Simplified synthesis of N‐(3‐[18F]fluoropropyl)‐2β‐carbomethoxy‐3β‐(4‐fluorophenyl)nortropane ([18F]β‐CFT‐FP) using [18F]fluoropropyl tosylate as the labelling reagent

A synthesis method has been developed for the labelling of N‐(3‐[¹⁸F]fluoropropyl)‐2β‐carbomethoxy‐3β‐(4‐fluorophenyl)nortropane ([¹⁸F]β‐CFT‐FP), a potential radioligand for visualization of the dopamine transporters by positron emission tomography. The two‐step synthesis includes preparation of [¹⁸F]fluoropropyl tosylate and its use without purification in the fluoroalkylation of 2β‐carbomethoxy‐3β‐(4‐fluorophenyl)nortropane (nor‐β‐CFT). The final product is purified by HPLC. Optimization of the two synthesis steps resulted in a greater than 30% radiochemical yield of [¹⁸F]β‐CFT‐FP (decay corrected to end of bombardment). The synthesis time including HPLC‐purification was approximately 90 min. The radiochemical purity of the final product was higher than 99% and the specific radioactivity at the end of synthesis was typically 20 GBq/µmol. In comparison to alkylation by [¹⁸F]fluoropropyl bromide, the procedure described here results in an improved overall radiochemical yield of [¹⁸F]β‐CFT‐FP in a shorter time. Copyright © 2005 John Wiley & Sons, Ltd.     

Synthesis of [guanido‐13C]‐γ‐hydroxyarginine

This report describes an efficient method of synthesizing [guanido‐¹³C]‐γ‐hydroxyarginine HCl salt. Iodolactonization of N‐Boc‐protected allylglycine mainly provided the cis iodo compound 2. This was converted to an amine through azide 4. The amine 5 was reacted with N‐Boc‐protected [¹³C]thiourea to afford N‐Boc‐protected [¹³C]guanidine 6, which underwent base catalyzed ring opening. Removal of the N‐Boc group afforded [guanido‐¹³C]‐γ‐hydroxyarginine HCl salt 7 giving a 30% overall yield of the final product from N‐Boc protected allylglycine 1 in five steps. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of 1‐(2′‐deoxy‐2′‐fluoro‐β‐D‐arabinofuranosyl)‐[Methyl‐11C]thymine ([11C]FMAU) via a Stille cross‐coupling reaction with [11C]methyl iodide

1‐(2′‐deoxy‐2′‐fluoro‐β‐D‐arabinofuranosyl)‐[methyl‐¹¹C]thymine ([¹¹C]FMAU) [¹¹C]‐ 1 was synthesised via a palladium‐mediated Stille coupling reaction of 1‐(2′‐deoxy‐2′‐fluoro‐β‐D‐arabinofuranosyl)‐5‐(trimethylstannyl)uracil 2 with [¹¹C]methyl iodide in a one‐pot procedure. The reaction conditions were optimized by screening various catalysts and solvents, and by altering concentrations and reaction temperatures. The highest yield was obtained using Pd₂(dba)₃ and P(o‐tolyl)₃ in DMF at 130°C for 5 min. Under these conditions the title compound [¹¹C]‐ 1 was obtained in 28±5% decay‐corrected radiochemical yield calculated from [¹¹C]methyl iodide (number of experiments=7). The radiochemical purity was >99% and the specific radioactivity was 0.1 GBq/μmol at 25 min after end of bombardment. In a typical experiment 700–800 MBq of [¹¹C]FMAU [¹¹C]‐ 1 was obtained starting from 6–7 GBq of [¹¹C]methyl iodide. A mixed ¹¹C/¹³C synthesis to yield [¹¹C]‐ 1 /(¹³C)‐ 1 followed by ¹³C‐NMR analysis was used to confirm the labelling position. The labelling procedure was found to be suitable for automation. Copyright © 2002 John Wiley & Sons, Ltd.     

β-Endorphin

Three β_h-EP analogs which show different extents of alteration in analgesic potency by substitution of a single amino acid residue were assayed for their peripheral opioid activity and the binding to opioid μ-receptor to determine the relationships among the opioid activities obtained from different assays. In the guinea pig ileum assay, [Gln⁸]-β_h-EP showed a higher inhibitory activity than the parent peptide. [Tyr³¹]-analog had the same potency as β_h-EP, while [Trp²⁷]-analog retained only one fourth the potency of β_h-EP. Assayed on the vas deferens of the mouse and the rat, all three substituted β_h-EP analogs exhibited a lower potency than their parent peptide. Receptor binding assay using [³H]-dihydromorphine as the primary ligand showed that [Gln⁸]-analog had a binding potency 1.5-fold that of β_h-EP, while the potencies of [Tyr³¹]- and [Trp²⁷]-analogs were not significantly different from that of the parent peptide. No correlation in relative potency was found between vas deferens assays and their μ-receptor binding or analgesic activity. However, the relative potencies of binding to μ-receptor in [Gln⁸]- and [Tyr³¹]-analogs were found to be consistent with those of analgesic and guinea pig ileum assays, whereas the binding to β-EP receptor of all analogs appeared to be related to the charge properties of β-EP molecule.     

Synthesis of N‐(3‐[18F]Fluoropropyl)‐2β‐carbomethoxy‐3β‐(4‐iodophenyl)nortropane ([18F]FP‐β‐CIT)

N‐(3‐[¹⁸F]fluoropropyl)‐2β‐carbomethoxy‐3β‐(4‐iodophenyl)nortropane ([¹⁸F]FP‐β‐CIT) was synthesized in a two‐step reaction sequence. In the first reaction, 1‐bromo‐3‐(nitrobenzene‐4‐sulfonyloxy)‐propane was fluorinated with no‐carrier‐added fluorine‐18. The resulting product, 1‐bromo‐3‐[¹⁸F]‐fluoropropane, was distilled into a cooled reaction vessel containing 2β‐carbomethoxy‐3β‐(4‐iodophenyl)‐nortropane, diisopropylethylamine and potassium iodide. After 30 min, the reaction mixture was subjected to a preparative HPLC purification. The product, [¹⁸F]FP‐β‐CIT, was isolated from the HPLC eluent with solid‐phase extraction and formulated to yield an isotonic, pyrogen‐free and sterile solution of [¹⁸F]FP‐β‐CIT. The overall decay‐corrected radiochemical yield was 25 ± 5%. Radiochemical purity was > 98% and the specific activity was 94 ± 50 GBq/µmol at the end of synthesis. Copyright © 2006 John Wiley & Sons, Ltd.     

Functional activation of Gαq/11 protein via α1‐adrenoceptor in rat cerebral cortical membranes

Although it is recognized that α₁‐adrenoceptors are coupled to diverse intracellular signalling pathways, its primary transduction mechanisms are evoked by activating phospholipase C in the cell membrane through Gα_q/11, resulting in production of inositol 1,4,5‐trisphosphate and diacylglycerol. However, there have been few studies that indicate directly the involvement of Gα_q/11 proteins in this signalling pathway in the central nervous system. In the current study, we tried to pharmacologically characterize (?)‐adrenaline‐stimulated [³⁵S]GTPγS binding to Gα_q/11 in rat brain membranes. Functional activation of Gα_q/11 coupled to α₁‐adrenoceptor was investigated by using [³⁵S]GTPγS binding/immunoprecipitation assay in the membranes prepared from rat cerebral cortex, hippocampus, and striatum. The specific [³⁵S]GTPγS binding to Gα_q/11 was stimulated by (?)‐adrenaline in a concentration‐dependent and saturable manner in rat cerebral cortical membranes. In hippocampal or striatal membranes, the stimulatory effects of (?)‐adrenaline were scarce. The effect of (?)‐adrenaline was potently inhibited by prazosin, a potent and selective α₁‐adrenoceptor antagonist, but not by yohimbine, a selective α₂‐adrenoceptor antagonist. The response was mimicked by cirazoline, but not by R(?)‐phenylephrine. Although oxymetazoline also stimulated the specific [³⁵S]GTPγS binding to Gα_q/11 as an apparent “super‐agonist”, detailed pharmacological characterization revealed that its agonistic properties in this experimental system were derived from off‐target effects on 5‐HT_2A receptors, but not via α₁‐adrenoceptors. In conclusion, functional coupling of α₁‐adrenoceptors to Gα_q/11 proteins are detectable in rat brain membranes by means of [³⁵S]GTPγS binding/immunoprecipitation assay. It is necessary to interpret the experimental data with caution when oxymetazoline is included as an agonist at α₁‐adrenoceptors.     

Efficient O‐ and N‐(β‐fluoroethylation)s with NCA [18F]β‐fluoroethyl tosylate under microwave‐enhanced conditions

Reactions of no‐carrier‐added (NCA) [¹⁸F]β‐fluoroethyl tosylate with amine, phenol or carboxylic acid to form the corresponding [¹⁸F]N‐(β‐fluoroethyl)amine, [¹⁸F]β‐fluoroethyl ether or [¹⁸F]β‐fluoroethyl ester, were found to be rapid (2–10 min) and efficient (51–89% conversion) under microwave‐enhanced conditions. These conditions allow reactants to be heated rapidly to 150°C in a low boiling point solvent, such as acetonitrile, and avoid the need to use high boiling point solvents, such as DMSO and DMF, to promote reaction. The microwave‐enhanced reactions gave about 20% greater radiochemical yields than thermal reactions performed at similar temperatures and over similar reaction times. With a bi‐functional molecule, such as DL‐pipecolinic acid, [¹⁸F]β‐fluoroethyl tosylate reacts exclusively with the amino group. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis of deoxyadenosine‐5′‐(35S)‐thiomonophosphate [dAMP(35S)] of high specific activity – a key intermediate for the synthesis of Sp‐deoxyadenosine‐5′‐(alpha;‐35S)‐thiotriphosphate [Sp‐dATP (α‐35S)]

Unprotected deoxyadenosine 1 was treated with an excess of phosphorus acid and stoichiometric proportions of N, N′‐di‐p‐tolylcarbodiimide in anhydrous pyridine to give deoxyadenosine‐5′‐monophosphite 2 . The latter was activated with trimethylsilyl chloride followed by sulphurisation with elemental ³⁵S (specific activity>1000 Ci/mmol) in toluene solution to give deoxyadenosine‐5′‐(³⁵S)‐thiomonophosphate [dAMP(³⁵S)] 3 . Enzymatic conversion of deoxyadenosine‐5′‐(³⁵S)‐thiomonophosphate to Sp‐deoxyadenosine‐5′‐(α‐³⁵S)‐thiotriphosphate [Sp‐dATP (α‐³⁵S)] 5 was carried out following a standard reaction protocol. Copyright © 2001 John Wiley & Sons, Ltd.     

Synthesis of a non‐peptidic PET tracer designed for α5β1 integrin receptor

Arginine–glycine–aspartic acid (RGD)‐containing peptides have been traditionally used as PET probes to noninvasively image angiogenesis, but recently, small selective molecules for α₅β₁ integrin receptor have been developed with promising results. Sixty‐one antagonists were screened, and tert‐butyl (S)‐3‐(2‐((3R,5S)‐1‐(3‐(1‐(2‐fluoroethyl)‐1H‐1,2,3‐triazol‐4‐yl)propanoyl)‐5‐((pyridin‐2‐ylamino)methyl)pyrrolidin‐3‐yloxy)acetamido)‐2‐(2,4,6‐trimethylbenzamido)propanoate (FPMt) was selected for the development of a PET tracer to image the expression of α₅β₁ integrin receptors. An alkynyl precursor (PMt) was initially synthesized in six steps, and its radiolabeling was performed according to the azide–alkyne copper(II)‐catalyzed Huisgen's cycloaddition by using 1‐azido‐2‐[¹⁸F]fluoroethane ([¹⁸F]12). Different reaction conditions between PMt and [¹⁸F]12 were investigated, but all of them afforded [¹⁸F]FPMt in 15 min with similar radiochemical yields (80–83%, decay corrected). Overall, the final radiopharmaceutical ([¹⁸F]FPMt) was obtained after a synthesis time of 60–70 min in 42–44% decay‐corrected radiochemical yield. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis of acyl[35S]sulfonamides: Coupling of high specific activity [35S]methane sulfonamide with acids and acid chlorides

Direct coupling of high specific activity [³⁵S]methanesulfonamide, generated from [³⁵S]methanesulfonyl chloride and ammonia, with acids and acid chlorides afforded the corresponding [³⁵S]acyl sulfonamides in excellent yields. Examples of high specific activity [³⁵S]acyl sulfonamides were prepared containing functionality that can be further elaborated through carbon–carbon or carbon–nitrogen bond forming reactions.     

Synthesis of tracer labelled [11,12‐3H]‐β‐carotene

The synthesis of tracer labelled [11,12‐³H]‐β‐carotene is described. The procedure uses Wittig condensation of tracer labelled ³H‐retinal (retinal spiked with [11,12‐³H]‐retinal) with retinyl triphenylphosphonium bromide. The preparation of tracer labelled[³H]‐β‐carotene is suitable for studies involving bioavailability and bioconversion of β‐carotene to vitamin A. Copyright © 2003 John Wiley & Sons, Ltd.     

Interaction of morphine but not fentanyl with cerebral α2‐adrenoceptors in α2‐adrenoceptor knockout mice

Objectives α₂‐Adrenergic and μ‐opioid receptors belong to the rhodopsin family of G‐protein coupled receptors and mediate antinociceptive effects via similar signal transduction pathways. Previous studies have revealed direct functional interactions between both receptor systems including synergistic and additive effects. To evaluate underlying mechanisms, we have studied whether morphine and fentanyl interacted with α₂‐adrenoceptor‐subtypes in mice lacking one individual α₂‐adrenoceptor‐subtype (α₂‐adrenoceptor knockout). Methods Opioid interaction with α₂‐adrenoceptors was investigated by quantitative receptor autoradiography in brain slices of α_2A‐, α_2B‐ or α_2C‐adrenoceptor deficient mice. Displacement of the radiolabelled α₂‐adrenoceptor agonist [¹²⁵I]paraiodoclonidine from α₂‐adrenoceptors in different brain regions by increasing concentrations of morphine, fentanyl and naloxone was analysed. The binding affinity of both opioids to α₂‐adrenoceptor subtypes in different brain regions was quantified. Key findings Morphine but not fentanyl or naloxone provoked dose‐dependent displacement of [¹²⁵I]paraiodoclonidine from all α₂‐adrenoceptor subtypes in the brain regions analysed. Binding affinity was highest in cortex, medulla oblongata and pons of α_2A‐adrenoceptor knockout mice. Conclusions Our results indicated that morphine interacted with α₂‐adrenoceptors showing higher affinity for the α_2B and α_2C than for the α_2A subtype. In contrast, fentanyl and naloxone did not show any relevant affinity to α₂‐adrenoceptors. This effect may have an impact on the pharmacological actions of morphine.     

Synthesis of C‐14 and C‐13, H‐2‐labeled IKK inhibitor: [14C] and [13C4,D3]‐N‐(6‐chloro‐7‐methoxy‐9H‐pyrido[3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide

[¹⁴C]‐N‐(6‐Chloro‐7‐methoxy‐9H‐pyrido [3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide (5B ), an IKK inhibitor, was synthesized from [¹⁴C]‐barium carbonate in two steps in an overall radiochemical yield of 41%. The intermediate, [carboxyl‐¹⁴C]‐2‐methylnicotinic acid, was prepared by the lithiation and carbonation of 3‐bromo‐2‐methylpyridine. [¹³C₄,D₃]‐N‐(6‐chloro‐7‐methoxy‐9H‐pyrido [3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide (5C ) was synthesized from [1,2,3,4‐¹³C₄]‐ethyl acetoacetate and [D₄]‐methanol in six steps in an overall yield of 2%. [¹³C₄]‐2‐methylnicotic acid, was prepared by condensation of [¹³C₄]‐ethyl 3‐aminocrotonate and acrolein, followed by hydrolysis with lithium hydroxide. Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis of [18F]3‐[1‐(3‐fluoropropyl)‐(S)‐pyrrolidin‐2‐ylmethoxy]pyridine ([18F]NicFP): a potential α4β2 nicotinic acetylcholine receptor radioligand for PET

Nicotinic acetylcholine receptors are widely distributed throughout the human brain and are believed to play a role in several neurological and psychiatric disorders. In order to identify an effective PET radioligand for in vivo assessment of the α4β2 subtype of nicotinic receptor, we synthesized [¹⁸F]3‐[1‐(3‐fluoropropyl)‐(S)‐pyrrolidin‐2‐ylmethoxy]pyridine (NicFP). The in vitro K_D of NicFP was determined to be 1.1 nM, and the log P value obtained by HPLC analysis of the unlabelled standard was found to be 2.2. The radiosynthesis of [¹⁸F]NicFP was carried out by a nucleophilic substitution reaction of anhydrous [¹⁸F]fluoride and the corresponding mesylate precursor. After purification by HPLC, the radiochemical yield was determined to be 11.3±2.1% and the specific activity was 0.47±0.18 Ci/μmol (EOS, n = 3). The time of synthesis and purification was 99±2 min. The final product was prepared as a sterile saline solution suitable for in vivo use. Copyright © 2003 John Wiley & Sons, Ltd.     

An affinity label for δ‐opioid receptors derived from [d‐Ala2]deltorphin I*

Abstract: A series of potential affinity label derivatives of the amphibian opioid peptide [d ‐Ala²]deltorphin I were prepared by incorporation at the para position of Phe³ (in the ‘message’ sequence) or Phe⁵ (in the ‘address’ sequence) of an electrophilic group (i.e. isothiocyanate or bromoacetamide). The introduction of the electrophile was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide, followed later in the synthesis by selective deprotection of the Alloc group and modification of the resulting amine. While para substitution decreased the δ‐opioid receptor affinity, selected analogs retained nanomolar affinity for δ receptors. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited moderate affinity (IC₅₀ = 83 nm ) and high selectivity for δ receptors, while the corresponding amine and bromoacetamide derivatives showed pronounced decreases in δ‐receptor affinity (80‐ and >1200‐fold, respectively, compared with [d ‐Ala²]deltorphin I). In the ‘address’ sequence, the Phe(p‐NH₂)⁵ derivative showed the highest δ‐receptor affinity (IC₅₀ = 32 nm ), while the Phe(p‐NHCOCH₂Br)⁵ and Phe(p‐NCS)⁵ peptides displayed four‐ and tenfold lower δ‐receptor affinities, respectively. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited wash‐resistant inhibition of [³H][d ‐Pen²,D‐Pen⁵]enkephalin (DPDPE) binding to δ receptors at a concentration of 80 nm . [d ‐Ala², Phe(p‐NCS)³]deltorphin I represents the first affinity label derivative of one of the potent and selective amphibian opioid peptides, and the first electrophilic affinity label derivative of an agonist containing the reactive functionality in the ‘message’ sequence of the peptide.     

Metformin inhibits Aβ25‐35‐induced apoptotic cell death in SH‐SY5Y cells

Metformin, a first‐line drug for type‐2 diabetes, plays a potentially protective role in preventing Alzheimer's disease (AD), but its underlying mechanism is unclear. In this study, Aβ_25‐35‐treated SH‐SY5Y cells were used as a cell model of AD to investigate the neuroprotective effect of metformin, as well as its underlying mechanisms. We found that metformin decreased the cell apoptosis rate and death, ratio of Bcl‐2/Bax, and expression of NR2A and NR2B, and increased the expression of LC3 in Aβ_25‐35‐treated SH‐SY5Y cells. Metformin also reduced intracellular and extracellular Glu concentrations, as well as the intracellular concentration of Ca²⁺ and ROS in Aβ_25‐35‐treated SH‐SY5Y cells. These findings suggest that metformin inhibits Aβ_25‐35‐treated SH‐SY5Y cell death by inhibiting apoptosis, decreasing intracellular Ca²⁺ and ROS by reducing neurotoxicity of excitatory amino acids, and by possibly reversing autophagy disorder via regulating autophagy process.     

Evaluation of [18F]2FP3 in pigs and non‐human primates

So far, no suitable 5‐HT₇R radioligand exists for clinical positron emission tomography (PET) imaging. [¹⁸F]2FP3 was first tested in vivo in cats, and the results were promising for further evaluations. Here, we evaluate the radioligand in pigs and non‐human primates (NHPs). Furthermore, we investigate species differences in 5‐HT₇R binding with [³H]SB‐269970 autoradiography in post‐mortem pig, NHP, and human brain tissue. Specific binding of [¹⁸F]2FP3 was investigated by intravenous administration of the 5‐HT₇R specific antagonist SB‐269970. [³H]SB‐269970 autoradiography was performed as previously described. [¹⁸F]2FP3 was synthesized in an overall yield of 35% to 45%. High brain uptake of the tracer was found in both pigs and NHPs; however, pretreatment with SB‐269970 only resulted in decreased binding of 20% in the thalamus, a 5‐HT₇R–rich region. Autoradiography on post‐mortem pig, NHP, and human tissues revealed that specific binding of [³H]SB‐269970 was comparable in the thalamus of pig and NHP. Despite the high uptake of [¹⁸F]2FP3 in both species, the binding could only be blocked to a limited degree with the 5‐HT₇R antagonists. We speculate that the affinity of the radioligand is too low for imaging the 5‐HT₇Rs in vivo and that part of the PET signal arises from targets other than the 5‐HT₇R.     

Synthesis of [2‐13C, 4‐13C]‐(2R,3S)‐catechin and [2‐13C, 4‐13C]‐(2R,3R)‐epicatechin

The first synthesis of doubly labeled, [2‐¹³C, 4‐¹³C]‐(2R,3S)‐catechin 15 and [2‐¹³C, 4‐¹³C]‐(2R,3R)‐epicatechin 18 starting from labeled 2‐hydroxy‐4, 6‐bis(benzyloxy)acetophenone 3 and labeled 3, 4‐bis(benzyloxy)‐benzaldehyde 7 are described. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of Ethanol on Recombinant Rat GABAA Receptors: [35S]t-Butylbicyclophosphorothionate ([35S]TBPS) Binding Study

Abstract: To determine the roles of the alternatively spliced short and long forms of the γ2 subunit in the effect of ethanol on the GABA_A receptor function, picrotoxin-sensitive [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding was studied in recombinant rat α1β2γ2 and α6β2γ2 receptors expressed in human embryonic kidney 293 cells. Ethanol (10–500 mM) in the absence of added GABA had only minor effects on [³⁵S]TBPS binding irrespective of the γ2 splice variant, its effects being greater in α6β2γ2 than in α1β2γ2 receptors. Ethanol (100 mM) decreased the binding in all four subunit combinations at various concentrations of GABA, again an effect independent of the γ2 variant. The two γ2 variants had different effects on GABA modulation of the binding, with the long γ2 variant decreasing the efficiency of GABA inhibition in α6β2γ2 receptors and enhancing the biphasic GABA stimulation and inhibition in α1β2γ2 receptors. The findings confirm the importance of the α subunits in the allosteric interactions between the convulsant binding site and other effector sites, which can be modified only to a minor extent by the type of the γ2 splice variant.     

Agonist function of the recombinant α4β3δ GABAA receptor is dependent on the human and rat variants of the α4‐subunit

1. It is known that the α₄‐subunit is likely to occur in the brain predominantly in α₄β₃δ receptors at extrasynaptic sites. Recent studies have revealed that the α₁‐, α₄‐, γ₂‐ and δ‐subunits may colocalize extrasynaptically in dentate granule cells of the hippocampus. In the present study, we characterized a series of recombinant GABA_A receptors containing human (H) and rat (R) α₁/α₄‐, β₂/β₃‐ and γ_2S/δ‐subunits in Xenopus oocytes using the two‐electrode voltage‐clamp technique. 2. Both Hα₁β₃δ and Hα₄β₃γ_2S receptors were sensitive to activation by GABA and pentobarbital. Contrary to earlier findings that the α₄β₃δ combination was more sensitive to agonist action than the α₄β₃γ_2S receptor, we observed extremely small GABA‐ and pentobarbital‐activated currents at the wild‐type Hα₄β₃δ receptor. However, GABA and pentobarbital activated the wild‐type Rα₄β₃δ receptor with high potency (EC₅₀ = 0.5 ± 0.7 and 294 ± 5 μmol/L, respectively). 3. Substituting the Hα₄ subunit with Rα₄ conferred a significant increase in activation on the GABA and pentobarbital site in terms of reduced EC₅₀ and increased I_max. When the Hα₄ subunit was combined with the Rβ₃ and Rδ subunit in a heteropentameric form, the amplitude of GABA‐ and pentobarbital‐activated currents increased significantly compared with the wild‐type Hα₄β₃δ receptor. 4. Thus, the results indicate that the Rα₄β₃δ, Hα₁β₃δ and Hα₄β₃γ_2S combinations may contribute to functions of extrasynaptic GABA_A receptors. The presence of the Rα₄ subunit at recombinant GABA_A receptors containing the δ‐subunit is a strong determinant of agonist action. The recombinant Hα₄β₃δ receptor is a less sensitive subunit composition in terms of agonist activation.