxin-free AEQ DNA was used in the transient transfection protocol described above. Harvest Forty-eight hours post transfection, the media were removed, the plates were washed with PBS, and then PBS containing 5 mM EDTA was added to remove cells. The cells were counted and each variation was pooled at 2 107 cells/ml using 10% FBS/90% Ham’s F-12 media. An equal volume of 80% FBS/20% DMSO was added to the cell suspension just prior to freezing in Cryo 1 °C freezing containers (Nalgene) at 80 °C. The following day the vials were placed in vapor phase above liquid nitrogen for storage until used for assay. Aequorin assays Luminescence measures were performed on a FDSS6000 (Hamamatsu; Bridgewater, NJ) reader which is a fully integrated system containing internal liquid handling capacity. Assay buffer consisted of DMEM/Ham’s F-12 50/50 mix, no phenol red containing L-glutamine (MediaTech), 15 mM HEPES, and 0.1% BSA. The functional luminescence-based assay was performed as follows. Frozen cells were removed from liquid nitrogen storage and rapidly thawed in a 37 °C water bath for 2 min. The cells were loaded with coelenterazine-h by washing and adding 15 ml of assay buffer to 1 ml of cells and centrifuged at 276g for 3 min. The pellet was resuspended at 1.7 106 cells/ml of assay buffer containing 5 lM coelenterazine-h. Aliquots of 15 ml of cells were placed into 15-ml conical bottom tubes, completely filling the tube with assay buffer, and incubated overnight for 18 h at 22 °C in the dark with gentle rotation (Labnet H5100; Edison, NJ, 10 rpm) in order to maintain the cells in suspension. The cells were then removed from the rotator and diluted to the final cell concentration with assay buffer at 22 °C. During the assay, cells were maintained in suspension by gentle stirring with a magnetic stir bar incorporated into a cell reservoir as part of the FDSS6000 liquid handling features. For the measurement of agonist-stimulated Ca2+ release, compounds were plated into 384-well plates and introduced into the FDSS6000 reader. After a 10 s basal determination, cells were added to the compound plate at 5000 cells per well. For agonist responses, the light emission was recorded for 90 s and the results reported as integrated relative luminescent units (IRLU; area under the response curve as function of time). For determination of antagonist activity, cells and compound were allowed to incubate for 3 min prior to the addition of an EC80 concentration of agonist. All cell and agonist additions were carried out using the internal liquid handling capacity of the FDSS6000. Digitonin Digitonin, which produces the maximal luminescent signal, was used as a reference response and included for each determination of agonist concentration response curves. The reference response was determined in the presence of 100 lM digitonin with light emission recorded for 90 s and reported as IRLUs. Data analysis Each of the two transfection strategies was repeated on three separate occasions. To evaluate each of these transfections, five
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separate determinations of agonist concentration–response curves as well as concentration–response curves of the inhibition of the EC80 response of agonist were completed. Data reported for each of the three separate transfections were the average for each of the five separate determinations of each individual transfection. All concentration response data were analyzed using GraphPad Prism 5 and values determined using a four-parameter logistic equation for the curve fitting. Agonist activity was reported as percentage of digitonin response obtained with 100 lM digitonin. EC80 was calculated for each concentration–response curve and the average of the five values was determined. This average was used as the EC80 value when determining inhibition by antagonists. To determine if the individual transfection events were similar from event to event, each agonist and antagonist profile was evaluated using a one-way analysis of variance with an a priori alpha level of 0.05. Similar analyses were used for the EC50 and IC50 values as well as the maximum response. Results The pharmacology of each transient transfection condition was characterized by agonist profiles using acetylcholine (ACh) and oxotremorine (Oxo), along with antagonist profiles in which the EC80 responses of ACh and Oxo were antagonized by 4-DAMP and atropine.
separate transfections (Fig. 1) with the relative average EC50 value of the agonists ACh and Oxo determined as 31 ± 8 and 35 ± 9 nM, respectively (Table 1). The difference in magnitude of the EC50 values for the three transfection events was 2.3-fold for ACh and 2.5fold for Oxo (Table 1). Some variability was noted from transfection event to transfection event (Fig. 1). The maximum response for ACh ranged from 10210 to 18147 IRLU, representing a 1.8-fold difference, with an average of 14060 IRLU. For Oxo, the range was 9952 IRLU to 17843 IRLU, representing a 1.8-fold difference, with an average of 13912 IRLU (Table 1). No significant differences were found in the agonist potency or maximum response for either the ACh- or Oxo-stimulated response among the three transfection events. The antagonist activity of 4-DAMP for the inhibition of the EC80 response of ACh was consistent in the three separate transfections (average IC50 value of 1.2 ± 0.2 nM; Table 2), which was slightly more potent than the antagonist activity against atropine (7.2 ± 0.8 nM; Table 2). The data obtained for inhibition of the EC80 response of Oxo were similar to those of ACh, with average IC50 values of 2.0 ± 0.6 and 8.3 ± 3 nM for 4-DAMP and atropine,
Table 1 Strategy 1: Values for the potency and maximum response for acetylcholine- and oxotremorine-stimulated responses in cells stably expressing AEQ and transiently transfected with hM1R. Acetylcholine
Strategy 1: Transient transfection of hM1R into CHO-A23 cells stably expressing AEQ Following transfection of the hM1R plasmid into cells stably expressing AEQ, both ACh and Oxo produced concentration-dependent increases in Ca2+ that were reproducible among the three
TT No. 1 TT No. 2 TT No. 3 Average
Oxotremorine
EC50 (nM)
Max (IRLU)
EC50 (nM)
Max (IRLU)
18 ± 3 33 ± 5 41 ± 11 31 ± 8
18147 ± 2912 10210 ± 3064 13822 ± 3591 14060 ± 2994
20 ± 4 34 ± 6 50 ± 17 35 ± 9
17843 ± 3089 9952 ± 3147 13941 ± 3638 13912 ± 2278
Values are mean ± SEM of five independent experiments.
Fig. 1. Strategy 1: Agonist responses for cells stably expressing apoAEQ protein and transiently transfected with hM1R. Data represent three independent transfections (TT No. 1, TT No. 2, and TT No. 3); with each curve the average of five separate experiments. ACh- (A) and Oxo- (C) stimulated responses are presented as percentage of digitonin response. Both agonists produced concentration-dependent increases in Ca2+ with average EC50 values of 31 ± 12 nM for ACh (A) and 35 ± 15 nM for Oxo (C). As a comparison the actual IRLUs are included for ACh- (B) and Oxo- (D) stimulated responses to illustrate potential variability in the maximal brightness. The maximal response for ACh (B) ranged from 10120 to 18147 IRLU whereas the maximal response for Oxo (D) ranged from 9952 to 17843. There were no significant differences in the maximal brightness among the three separate transient transfections.
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Aequorin, GPCRs and cryopreserved transiently transfected cells / B. Jones et al. / Anal. Biochem. 400 (2010) 184–189 Table 2 Strategy 1: IC50 values for the inhibition of agonist responses in cells stably expressing AEQ and transiently transfected with hM1R. Acetylcholine 4-DAMP (nM) TT No. 1 TT No. 2 TT No. 3 Average
1.5 ± 0.2 1.1 ± 0.1 1.0 ± 0.2 1.2 ± 0.2
Oxotremorine Atropine (nM) 8.7 ± 2 6.1 ± 1 6.8 ± 2 7.2 ± 0.8
4-DAMP (nM) 3.1 ± 0.6 1.5 ± 0.4 1.4 ± 0.3 2.0 ± 0.6
Table 3 Strategy 2: Values for the potency and maximum response for acetylcholine- and oxotremorine-stimulated responses in cells stably expressing hM1R and transiently transfected with AEQ. Acetylcholine
Atropine (nM) 11 ± 3 8.2 ± 2 5.8 ± 1 8.3 ± 3
Values are mean ± SEM of five independent experiments.
TT No. 4 TT No. 5 TT No. 6 Average
Oxotremorine
EC50 (nM)
Max (IRLU)
EC50 (nM)
Max (IRLU)
44 ± 12 36 ± 2 41 ± 2 40 ± 2
27272 ± 5107 46556 ± 6626 36451 ± 4792 36760 ± 5568
32 ± 3 43 ± 2 47 ± 4 41 ± 4
23336 ± 6395 47150 ± 7747 36433 ± 5964 35640 ± 6885
Values are mean ± SEM of five independent experiments.
respectively (Table 2). No significant differences were found in the antagonist potency for inhibition of either the ACh- or Oxo-stimulated response among the three transfection events.
Strategy 2: Transient transfection of AEQ into cells stably expressing M1R The transfection of the AEQ plasmid into cells stably expressing hM1R resulted in concentration-dependent increases in Ca2+ with both ACh and Oxo that were reproducible among the three separate transfections (Fig. 2). The agonists were equipotent with the relative average EC50 value for the three transfections of 40 ± 2 nM for ACh and 41 ± 4 nM for Oxo (Table 3). The differences in magnitude of the EC50 values for the three separate transfection events of 1.2- and 1.5-fold for ACh and Oxo, respectively, were not significant (Table 3). Again, the maximum responses did show some nonsignificant variability from transfection event to transfection event (Fig. 2). The range of responses for ACh was 27272 to 46556 IRLU (1.7-fold difference) with an average of 36760 IRLU. Similarly, the range of responses for Oxo was 23336 to 47150 IRLU
(2.0-fold difference) with an average of 35640 IRLU (Table 3). The inhibition of the EC80 response of ACh by 4-DAMP showed an average IC50 value of 3.4 ± 1 nM for the three transfections and was equipotent to atropine, which produced an average IC50 value of 4.4 ± 2 nM (Table 4). The IC50 values for the inhibition of the Oxo EC80 response were 4.7 ± 2 and 7.5 ± 3 nM for 4-DAMP and atropine, respectively (Table 4). No significant differences were found in the antagonist potency for inhibition of either the ACh- or the Oxo-stimulated response. Digitonin For the transfection of hM1R into AEQ-expressing cells (Strategy 1), the agonist-stimulated response was approximately 45% of the digitonin response, whereas the agonist response for the transfection of AEQ into the hM1R-bearing cells (Strategy 2) was approximately 60% of the digitonin response (Table 5). The maximum brightness, as determined by the digitonin response, for transfection Strategy 2 was approximately twice that of Strategy 1 (Table 5).
Fig. 2. Strategy 2: Agonist responses for cells stably expressing hM1R and transiently transfected with apoAEQ. Data represent three independent transfections (TT No. 4, TT No. 5, and TT No. 6); with each curve the average of five separate experiments. ACh- (A) and Oxo- (C) stimulated responses are presented as percentage of digitonin response. Both agonists produced concentration-dependent increases in Ca2+ with average EC50 values of 40 ± 4 nM for ACh (A) and 41 ± 8 nM for Oxo (C). As a comparison the actual IRLUs are included for ACh- (B) and Oxo- (D) stimulated responses to illustrate potential variability in the maximal brightness. The maximal response for ACh (B) ranged from 27272 to 46556 IRLU, whereas the maximal response for Oxo (D) ranged from 23336 to 47150. There were no significant differences in the maximal brightness among the three separate transient transfections.
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Table 4 Strategy 2: IC50 values for the inhibition of acetylcholine and oxotremorine responses in cells stably expressing hM1R and transiently transfected with AEQ. Acetylcholine
TT No. 4 TT No. 5 TT No. 6 Average
Oxotremorine
4-DAMP (nM)
Atropine (nM)
4-DAMP (nM)
Atropine (nM)
5.4 ± 2 2.4 ± 0.8 2.4 ± 0.5 4.3 ± 1
8.5 ± 3 2.7 ± 0.4 2.1 ± 0.4 4.4 ± 2
8.8 ± 3 2.8 ± 0.7 2.6 ± 0.9 4.7 ± 2
13 ± 3 6.3 ± 2 3.3 ± 1 7.5 ± 3
Values are mean ± SEM of five independent experiments.
Table 5 Maximal stimulated response for digitonin, acetylcholine, and oxotremorine.
Digitonin ACh Oxo
Strategy 1 (hM1R into stable AEQ cells)
Strategy 2 (AEQ into hM1Rbearing cells)
27437 ± 4629 12204 ± 1482 12090 ± 2064
52488 ± 10022 34125 ± 6714 31142 ± 5824
Values are mean ± SEM (IRLU) of the three individual transfections within each transfection strategy.
Discussion The role of Ca2+ as a second messenger is well understood. Likewise, the use of transient transfection technology is commonplace for the study of receptor function as well as calcium flux. The results presented above describe the successful use of cryopreserved transiently transfected cells and the more recently developed AEQ technology platform for the measurement of GPCR-mediated increases in intracellular Ca2+ [19–21]. The use of AEQ as a biosensor for the measurement of intracellular Ca2+ release has been well established [3,11,22–26]. With the cloning of apoAEQ cDNA [27,28], certain advantages of AEQ technology compared to the Ca2+-sensitive fluorescent dyes have emerged. Most important is the ability to measure intracellular Ca2+ in specific subcellular locations without background signal, toxicity, or potential to alter the Ca2+ dynamics. Likewise there are methodological advantages provided by use of AEQ that allow an increase of throughput with a decrease in reagent cost in comparison to the fluorescent dyes [21]. While the AEQ technology has some substantial advantages over fluorescent dye assays, it does require the use of cells expressing both the target of interest and the apoAEQ protein. The commercial availability of both the apoAEQ plasmid and the parental cells expressing apoAEQ provides an opportunity for the rapid development of reagents using transient transfections. Since both the plasmid and the cells are available, a strategy of either (1) transfecting the target of interest into cells expressing the apoAEQ or (2) transfecting the apoAEQ plasmid into cells expressing the target of interest could be employed. The present work demonstrates successful application of both strategies. The muscarinic M1 receptor is coupled to a pertussis toxininsensitive G protein of the Gaq family. Utilizing this system to examine the utility of combining transient transfections and the AEQ technology platform as a means for developing reagents has the advantages of direct Gaq coupling along with a well-characterized molecular pharmacology literature. The present study examined two strategies: the first (Strategy 1) involved transiently transfecting the hM1R into CHO-K1 cells stably expressing the apoAEQ protein, while the second (Strategy 2) utilized transient transfection of the AEQ plasmid into CHO-K1 cells stably expressing hM1R. The pharmacology was characterized by the use of the endogenous agonist ACh and the synthetic alkaloid agonist Oxo along with the antagonists 4-DAMP and atropine. For both trans-
fection conditions, ACh and Oxo provided concentration-dependent increases in calcium release. The EC50 values for both ACh and Oxo were similar in both transfection strategies (31 ± 8 and 35 ± 9 nM, respectively, for Strategy 1, and 40 ± 2 and 41 ± 4 nM, respectively, for Strategy 2). These values are well within the range of previously reported values for functional assay systems [6,29– 35] and compare well to the values obtained using other luminometers and plate formats on a double stable CHO-AEQ-M1 cell line (EC50 = 11.6 ± 4 nM for ACh and 7.2 ± 2 nM for Oxo; PerkinElmer, data not shown). The antagonists 4-DAMP and atropine were used to inhibit the EC80 response of both ACh and Oxo. For Strategy 1 (hM1R into stable AEQ cells), both 4-DAMP and atropine produced concentration-dependent inhibition of the ACh- and Oxo-induced Ca2+ flux consistent with previously reported literature values [6,35–39]. The 4-DAMP inhibition was slightly more potent than atropine on the ACh-mediated response (IC50 = 1.2 ± 0.2 and 7.2 ± 0.8 nM, respectively) as well as the Oxo-mediated response (IC50 = 2.0 ± 0.6 and 8.3 ± 3 nM, respectively). For Strategy 2 (AEQ plasmid into hM1R stables), both 4-DAMP and atropine again produced concentration-dependent inhibition of the Ca2+ flux consistent with literature values. In this case there was little difference in the inhibition values obtained with either antagonist for inhibition of the ACh or Oxo induced Ca2+ flux. There was no significant difference in the results obtained between the two different transfection strategies, supporting the suggestion that employing either strategy would yield appropriate pharmacological systems. As with most functional assays, the agonist-stimulated maximal response can vary from experiment to experiment as is the case herein. Modern reagents have increased the reproducibility of the transient transfection, but this technique still has inherent variability and is most probably the major source of observed experiment-to-experiment variations. As a consequence, the use of appropriate internal controls such as reference agonists and digitonin is recommended. For this study digitonin was included as the internal control. Digitonin is a glycoside obtained from Digitalis purporea that effectively water-solubilizes lipids, thus permeabilizing membranes. In doing so, digitonin allows influx of Ca2+ from the buffer, exposing all the available aequorin to Ca2+. This in turn causes the maximum possible luminescence (or maximal brightness) achievable by the cells to occur. For both strategies, the average maximal response produced by agonist stimulation was less than the maximal brightness obtainable with digitonin, indicating that agonist stimulation of hM1R did not lead to full consumption of the calcium tracer in the cells. Transient transfection of AEQ into the hM1R-bearing cells (Strategy 2) produced a maximal digitonin induced brightness that was nearly twofold higher than that obtained from cells prepared by the transfection of hM1R into the AEQ-expressing cells (Strategy 1). Likewise the average maximal agonist-stimulated response was greater in Strategy 2 compared to Strategy 1. There was nearly a 3fold increase in the agonist-stimulated response for Strategy 2 compared to Strategy 1. This is consistent with a greater expression of apoAEQ protein in the transient transfection compared to stables. The slightly lower ratio of agonist to digitonin response in Strategy 1 could be explained by the persistence of cells stably expressing apoAEQ that were not transfected by hM1R in the cell population, as usually occurs with the currently available transient transfection techniques. In contrast, Strategy 2 produces a cell population in which only the transfected cells are able to generate a luminescent signal. The ratio of agonist to digitonin response is therefore higher, since the nontransfected cells do not contribute to the digitonin signal. It is also noteworthy that all of these data were generated with cryopreserved ‘‘assay-ready” cells. This is becoming the norm for delivering stable cell lines for functional cell-based assays, but reports with cryopreserved transiently transfected cells are still
Aequorin, GPCRs and cryopreserved transiently transfected cells / B. Jones et al. / Anal. Biochem. 400 (2010) 184–189
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