Survey of Nonxanthine Derivatives as Adenosine Receptor Ligands

Abstract

The binding affinities at rat A₁, A_2a, and A₃ adenosine receptors of a wide range of heterocyclic derivatives have been determined. Mono-, bi-, tricyclic and macrocyclic compounds were screened in binding assays, using either [³H]PIA or [³H]CGS 21680 in rat brain membranes or [¹²⁵I]AB-MECA in CHO cells stably transfected with rat A₃ receptors. Several new classes of adenosine antagonists (e. g. 5- oxoimidazopyrimidines and a pyrazoloquinazoline) were identified. Various sulfonylpiperazines, 11- hydroxytetrahydrocarbazolenine, 4H-pyrido[1,2-a]pyrimidin-one, folic acid, and cytochalasin H and J bound to A₃ receptors selectively. Moreover, cytochalasin A, which bound to A₁ adenosine receptors with Ki value of 1.9 μM, inhibited adenylyl cyclase in rat adipocytes, but not via reversible A₁ receptor binding.

     

The effects of zolpidem treatment on GABA(A) receptors in cultured cerebellar granule cells: changes in functional coupling

AimsHypnotic zolpidem is a positive allosteric modulator of γ-aminobutyric acid (GABA) action, with preferential although not exclusive binding for α1 subunit-containing GABA_A receptors. The pharmacological profile of this drug is different from that of classical benzodiazepines, although it acts through benzodiazepine binding sites at GABA_A receptors. The aim of this study was to further explore the molecular mechanisms of GABA_A receptor induction by zolpidem.Main methodsIn the present study, we explored the effects of two-day zolpidem (10 μM) treatment on GABA_A receptors on the membranes of rat cerebellar granule cells (CGCs) using [³H]flunitrazepam binding and semi-quantitative PCR analysis.Key findingsTwo-day zolpidem treatment of CGCs did not significantly affect the maximum number (B_max) of [³H]flunitrazepam binding sites or the expression of α1 subunit mRNA. However, as shown by decreased GABA [³H]flunitrazepam binding, two-day exposure of CGCs to zolpidem caused functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptor complexes.SignificanceIf functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptors is the mechanism responsible for the development of tolerance following long-term administration of classical benzodiazepines, chronic zolpidem treatment may induce tolerance.     

Characterization of a low affinity binding site for N6-substituted adenosine derivatives in rat testis membranes

Abstract

The binding characteristics of radiolabeled N⁶-(cyclohexyl)adenosine ([³H]CHA), N⁶-(R-phenylisopropyl)adenosine ([³H]R-PIA), 5′-N-ethylcarboxamidoadenosine ([³H]NECA), and 2-[4-(2-carboxyethyl)phenyl]ethyl-amino-5′-N-ethylcarboxamidoadenosine ([³H]CGS 21680), to rat testis membranes were investigated. Specific binding of [³H]CGS 21680, a selective agonist for the A_2a adenosine receptor, was very modest whilst the nonselective agonist [³H]NECA bound to rat testis membranes showing high binding capacity. At least two types of binding sites for [³H]NECA could be identified in rat testis membranes: high affinity sites and high capacity sites. Selective agonists for the At adenosine receptor, [³H]CHA and [³H]R-PIA bound with high affinity to a single class of binding sites. This high affinity binding site showed the typical pharmacological specificity of the A₁ adenosine receptor with a potency order for agonists of CHA R-PIA > NECA > N⁶-(S-phenylisopropyl)adenosine (S-PIA). In order to detect the presence of the A₃ adenosine receptor in these membranes we selectively blocked the A₁ receptor with a large molar excess of a xanthine antagonist, either 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or xanthine amine congener (XAC). In the presence of an antagonist a low affinity binding site for [³H]CHA and [³H]R-PIA was detected. This low affinity binding site showed a different pharmacological specificity than the high affinity binding site. In fact the potency order for agonists was CHA NECA = R-PIA > S-PIA. This finding suggests that the low affinity binding site represents the A₃ adenosine receptor.     

GTP and IAP Shifts Adenosine A1-Agonist Binding in Smooth Muscle Membranes to the Low Affinity State,Similar to the one Found in Intact Cells

Abstract

Adenosine A₁ receptors in the smooth muscle cell line DDT₁ MF-2 were characterized by radioligand binding using the antagonist [³H]-8-cyclopentyl-1,3-dipropylxanthine ([³H]DPCPX) as the ligand. Binding properties of adenosine agonists and antagonists to both intact cells and membranes were investigated.     

Autoradiographic Visualization of Kappa Opioid Receptors with Labelled Dynorphins in Guinea Pig Brain

Abstract

The distribution of kappa opioid receptors in guinea pig brain was measured by in vitro receptor autoradiography using [³H]dynorphin A_1–9, [³H]dynorphin A_1–8 and [³H]bremazocine as ligands. The sites labelled by the two dynorphins had identical, heterogeneous distributions in brain sections. High levels of kappa receptors were seen in striatum, claustrum, nucleus accumbens and laminae V and VI of the cerebral cortex. The substantia nigra and superior colliculus also had high dynorphin binding levels. The [³H]dynorphin autoradiographs were closely similar to those obtained using [³H]bremazocine in the presence of mu and delta receptor displacers. It is concluded that tritiated dynorphin A fragments can be used for autoradiographic studies of kappa opioid receptors in brain.     

Subchronic Treatment with Methamphetamine and Phencyclidine Differentially Alters the Adenosine A1 and A2A Receptors in the Prefrontal Cortex,Hippocampus, and Striatum of the Rat

Subchronic treatment with MAP (4.6 mg/kg, i.p., once daily for 11 days) significantly decreased the K_d, but not B_max, values of [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) binding to adenosine A₁ receptors in the prefrontal cortex and hippocampus, but not striatum, of rat brain. However, subchronic treatment with PCP (10 mg/kg, i.p., once daily for 11 days) did not alter the K_d and B_max values of [³H]DPCPX binding to adenosine A₁ receptors in these three regions. Subchronic treatment with MAP or PCP did not alter the B_max and K_d values of [³H]2-p-(2-carboxyehyl)phenethylamino-5-N-ethylcarboxyamidoadenosine ([³H]CGS21680) binding to adenosine A_2A receptors in the striatum. Furthermore, subchronic treatment with MAP or PCP significantly decreased the specific binding of [³H]CGS21680 to adenosine A_2A receptors in the hippocampus, but not in the prefrontal cortex. Thus, these results suggest that MAP and PCP may produce differential effects on the adenosine A_2A receptors, but not adenosine A₁ receptors in rat brain.     

Characterization of the Binding of a Novel Non-Xanthine Adenosine Antagonist Radioligand, [3H]CGS 15943, to Multiple Affinity States of the Adenosine A1 Receptor in the Rat Cortex

Abstract

The use of xanthine adenosine receptor antagonists such as 1,3-dipropyl-8-phenylxanthine (DPX) as radioligands for the characterization of adenosine receptor Pharmacology have been limited by their high lipophilicity, low specific activity, and their general lack of selectivity and affinity for adenosine receptors. Recent attempts to address the technical problems associated with this class of compounds has resulted in the development of several xanthine derivatives (e.g. the functionalized xanthine congeners [³H]XCC and [³H]XAC², and [³H]CPX³) which bind with high and selective affinity to the adenosine A₁ receptor subtype. Based on efforts to optimize non-xanthine adenosine receptor antagonists, CGS 15943, a derivative of the pyrazoloquinazoline benzodiazepine receptor inverse agonist CGS 8216⁵, represents the first reported non-xanthine structure that potently blocks adenosine receptors⁶. CGS 15943 has nanomolar affinity for both A₁ and A₂ receptor subtypes⁶. However, in contrast to many of the xanthine adenosine receptor antagonists, CGS 15943 is not a phosphodiesterase inhibitor and does not interact with adenosine transporter sites⁶. This compound is a potent and selective adenosine receptor antagonist in vivo ⁷ with a solubility/affinity ratio of greater than 1000⁷. In the present studies, the binding of [³H]CGS 15943 to the adenosine A₁ receptor was characterized.     

Photoaffinity Labelling and Solubilization of the Central 5-HT1A Receptor Binding Site

Abstract

Two complementary approaches, covalent labelling and solubilization, have been used to study the biochemical properties of the central 5-HT_1A receptor binding site. We have first designed a photoaffinity ligand containing the structure of 8-OH-DPAT, a potent and specific agonist of 5-HT_1A sites. Thus, 8-methoxy-2[N-n-propyl,N-3-(2-nitro-4-azido-phenyl)- aminopropyl]aminotetralin or 8-methoxy-3'-NAP-amino-PAT, was found to displace, in the dark, [³H]8-OH-DPAT from 5-HT_1A sites in rat hippocampal membranes with an IC₅₀ of 6.6 nM. Under two cumulative UV irradiations (366 nm, for 20 min at 4°C), 8-methoxy-3-'-NAP-amino-PAT (30 nM) blocked irreversibly 55-60% of 5-HT_1A binding sites. This blockade was specific of 5-HT_1A sites since the other serotoninergic sites, 5-HT_1B, 5-HT₂ and also the presynaptic 5-HT₃ sites were not affected by the treatment. In addition, the binding of [³H]Spiperone and [³H]7-OH-DPAT to striatal dopamine sites remained unchanged under similar photolysis conditions. The tritiated derivative of the photoaffinity ligand (92 Ci/mmol) was then synthesized for the identification of the covalently bound protein(s). SDS-PAGE of solubilized membranes irradiated in the presence of 20 nM ³H-8-methoxy-3'-NAP-amino-PAT allowed the detection of a 63 kD protein whose labelling appeared specific. Thus, ³H-incorporation into the 63 kD band could be prevented by uM concentrations of 5-HT, 8-OH-DPAT and other selective 5-HT_1A ligands such as isapirone. In contrast, the 5-HT₂ antagonist ketanserin, norepinephrine and dopamine-related ligands (including 7-OH-DPAT) were ineffective. Direct solubilization of 5-HT_1A receptor binding sites was also attempted from rat hippocampal membranes. The best results were obtained using CHAPS (10 mM) plus NaCl (0.2 M), which led to 50 % recovery of 5-HT_1A sites in the 100,000 g supernatant. The pharmacological properties and sensitivity to N-ethyl-maleimide and GppNHp of soluble sites appeared near identical to those of membrane-bound 5-HT_1A sites.     

[3H]Spiroxatrine labels a serotonin1A-like site in the rat hippocampus

[³H]Spiroxatrine was examined as a potential ligand for the labeling of 5-HT_1A sites in the rat hippocampus. Analysis of the binding of [³H]spiroxatrine in the absence and presence of varying concentrations of three monoamine neurotransmitters revealed that serotonin (5-HT) had high affinity (IC₅₀= 20.7 nM for the [³H]spiroxatrine binding sites, consistent with the labeling of 5-HT₁ sites, while dopamine and norepinephrine had very low affinity (IC₅₀=57600 nM and >10⁻⁴ M respectively). Saturation studies of the binding of [³H]spiroxatrine revealed a single population of sites with a K_d=2.21 nM. Further pharmacologic characterization with the 5-HT_1A ligands 8-hydroxy-2-(di-n-propylamino) tetralin, ipsapirone, and WB4101 and the butyrophenone compounds spiperone and haloperidol gave results that were consistent with [³H]spiroxatrine labeling 5-HT_1A sites. This ligand produced stable, reproducible binding with a good ratio of specific to nonspecific binding. The binding of [³H]spiroxatrine was sensitive to GTP, suggesting that this ligand may act as an agonist. This was supported by the finding that spiroxatrine inhibits forskolin-stimulated adenylate cyclase activity (a proposed 5-HT_1A receptor model) in the rat hippocampus. Since [³H]spiroxatrine is structurally distinct from other currently available radioligands for the 5-HT_1A site, it should provide new information about the properties of this putative serotonergic receptor.     

Excitatory and Inhibitory Effects of A1 and A2A Adenosine Receptor Activation on the Electrically Evoked [3H]Acetylcholine Release from Different Areas of the Rat Hippocampus

Abstract: The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [³H]acetylcholine ([³H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A₁/A₂ agonist 2-chloroadenosine (CADO; 2–10 µM) inhibited, in a concentration-dependent manner, the release of [³H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 µM) on [³H]ACh release was prevented by the A₁ antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A_2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [³H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 µM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [³H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [³H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine. In the CA1 area, only A₁ inhibitory receptors modulate ACh release, whereas in the CA3 area, both A_2A excitatory and A₁ inhibitory adenosine receptors modulate ACh release. In the dentate gyrus, both A₁ inhibitory and A_2A excitatory adenosine receptors are present, but endogenous adenosine does not activate them.     

Novel selective antagonist radioligands for the pharmacological study of A2B adenosine receptors

The adenosine A_2B receptor is the least well characterized of the four adenosine subtypes due to the lack of potent and selective agonists and antagonists. Despite the widespread distribution of A_2B receptor mRNA, little information is available with regard to their function. The characterization of A_2B receptors, through radioligand binding studies, has been performed, until now, by using low-affinity and non-selective antagonists like 1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX),(4-(2-[7-amino-2-(2-furyl)-[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)-phenol ([³H]ZM 241385) and 3-(3,4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propyl-xanthine ([¹²⁵I]ABOPX). Recently, high-affinity radioligands for A_2B receptors, [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide ([³H]MRS 1754), N-(2-(2-Phenyl-6-[4-(2,2,3,3-tetratritrio-3-phenylpropyl)-piperazine-1-carbonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl)-acetamide ([³H]OSIP339391) and N-benzo[1,3]dioxol-5-yl-2-[5-(1,3-dipropyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide] ([³H]MRE 2029F20), have been introduced. This minireview offers an overview of these recently developed radioligands and the most important applications of drugs towards A_2B receptors.     

Differentiating Adenosine Receptors and Adenosine Uptake Sites in Brain

Abstract

The characteristics of adenosine receptors and adenosine uptake sites in brain are presented. High affinity adenosine receptors of the A₁ type bind [³H]cyclohexyladenosine ([³H]CHA) and [³ H]diethyl-phenyl-xanthine ([³H]DPX) with 10^?9 potency while adenosine uptake sites are labeled 10^?10 potency with [³ H]nitrobenzyl-thioinosine ([³H]NBI). NBI does not inhibit either [³H]CHA (agonist) or [³H]DPX (antagonist) binding to adenosine receptors in brain cortical membranes and conversely CHA and other adenosine receptor ligands are very poor inhibitors of [³H]NBI binding to adenosine uptake sites. A number of other differences between the receptor and uptake site are discussed which provide rather strong evidence that these two sites are quite distinct and that the labeled ligands used represent specific probes for each site.     

Characterization of adenosine receptors in Mullus surmuletus

The intent of the present study was to investigate adenosine receptor sites in brain membranes of the saltwater teleost fish, Mullus surmuletus, using the A₁ receptor selective agonist, [³H]CHA, and A_2a receptor selective agonist [³H]CGS 21680. The A₁ selective agonist, [³H]CHA, bound saturably, reversibly and with high affinity to a single-class of binding sites (K_d 1.47 nM; B_max 100–190 fmol/mg protein, dependent on fish length). The A_2a selective agonist, [³H]CGS 21680, also bound saturably, reversibly and with relative high affinity to a single-class of binding sites (K_d 44.2 nM; B_max 150–300 fmol/mg protein dependent on fish length). In equilibrium competition experiments, adenosine analogous, NECA, CGS 21680, CHA, CPA, S-PIA, R-PIA, CPCA, DPMA, and xanthine antagonists, DPCPX, XAC, and THEO all displaced [³H]CHA and [³H]CGS 21680 specifically bound to brain membranes from Mullus surmuletus. Specific binding of both [³H]CHA and [³H]CGS 21680 was inhibited by GDPβS. For [³H]CHA the IC₅₀ value was 2.5 ± 0.1 μM, while for [³H]CGS 21680 the IC₅₀ value was 7.7 ± 0.3 μM. Our results indicate that the high affinity binding sites for [³H]CHA have some pharmacological characteristics of mammalian A₁ adenosine receptors, while the binding sites for [³H]CGS 21680 appear to be virtually identical to the binding sites for [³H]CHA.     

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.     

Tritium-labeled agonists as tools for studying adenosine A<Subscript>2B</Subscript> receptors

A selective agonist radioligand for A_2B adenosine receptors (A_2BARs) is currently not available. Such a tool would be useful for labeling the active conformation of the receptors. Therefore, we prepared BAY 60-6583, a potent and functionally selective A_2BAR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [³H]BAY 60-6583. This is probably due to its high non-specific binding and its moderate affinity, which had previously been overestimated based on functional data. As an alternative, we evaluated the non-selective A_2BAR agonist [³H]NECA for its potential to label A_2BARs. [³H]NECA showed specific, saturable, and reversible binding to membrane preparations of Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells stably expressing human, rat, or mouse A_2BARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [³H]NECA than versus the A_2B-antagonist radioligand [³H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an A_2BAR agonist, it displayed higher affinity versus [³H]PSB-603 than versus [³H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the A_2BAR. In conclusion, [³H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active A_2BAR conformation.     

A 2′←5′-Adenylate Trimer With a Positively Charged 2′(3′)-Terminal 8-(4-Aminobunl)Aminoadenosine Residue: Synthesis,Conformation and RNase L Binding

Abstract

An analogue of the 2-5A core trimer containing an 8-(4-aminobutyl)-aminoadenosine (1; A) residue at the 2′(3′)-terminus [2; (2′,5′)A₂A^?] was synthesized. The conformation of (2′,5′)A₂A^? was studied by ₁H, ¹³C-NMR, and CD spectroscopy. The (2′,5′)A₂A^? exhibits very low binding ability to the RNase L of mouse L cells, but slightly enhanced resistance to digestion by SVPD compared to the parent trimer.     

Internode length in Zea mays L.

[¹³C, ³H]Gibberellin A₂₀ (GA₂₀) has been fed to seedlings of normal (tall) and dwarf-5 and dwarf-1 mutants of maize (Zea mays L.). The metabolites from these feeds were identified by combined gas chromatography-mass spectrometry. [¹³C, ³H]Gibberellin A₂₀ was metabolized to [¹³C, ³H]GA₂₉-catabolite and [¹³C, ³H]GA₁ by the normal, and to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₁ by the dwarf-5 mutant. In the dwarf-1 mutant, [¹³C, ³H]GA₂₀ was metabolized to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₂₉-catabolite; no evidence was found for the metabolism of [¹³C, ³H]GA₂₀ to [¹³C, ³H]GA₁. [¹³C, ³H]Gibberellin A₈ was not found in any of the feeds. In all feeds no dilution of ¹³C in recovered [¹³C, ³H]GA₂₀ was observed. Also in the dwarf-5 mutant, the [¹³C]label in the metabolites was apparently undiluted by endogenous [¹³C]GAs. However, dilution of the [¹³C]label in metabolites from [¹³C, ³H]GA₂₀ was observed in normal and dwarf-1 seedlings. The results from the feeding studies provide evidence that the dwarf-1 mutation of maize blocks the conversion of GA₂₀ to GA₁.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - RP reverse phase     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

Development of Stable Cell Lines Expressing Different Subtypes of GabaA Receptors

Abstract

The experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABA_A receptors. For this, we developed an original two step selection strategy, in which the first step consisted of transfecting HEK 293 cells with rat GABA_A receptor α and β subunits. G 418 resistant colonies isolated at this step were screened for [³H] muscimol binding to select for those that coexpressed α- and β-subunits. The best α and β subunit expressing colony was then supertransfected with a plasmid coding for the γ rat GABA_A receptor subunit and a mutant DHFR gene. After a second round of selection, this time in presence of methotrexate, those colonies that coexpressed ternary αβγ GABA_A receptor combinations were distinguished using [³H] flumazenil as a probe. This strategy was applied to the isolation of 3 GABA_A receptor clones, α₁β₂γ_2S, α₁β₂γ_2S and α₁β₂γ_2S, that expressed relatively high levels of these proteins. These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected with equivalent subunit combinations. These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and potency of compounds at recombinant GABA_A receptor subtypes.     

Synthesis of 2H,13C-Labelled 2′-Deoxynucleosides and Their Site Specific Incorporation into Oligo-DNA for Structural Studies via Relaxation Time Measurements

Abstract

We have recently shown¹ the usefulness of ²H, ¹³C-labelled 2′-deoxynu-cleoside building blocks for structural studies via relaxation time measurements. The synthesis of phosphoramidite blocks 11 and 12 for their site-specific incorporation (indicated by underlines) into the d⁵′(¹C²G³ A ⁴ T ⁵ T ⁶ A ⁷ A ⁸ T ⁹C¹⁰G)₂ ³′ is briefly described for studying the T₁ and T_1[sgrave] relaxations of ²H and ¹³C at specific deuterated carbons in a large molecule.