Abstract
Human 5-HT7A receptors positively modulated adenylyl cyclases via Gs subtypes of G proteins in human embryonic kidney 293 cells, and bound 5-hydroxytryptamine (HT) with high and low affinity (K I values of 1.5 ± 0.3 and 93 ± 4 nM). More than 60% of 5-HT7A receptors, however, displayed the high-affinity 5-HT binding with no sensitivity to 5′-guanylylimidodiphosphate. In this study, we found that select amphipathic agents affected the high-affinity 5-HT binding to 5-HT7A. Oleic acid at low concentrations (<15 μM), but not palmitic, stearic, and arachidonic acids, increased maximal [3H]5-HT binding without affecting itsK D value and [3H]mesulergine (antagonist) binding. Fatty acid-free bovine serum albumin (FF-BSA), a scavenger of fatty acids and lipid metabolites, substantially reduced maximal [3H]5-HT binding (no change inK D value and antagonist binding) but lost its action upon treatment with inactive stearic acid. FF-BSA and oleic acid produced no appreciable effects on [3H]5-HT binding to analogous 5-HT receptors 5-HT1D and 5-HT2C. Among various lysophospholipids, lysophosphatidyl choline (50 μM) decreased maximal [3H]5-HT binding, and a similar zwitterion, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; 0.1%), increased it (no change inK D). Functionally, 5-HT–induced guanosine-5′-O-(3-[35S]thio)triphosphate (GTPγ35S) binding was enhanced by oleic acid and CHAPS, but reduced by FF-BSA and lysophosphatidyl choline; the amphipathic agents and FF-BSA did not affect dopamine-induced GTPγ35S binding at D1, a prototypic Gs-coupled receptor. At 5-HT7A, oleic acid, FF-BSA, CHAPS, and lysophosphatidyl choline also brought about corresponding changes in the half-maximal 5-HT concentration for cAMP production, without affecting the maximal and basal levels. We propose that endogenous, amphipathic lipid metabolites may modulate 5-HT7A receptors allosterically to promote high-affinity 5-HT binding and to enable receptors to couple more efficiently to Gs subtypes of G proteins.
5-HT7Receptors are G protein-coupled receptors with seven transmembrane segments, (Bard et al., 1993; Ruat et al., 1993; Shen et al., 1993), and exist in three alternatively spliced isoforms (Heidmann et al., 1997; Jasper et al., 1997; Stam et al., 1997; Heidmann et al., 1998). The predominant isoform, 5-HT7A, is localized in discrete limbic regions (thalamus and hypothalamus) of the brain as well as peripheral tissues, including coronary artery and certain gastrointestinal tissues (Meyerhof et al., 1993; To et al., 1995; Leung et al., 1996). Multiple physiological roles have been proposed for the receptor, such as regulation of circadian rhythms [because of its presence in the suprachiasmatic nucleus of the hypothalamus (Lovenberg et al., 1993; Kawahara et al., 1994)], mood and emotions [because of its limbic location and its high-affinity interactions with antipsychotics and antidepressants (Adham et al., 1998; Meyerhof et al., 1993; To et al., 1995)], and also migraine genesis [because of its smooth muscle relaxing activity (Leung et al., 1996; Cushing et al., 1996)]. At cellular level, the 5-HT7Areceptor, when expressed heterologously in mammalian cells, positively modulates adenylyl cyclases (Bard et al., 1993; Ruat et al., 1993; Shen et al., 1993; Adham et al., 1998) via activation of Gs subtypes of G proteins. We confirmed here that the human 5-HT7A receptor as expressed heterologously in human embryonic kidney (HEK) 293 cells mediates the cholera toxin-sensitive cAMP production. At the same time, we observed that most receptors (> 60%) bound 5-HT with high affinity. Classically, high-affinity agonist binding represents the phenotype for G protein-bound receptors, but was unusual for 5-HT7A in its marked abundance and insensitivity to 5′-guanylylimidodiphosphate (GppNHp), as observed here. In this study, we explored whether phospholipid metabolites and similar amphipathic compounds could modulate the 5-HT7Areceptor, a membrane-embedded receptor. Such possibilities were hinted at by earlier reports that oleamide and oleic acid influenced 5-HT7A receptors expressed in HeLa cells (Hedlund et al., 1999). In addition, our preliminary experiments showed that fatty acid-free bovine serum albumin (FF-BSA), a scavenger of lipid metabolites, markedly affected binding of [3H]5-HT, but not [3H]mesulergine (antagonist), to 5-HT7A.
Materials and Methods
The cDNA for the human 5-HT7A receptor has been cloned into a PCI-Neo mammalian expression vector. The vector was used for transfection of HEK293 cells, using Ca2+phosphate precipitation techniques. Cells were selected for a month in the presence of G-418 (400 μg/ml). Transcripts for 5-HT7A were robustly detected with reverse transcription-polymerase chain reaction (using 3′ rapid amplification of cDNA ends reaction) from transfected but not naive cells. Cell membranes were prepared by standard procedures including homogenization and differential centrifugation as described elsewhere (Pregenzer et al., 1993). Binding of radioactive ligands was measured in membranes expressing recombinant receptors, using filtration techniques as described elsewhere (Pregenzer et al., 1993). Briefly, [3H]mesulergine and [3H]5-HT binding were measured in the medium containing 100 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 20 mM HEPES/Tris, pH 7.4, the radioactive ligand at varying concentrations (0.1 to 20 nM for typical binding profiles), and 20 μg of membrane protein, in a total volume of 500 μl at 23°C for 60 min. Reaction mixtures were filtered over Whatman GF/B filters under vacuum. Filters were washed three times with 4 ml of ice-cold 50 mM Tris/HCl buffer, pH 7.4. Nonspecific binding was estimated in the presence of excess unlabeled mesulergine (10 μM). Competition experiments with [3H]mesulergine (2 nM) were carried out in the presence of test compounds at various concentrations under the same conditions.
GTPγ35S binding was measured with the use of the procedures reported earlier (Chabert et al., 1994; Pregenzer et al., 1997), in the medium containing 25 mM HEPES, pH 8.0, 100 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 0.5 mM dithiothreitol, 0.003% digitonin, 2 nM GTPγ35S (5 to 3 × 105 cpm/assay), and 10 μg of membrane protein in a volume of 200 μl. Digitonin was used at trace amounts here, solely to make the membranes permeable to GTPγ35S. Test ligands were included at 10 μM, unless indicated otherwise. Membranes were preincubated with 100 μM 5′-adenylylimidodiphosphate for 30 min, 10 μM GDP for 10 min on ice, and then were added to the rest of reaction components. In some experiments, membranes were treated with N-ethylmaleimide (NEM) at 100 μM, and excess NEM was neutralized with β-mercaptoethanol at the end of 30-min incubation. Reaction mixtures were incubated at 30° for 30 min and were filtered over Whatman GF/B filters under vacuum. Filters were washed three times with 4 ml of ice-cold buffer containing 100 mM NaCl, 20 mM Tris/HCl, pH 8.0, and 25 mM MgCl2. Agonist-induced GTPγ35S binding was obtained by subtracting that observed without agonists. The binding data were analyzed using nonlinear regression method (Sigma Plot), and presented with mean values ± S.E.
Cellular changes in cAMP were measured using a FlashPlate assay kit from PerkinElmer Life Science Products (Boston, MA). Briefly, cells were grown in a 96-well plate to about 80%confluence, washed three times with phosphate-buffered saline, and treated with test ligands for 30 min. cAMP in cell lysates were measured using the competition between 125I-cAMP and nonradioactive antigen for a fixed number of antibody binding sites in microplates coated with solid scintillant.
GTPγ35S-bound Gα subunits were identified following the method described elsewhere (Okamoto et al., 1992) with a modification (Alberts, et al., 1999): the activation of receptors with serotonin in the presence of GTPγ35S before membrane solubilization with detergents. Briefly, membranes were incubated in the presence of GTPγ35S (4 nM) and serotonin (10 μM) under conditions identical to those for GTPγ35S binding as described above. Treated membranes were solubilized with an equal volume of a buffer containing 100 mM Tris/HCl, pH 8.0, 10 mM MgCl2, 100 mM NaCl and 0.6% CHAPS for 30 min on ice, and were diluted to a final CHAPS concentration of 0.125%. An aliquot of the mixtures (typically 300 μl) was transferred to a well in a 96-well plate that had been coated successively with goat anti-rabbit antibodies (1:100 dilution), bovine serum albumin (5 mg/ml), and one of the affinity-purified rabbit antibodies for various Gα subunits (1:200 dilution). After incubation for 1 h at room temperature and washing, each well was counted for35S using a standard scintillation cocktail and a β-counter. The antibodies we used here include those specific for Gαi (the C-terminal sequence, 345–354), Gαs (the C-terminal sequence, 385–394), Gα q/11 (the common C-terminal sequence, QLNLKEYNLV), or Gα13 (the sequence 367–377) from Calbiochem (San Diego, CA). The mouse monoclonal antibody raised against bovine Go protein was obtained from Chemicon (Temecula, CA). Agonist-induced GTPγ35S binding was computed by subtracting the level observed without test agonists. Stock solutions were prepared in 0.1% ascorbic acid for monoamines, in dimethyl sulfoxide for lysophospholipids, and in alcohol for fatty acids and oleamide. Dilutions were made in borosilicate glass tubes, and final concentrations of dimethyl sulfoxide or alcohol were less than 0.5%. CHAPS and FF-BSA were prepared in the assay buffer. Reactions were carried out in polypropylene microtubes.
Results
Binding of [3H]5-HT and [3H]mesulergine (antagonist) to the human 5-HT7A receptor, when expressed heterologously in HEK293 cells, fitted well to a one-site binding model (linearity) with dissociation constants (K D) of 1.5 ± 0.1 and 4.3 ± 0.1 nM, respectively, and maximal binding values of 1.7 ± 0.2 (Fig. 1) and 2.6 ± 0.1 pmol/mg of protein, respectively (Fig. 1). With naive HEK293 cells, we observed no detectable levels of [3H]mesulergine and [3H]5-HT binding under the same conditions. We also carried out competition experiments with [3H]mesulergine at 4 nM and 5-HT at various concentrations (Fig. 1). The displacement data fitted to a model of two binding sites, high-affinity sites with aK I value of 1.8 ± 0.3 nM (similar to the K D for 5-HT), accounting for 64% of [3H]mesulergine binding sites, and low-affinity sites with a K I value of 93 ± 4 nM. 5-Carboxamidotryptamine maleate (5-CT), another agonist, displayed a similar biphasic profile, with high-affinity sites with aK I value of 1.6 ± 0.2 nM accounting for 65% of [3H]mesulergine binding sites and low-affinity sites with a K I value of 76 ± 3 nM (Table 1). This indicates that more than 60% of the total receptors, when estimated from maximal [3H]mesulergine (an antagonist) binding, displayed the high-affinity 5-HT binding. Classically, this phenotype represents G protein-associated receptors but was unusual for 5-HT7A in its marked abundance. Several antagonists, on the other hand, displaced [3H]mesulergine binding in monophasic patterns (e.g., methiothepin, lisuride, and metergoline, withK I values of 0.25 ± 0.04, 0.33 ± 0.05, and 0.97 ± 0.05 nM, respectively) (Table 1). GppNHp (10 μM), a nonhydrolyzable GTP analog, often uncouples receptor-G protein interactions, but it produced no appreciable changes in the relative populations of low- and high-affinity sites for 5-HT, as shown in the biphasic displacement pattern of [3H]mesulergine by 5-HT (Fig. 1). On the other hand, GppNHp abolished high-affinity dopamine binding to the D1 dopamine receptor (a prototypic Gs coupled receptor) when expressed in the same cell line (HEK293 cells) at a similar receptor density, 2.5 ± 0.2 pmol/mg protein. Dopamine reduced [3H]SCH23390 (antagonist with aK D value of 0.66 ± 0.05 nM) binding to D1 receptors by 12 ± 2% with a K Ivalue of 53 ± 16 nM, but this high-affinity site disappeared in the presence of GppNHp, leaving only low-affinity sites with aK I value of 7576 ± 574 nM (Fig. 1).
We also examined agonist-induced GTPγ35S binding to Gα subunits. At 5-HT7A, 10 μM 5-HT–induced GTPγ35S binding increased as a function of GTPγ35S concentrations (0.5 to 20 nM), with an EC50 value of 2.2 ± 0.8 nM and maximal binding of 270 ± 32 fmol/mg of protein (Fig.2). Similar values were observed with dopamine-activated D1 dopamine receptors (2.6 ± 0.3 nM and 330 ± 53 fmol/mg of protein) (Fig. 2). Methiothepin (100 μM) by itself produced no effect on the basal level, but blocked 1 μM 5-HT–induced GTPγ35S binding at 5-HT7A (24 ± 4% over the basal) whereas 100 μM SCH23390 blocked dopamine-induced GTPγ35S binding at D1 (Fig. 2). Moreover, at 5-HT7A, the methiothepin-sensitive GTPγ35S binding was primarily associated with Gαs, as detected with the immobilization method with various Gα-specific antibodies (Okamoto et al., 1992). The relative levels of GTPγ35S association with the antibodies specific for Gαs, Gαi, Gαo, Gαq, and Gα13 were 24 ± 3, 5 ± 2, 1 ± 1, 4 ± 4, and 0 ± 1%, respectively, above the basal binding (no 5-HT in anti-Gαs-coated wells) (Fig. 2).
Generally, Gi/Go subtypes are the most abundant and active cellular G proteins contributing to GTPγ35S binding. To evaluate independently the potential involvements of Gi/Go subtypes at 5-HT7A, membranes were treated with 100 μM NEM, an established, selective inhibitor of Gi/Go subtypes (Winslow et al., 1987; Nakajima et al., 1990; Alberts et al., 1999). NEM treatment decreased the basal GTPγ35S (2 nM) binding by 21 ± 3, but with no appreciable effect on the net 5-HT-induced GTPγ35S binding (Fig. 2). This further supports the primary association of 5-HT7A with Gs but not Gi/Go subtypes.
We examined the effects of lipid metabolites and similar amphipathic compounds on the 5-HT7A receptor, because such agents have been reported to allosterically modulate various membrane receptors (Koenig and Martin, 1992; Hedlund et al., 1999). FF-BSA, a scavenger of lipid metabolites, produced a noticeable, concentration-dependent reduction in [3H]5-HT binding, with little effect on [3H]mesulergine (antagonist) binding to 5-HT7A (Fig.3). At 3%, FF-BSA decreased [3H]5-HT (2 nM) binding by 46 ± 4%. Scatchard analysis showed a decrease in maximal 5-HT binding from 1.7 ± 0.2 to 1.1 ± 0.1 pmol/mg of protein, with no appreciable change in its K D value (1.2 ± 0.2 nM; Table 2 and Fig.4). Among fatty acids tested, only oleic acid at concentrations less than 15 μM increased 2 nM [3H]5-HT binding by 24 ± 5% without affecting [3H]mesulergine binding. Oleic acid at 15 μM increased maximal [3H]5-HT binding from 1.7 ± 0.2 to 2.2 ± 0.2 pmol/mg of protein but showed no effect on its K D value (1.3 ± 0.2 nM) (Fig. 4). At higher concentrations, however, oleic acid gradually inhibited both [3H]5-HT and [3H]mesulergine (4 nM) binding. This could arise from its ability to disturb ligand binding sites as an amphipathic compound. Stearic acid at 30 μM showed no effects, but at higher concentrations, it decreased both [3H]5-HT and [3H]mesulergine binding (Fig. 3). Similar profiles were observed with palmitic, arachidonic, and myristic acids (data not shown). It seems that FF-BSA could decrease [3H]5-HT binding by two ways, scavenging lipid metabolites such as oleic acid or possible protein-protein interactions with receptors. When fatty acid binding sites at FF-BSA were saturated with innocuous stearic acid (10 μM), FF-BSA lost its action on [3H]5-HT binding (Fig. 3). This indicates that the FF-BSA action may arise from its scavenging of stimulatory lipid metabolites. Furthermore, oleic acid and FF-BSA produced no appreciable effects on [3H]5-HT binding to analogous 5-HT receptors (i.e., gorilla 5-HT1D and human 5-HT2C receptors) when expressed heterologously in HEK293 cells. In the presence of oleic acid at 15 μM, for example, [3H]5-HT binding to 5-HT1D and 5-HT2C was 104 ± 4 and 100 ± 4% of control, respectively; with FF-BSA at 3%, binding was 97 ± 2 and 104 ± 5%, respectively.
It has been reported that oleamide and oleic acid at 0.1 μM decrease allosterically the affinity of [3H]5-HT for rat 5-HT7A receptors by 2- to 3-fold in HeLa cells, without affecting maximal binding (Hedlund et al., 1999). Here oleamide and oleic acid at the concentration range 0.01 to 1 μM showed no appreciable effects on [3H]5-HT and [3H]mesulergine binding to human 5-HT7A receptors in HEK293 cells. At micromolar concentrations, oleic acid (<15 μM) stimulated [3H]5-HT binding in HEK293 cells, and oleamide (>50 μM) blocked gradually both [3H]5-HT and [3H]mesulergine binding (Fig. 3). These differences in receptor sensitivity could be ascribed to various factors, including species variations in rat and human receptors and possibly different surrounding lipid environments in the HeLa and HEK293 cells. In fact, the rat 5-HT7A in HeLA cells displayed a K D value for 5-HT (12.68 nM) nearly 10-fold greater than the human counterpart in HEK293 cells (1.5 nM) (Hedlund et al., 1999).
We also studied lysophospholipids, which are also generated from hydrolysis of phospholipids by phospholipases. Various lysophospholipids (oleoyl and stearyl) at 15 μM or less showed no selective effects on [3H]5-HT or mesulergine binding. Even at higher concentrations (50 μM), only lysophosphatidyl choline decreased selectively [3H]5-HT binding (no effect on [3H]mesulergine binding) by reducing maximal [3H]5-HT binding from 1.7 ± 0.2 to 1.2 ± 0.2 pmol/mg of protein, but not itsK D value (1.4 ± 0.1 nM) (Table 2). Lysophosphatidyl glycerol decreased both [3H]5-HT and [3H]mesulergine binding (Fig. 3), and similar profiles were observed with lysophosphatidyl inositol and lysophosphatidyl serine (data not shown). Lysophosphatidyl ethanolamine and lysophosphatidic acid at concentrations up to 100 μM marginally inhibited [3H]5-HT and mesulergine binding, with decreases of less than 20%.
Another nondenaturing zwitterionic detergent, CHAPS at 0.2% or less, markedly increased [3H]5-HT binding, but marginally decreased [3H]mesulergine binding (Fig. 3). In the presence of 0.1% CHAPS, maximal [3H]5-HT binding increased from 1.7 ± 0.2 to 2.6 ± 0.1 pmol/mg of protein, with no appreciable change in its K D value (1.4 ± 0.1 nM compared with 1.5 ± 0.1 nM). For [3H]mesulergine binding, 0.1% CHAPS showed no effect on maximal binding (2.5 ± 0.2 compared with 2.6 ± 0.1 pmol/mg of protein) but increased itsK D value from 4.3 ± 0.1 to 6.9 ± 0.4 nM. The opposite effects by lysophaphatidyl choline and CHAPS, two similar zwitterions, suggest that their actions arise from specific interactions with receptors, not from general membrane perturbations.
Functionally, these agents also affected 5-HT-induced GTPγ35S binding. FF-BSA (3%) and 50 μM lysophosphatidyl choline reduced 10 μM 5-HT–induced GTPγ35S binding to 56 ± 4 and 55 ± 10%, respectively, as normalized to that observed with 10 μM 5-HT, whereas oleic acid and CHAPS increased the GTPγ35S binding to 137 ± 5 and 133 ± 7%, respectively. In the analogous Gs-coupled D1 receptor, however, 15 μM oleic acid, 3% FF-BSA, 50 μM lysophosphatidyl choline, and 0.1% CHAPS produced no appreciable effects on 10 μM dopamine-induced, SCH-23390-sensitive GTPγ35S binding (96 ± 28, 95 ± 3, 83 ± 16, and 109 ± 22%, respectively).
We also examined how these agents affect cAMP accumulation at 5-HT7A in the HEK293 cell. Typically, 5-HT increased cAMP production in a concentration-dependent manner with an EC50 value of 8.1 ± 0.7 nM and maximal stimulation of 9 ± 1 pmol/well in a 96-well plate (Fig. 4). With cholera toxin treatment (5 μg/ml culture for overnight), the basal level of cAMP increased from 0.1 to 4 pmol/well, but 5-HT produced no responses; the covalent modification of Gssubtypes by cholera toxin (ADP-ribosylation) rendered them into permanently activated states (Milligan, 1988). FF-BSA at 3% and lysophosphatidyl choline at 50 μM showed no effects on the basal cAMP production (<0.2 pmol/well) but increased the 5-HT concentration (EC50) from 8.1 to 13.9 ± 1.2 and 15.8 ± 1.6 nM, respectively, without affecting maximal cAMP production (9.1 ± 0.8 and 8.3 ± 0.3 pmol/well, respectively; Fig. 4and Table 2). On the other hand, 15 μM oleic acid and 0.1% CHAPS decreased the EC50 value from 8.1 ± 0.7 to 3.7 ± 0.4 and 4 ± 1 nM, respectively, but with little effect on maximal production (8.4 ± 0.3 and 9 ±1 pmol/well, respectively) and on basal cAMP production.
Discussion
The human 5-HT7A receptor is a Gs-coupled receptor; when expressed heterologously in HEK293 cells, more than 60% of its population displayed high-affinity [3H]5-HT binding. We discovered here that the high-affinity agonist binding decreased in the presence of FF-BSA and increased in the presence of oleic acid without appreciable effects on antagonist binding. The actions of FF-BSA and oleic acid seem to be highly selective for 5-HT7A, judging from the following observations. 1) FF-BSA and oleic acid showed no appreciable effects on [3H]5-HT binding to analogous 5-HT receptors (5-HT1D and 5-HT2C). 2) Structural analogs of oleic acid, such as palmitic, stearic, and arachidonic acids, were not effective. 3) FF-BSA lost its action when preincubated with inactive 10 μM stearic acid, supporting its role in scavenging endogenous lipid metabolites rather than protein-protein interaction. 4) FF-BSA and oleic acid reduced maximal binding for 5-HT, not the K D value, indicating their selective action at receptors. 5) Oleic acid was effective at concentrations (<15 μM) too low to form micellar structures, and its action was not mimicked by lysophospholipids, another class of amphipathic lipid metabolites. 6) In general, the human 5-HT7A receptor seems to be susceptible to allosteric modulations by amphipathic agents. For example, lysophosphatidyl choline decreased maximal 5-HT binding for 5-HT (not antagonist binding), and CHAPS, a similar zwitterion, increased it. From these results, we propose that endogenous amphipathic compounds, probably including oleic acid and others that remain unidentified, may allosterically modulate 5-HT7A to increase high-affinity 5-HT binding.
In addition, the amphipathic compounds at concentrations that selectively affected [3H]5-HT binding also influenced 5-HT-induced GTPγ35S binding at 5-HT7A. Oleic acid and CHAPS increased and FF-BSA and lysophosphatidyl choline decreased 5-HT-induced GTPγ35S binding. No similar actions by these agents at D1 (a prototypic Gs-coupled receptor) also support the view of their specific interactions with 5-HT7A, but not with G proteins in the signaling pathways.
In this study, we also observed that GppNHp produced no appreciable effect on the high-affinity agonist binding to 5-HT7A but abolished high-affinity dopamine binding to D1. Two possibilities can be cited: 1) At 5-HT7A, a certain portion could be sensitive to GppNHp but not detectable because the overwhelming majority of high-affinity binding was insensitive to the guanine nucleotide. 2) 5-HT7A receptors could form a very tight complex with Gs that could not be destabilized by GppNHp. A similar situation has been proposed for Mel1a-metatonin receptor and Gi (Roka et al., 1999). Further study will be needed.
In summary, endogenous amphipathic lipid metabolites that could be scavenged by FF-BSA, such as oleic acid, seem to allosterically modulate 5-HT7A to enhance its high-affinity agonist binding, at least in part, and to confer more efficient coupling with Gs subtypes of G proteins. Their interactions with 5-HT7A seem to be specific, judging from the narrow structural requirement for modulators (oleic acid and CHAPS) and their inactivity at analogous 5-HT and dopamine receptors.
Footnotes
- Received April 24, 2001.
- Accepted September 7, 2001.
Abbreviations
- HT
- hydroxytryptamine
- GppNHp
- 5′-guanylylimidodiphosphate
- HEK
- human embryonic kidney
- FF-BSA
- fatty acid-free bovine serum albumin
- GTPγ35S
- guanosine-5′-O-(3-[35S]thio)triphosphate
- NEM
- N-ethylmaleimide
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
- 5-CT
- 5-carboxamidotryptamine maleate
- The American Society for Pharmacology and Experimental Therapeutics