Potency of GABA at human recombinant GABAA receptors expressed in Xenopus oocytes: a mini review

GABA_A receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure–function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA_A receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA_A receptors, α4/δ-containing GABA_A receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.     

Stigmasterol can be new steroidal drug for neurological disorders: Evidence of the GABAergic mechanism via receptor modulation

BackgroundGamma-aminobutyric acid A (GABA_A) receptors have been implicated in anxiety and epileptic disorders.Hypothesis/PurposeThis study aimed to investigate the effects of stigmasterol, a plant sterol (phytosterol) isolated from Artemisia indica Linn on neurological disorders.MethodsStigmasterol was evaluated on various recombinant GABA_A receptor subtypes expressed in Xenopus laevis oocytes and its anxiolytic and anticonvulsant potential was assessed using the elevated plus maze (EPM), light-dark box (LDB) test, and pentylenetetrazole- (PTZ-) induced seizure paradigms. Furthermore, computational modeling of α2β2γ2L, α4β3δ, and α4β3 subtypes was performed to gain insights into the GABAergic mechanism of stigmasterol. For the first time, a model of GABAδ subtype was generated. Stigmasterol was targeted to all the binding sites (neurotransmitters, positive and negative modulator binding sites) of GABA_A α2β2γ2L, α4β3, and α4β3δ complexes by in silico docking.ResultsStigmasterol enhanced GABA-induced currents at ternary α2β2γ2L, α4β3δ, and binary α4β3 GABA_AR subtypes. The potentiation of GABA-induced currents at extrasynaptic α4β3δ was significantly higher compared to the binary α4β3 subtype, indicating that the δ subunit is important for efficacy. Stigmasterol was found to be a potent positive modulator of the extrasynaptic α4β3δ subtype, which was also confirmed by computational analysis. The computational analysis reveals that stigmasterol preferentially binds at the transmembrane region shared by positive modulators or a binding site constituted by the M2-M3 region of α4 and M1-M2 of β3 at α4β3δ complex. In in vivo studies, Stigmasterol (0.5–3.0 mg/kg, i.p.) exerted significant anxiolytic and anticonvulsant effects in an identical manner of allopregnanolone, indicating the involvement of a GABAergic mechanism.ConclusionTo our knowledge, this is the first study reporting the positive modulation of GABA_A receptors, anxiolytic and anticonvulsant potential of stigmasterol. Thus, stigmasterol is considered to be a candidate steroidal drug for the treatment of neurological disorders due to its positive modulation of GABA receptors.     

Potentiating effect of glabridin from Glycyrrhiza glabra on GABAA receptors

Extracts from Glycyrrhiza are traditionally used for the treatment of insomnia and anxiety. Glabridin is one of the main flavonoid compounds from Glycyrrhiza glabra and displays a broad range of biological properties. In the present work, we investigated the effect of glabridin on GABA_A receptors. For this purpose, we employed the two-electrode voltage-clamp technique on Xenopus laevis oocytes expressing recombinant GABA_A receptors. Through this approach, we observed that glabridin presents a strong potentiating effect on GABA_A α1β(1?3)γ2 receptors. The potentiation was slightly dependent on the β subunit and was most pronounced at the α1β2γ2 subunit combination, which forms the most abundant GABA_A receptor in the CNS. Glabridin potentiated with an EC₅₀ of 6.3±1.7 µM and decreased the EC₅₀ of the receptor for GABA by approximately 12-fold. The potentiating effect of glabridin is flumazenil-insensitive and does not require the benzodiazepine binding site. Glabridin acts on the β subunit of GABA_A receptors by a mechanism involving the M286 residue, which is a key amino acid at the binding site for general anesthetics, such as propofol and etomidate. Our results demonstrate that GABA_A receptors are strongly potentiated by one of the main flavonoid compounds from Glycyrrhiza glabra and suggest that glabridin could contribute to the reported hypnotic effect of Glycyrrhiza extracts.     

Activation of Calcium-Phospholipid-Dependent Protein Kinase Enhances Benzodiazepine and Barbiturate Potentiation of the GABAA Receptor

Abstract: The effect of calcium-phospholipid-dependent protein kinase (PKC) on GABA_A receptor function was examined in Xenopus oocytes expressing recombinant human GABA_A receptor using two-electrode voltage-clamp measurements. Phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC, inhibited GABA-gated chloride currents by ～72% in oocytes expressing α_lβ₁γ_2L subunit cDNAs. Phorbol 12-monomyristate (PMM), a negative control analogue of PMA, did not alter GABA_A receptor responses. To investigate whether activation of PKC could alter the modulatory responses of the receptor complex, the effect of PMA on benzodiazepine and barbiturate potentiation of GABA responses was assessed. In oocytes expressing α_lβ₁γ_2s subunit cDNAs, diazepam (300 nM) potentiated GABA responses by ～160%. Following PMA (5-25 nM/) treatment, diazepam potentiation was significantly increased to 333%. No effect of the inactive phorbol ester PMM (25 nM) was observed on diazepam potentiation of GABA responses. PMA enhancement of diazepam potentiation of GABA responses was also observed in oocytes expressing α_lβ₁γ_2Ssubunit cDNAs, indicating that the unique PKC site present in the Tγ_2LL subunit is not required for observing the PMA effect. PMA (5-25 nM) also enhanced pentobarbital potentiation of GABA responses. In oocytes expressing α_lβ₁γ_2L subunit cDNAs, pentobarbital (25 μM) potentiated GABA receptor responses by ～97%. Following treatment with PMA (5-25 nM), pentobarbital potentiation of GABA responses increased to ～ 156%. The present results suggest that protein phosphorylation may alter the coupling between the allosteric modulatory sites within the GABA_A receptor complex.     

Altered GABAA Receptor Expression and Seizure Threshold Following Acute Ethanol Challenge in Mice Lacking the RIIβ Subunit of PKA

Ethanol causes pathological changes in GABA_A receptor trafficking and function. These changes are mediated in part by ethanol activation of protein kinase A (PKA). The current study investigated the expression of the GABA_A α1 and α4 subunits and the kinase anchoring protein AKAP150, as well as bicuculline-induced seizure threshold, at baseline and following acute injection of ethanol (3.5 g/kg IP) in a mouse line lacking the regulatory RIIβ subunit of PKA. Whole cerebral cortices were harvested at baseline, 1 h, or 46 h following injection of ethanol or saline and subjected to fractionation and western blot analysis. Knockout (RIIβ?/?) mice had similar baseline levels of PKA RIIα and GABA_A α1 and α4 subunits compared to wild type (RIIβ+/+) littermates, but had deficits in AKAP150. GABA_A α1 subunit levels were decreased in the P2 fraction of RIIβ?/?, but not RIIβ+/+, mice following 1 h ethanol, an effect that was driven by decreased α1 expression in the synaptic fraction. GABA_A α4 subunits in the P2 fraction were not affected by 1 h ethanol; however, synaptic α4 subunit expression was increased in RIIβ+/+, but not RIIβ?/? mice, while extrasynaptic α4 and δ subunit expression were decreased in RIIβ?/?, but not RIIβ+/+ mice. Finally, RIIβ knockout was protective against bicuculline-induced seizure susceptibility. Overall, the results suggest that PKA has differential roles in regulating GABA_A receptor subunits. PKA may protect against ethanol-induced deficits in synaptic α1 and extrasynaptic α4 receptors, but may facilitate the increase of synaptic α4 receptors.     

Immunochemical Localization of GABA<Subscript>A</Subscript> Receptor Subunits in the Freshwater Polyp <Emphasis Type="Italic">Hydra vulgaris</Emphasis> (Cnidaria,Hydrozoa)

γ-aminobutyric acid (GABA) receptors, responding to GABA positive allosteric modulators, are present in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa), one of the most primitive metazoans to develop a nervous system. We examined the occurrence and distribution of GABA_A receptor subunits in Hydra tissues by western blot and immunohistochemistry. Antibodies against different GABA_A receptor subunits were used in Hydra membrane preparations. Unique protein bands, inhibited by the specific peptide, appeared at 35, 60, ～50 and ～52 kDa in membranes incubated with α3, β1, γ3 or δ antibodies, respectively. Immunohistochemical screening of whole mount Hydra preparations revealed diffuse immunoreactivity to α3, β1 or γ3 antibodies in tentacles, hypostome, and upper part of the gastric region; immunoreactive fibers were also present in the lower peduncle. By contrast, δ antibodies revealed a strong labeling in the lower gastric region and peduncle, as well as in tentacles. Double labeling showed colocalization of α3/β1, α3/γ3 and α3/δ immunoreactivity in granules or cells in tentacles and gastric region. In the peduncle, colocalization of both α3/β1 and α3/γ3 immunoreactivity was found in fibers running horizontally above the foot. These data indicate that specific GABA_A receptor subunits are present and differentially distributed in Hydra body regions. Subunit colocalization suggests that Hydra GABA receptors are heterologous multimers, possibly sub-serving different physiological activities.     

Defined concatenated α6α1β3γ2 GABAA receptor constructs reveal dual action of pyrazoloquinolinone allosteric modulators

Pyrazoloquinolinones (PQs) have been extensively studied as modulators of GABA_A receptors with different subunit composition, exerting modulatory effects by binding at α+/β- interfaces of GABA_A receptors. PQs with a substituent in position R7 have been reported to preferentially modulate α6- subunit containing GABA_A receptors which are mostly expressed in the cerebellum but were also found in the olfactory bulb, in the cochlear nucleus, in the hippocampus and in the trigeminal sensory pathway. They are considered potentially interesting in the context of sensori-motor gating deficits, depressive-like behavior, migraine and orofacial pain. Here we explored the option to modify the lead ligands’ R7 position. In the compound series we observed two different patterns of allosteric modulation in recombinantly expressed α6β3γ2 receptors, namely monophasic and biphasic positive modulation. In the latter case the additional phase occurred in the nanomolar range, while all compounds displayed robust modulation in the micromolar range. Nanomolar, near silent binding has been reported to occur at benzodiazepine binding sites, but was not investigated at the diazepam insensitive α6+/γ2- interface. To clarify the mechanism underlying the biphasic effect we tested one of the compounds in concatenated receptors. In these constructs the subunits are covalently linked, allowing to form either the α6+/γ2- interface, or the α6+/β3- interface, to study the resulting modulation. With this approach we were able to ascribe the nanomolar modulation to the α6+/γ2- interface. While not all compounds display the nanomolar phase, the strong modulation at the α6+/β3 interface proved to be tolerant for all tested R7 groups. This provides the future option to introduce e.g. isotope labelled or fluorescent moieties or substituents that enhance solubility and bioavailability.     

Triton x-100 inhibits agonist-induced currents and suppresses benzodiazepine modulation of GABAA receptors in Xenopus oocytes

Changes in lipid bilayer elastic properties have been proposed to underlie the modulation of voltage-gated Na⁺ and L-type Ca²⁺ channels and GABA_A receptors by amphiphiles. The amphiphile Triton X-100 increases the elasticity of lipid bilayers at micromolar concentrations, assessed from its effects on gramicidin channel A appearance rate and lifetime in artificial lipid bilayers. In the present study, the pharmacological action of Triton-X 100 on GABA_A receptors expressed in Xenopus laevis oocytes was examined. Triton-X 100 inhibited GABA_A α₁β₃γ_2S receptor currents in a noncompetitive, time- and voltage-dependent manner and increased the apparent rate and extent of desensitization at 10 μM, which is 30 fold below the critical micelle concentration. In addition, Triton X-100 induced picrotoxin-sensitive GABA_A receptor currents and suppressed allosteric modulation by flunitrazepam at α₁β₃γ_2S receptors. All effects were independent of the presence of a γ_2S subunit in the GABA_A receptor complex. The present study suggests that Triton X-100 may stabilize open and desensitized states of the GABA_A receptor through changes in lipid bilayer elasticity.     

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.     

Identification of dihydrostilbenes in Pholidota chinensis as a new scaffold for GABAA receptor modulators

A dichloromethane extract of stems and roots of Pholidota chinensis (Orchidaceae) enhanced GABA-induced chloride currents (IGABA) by 132.75 ± 36.69% when tested at 100 μg/mL in a two-microelectrode voltage clamp assay, on Xenopus laevis oocytes expressing recombinant α₁β₂γ_2S GABA_A receptors. By means of an HPLC-based activity profiling approach, the three structurally related stilbenoids coelonin (1), batatasin III (2), and pholidotol D (3) were identified in the active fractions of the extract. Dihydrostilbene 2 enhanced I_GABA by 1512.19 ± 176.47% at 300 μM, with an EC₅₀ of 52.51 ± 16.96 μM, while compounds 1 and 3 showed much lower activity. The relevance of conformational flexibility for receptor modulation by stilbenoids was confirmed with a series of 13 commercially available stilbenes and their corresponding semisynthetic dihydro derivatives. Dihydrostilbenes showed higher activity in the oocyte assay than their corresponding stilbenes. The dihydro derivatives of tetramethoxy-piceatannol (12) and pterostilbene (20) were the most active among these derivatives, but they showed lower efficiencies than compound 2. Batatasin III (2) showed high efficiency but no significant subunit specificity when tested on the receptor subtypes α₁β₂γ_2s, α₂β₂γ_2s, α₃β₂γ_2s, α₄β₂γ_2s, α₅β₂γ_2s, α₁β₁γ_2s, and α₁β₃γ_2s. Dihydrostilbenes represent a new scaffold for GABA_A receptor modulators.     

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.     

Unique assignment of inter-subunit association in GABAA α1β3γ2 receptors determined by molecular modeling

Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA_A receptor (GABA_ARα1β3γ2). There is strong evidence that the heteropentameric receptor contains two α1, two β3, and one γ2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either γ2β3α1β3α1 or γ2α1β3α1β3 configurations. Here we use molecular modeling to thread the relevant GABA_AR subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA_A sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA_A sequences were threaded onto the AChBP template in the γ2β3α1β3α1 or γ2α1β3α1β3  arrangements. Only the γ2α1β3α1β3 arrangement satisfied three known criteria: (1) α1 His¹⁰² binds at the γ2 subunit interface in proximity to γ2 residues Thr¹⁴², Phe⁷⁷, and Met¹³⁰; (2) α1 residues 80-100 bind near γ2 residues 91-104; and (3) α1 residues 58-67 bind near the β3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.     

A new chromanone derivative isolated from Hypericum lissophloeus (Hypericaceae) potentiates GABAA receptor currents in a subunit specific fashion

A phytochemical investigation of the lipophilic extract of Hypericum lissophloeus (smoothbark St. John’s wort, Hypericaceae) was conducted, resulting in the isolation and identification of a new chromanone derivative: 5,7-dihydroxy-2,3-dimethyl-6-(3-methyl-but-2-enyl)-chroman-4-one (1). This compound was demonstrated to act as a potent stimulator of currents elicited by GABA in recombinant α₁β₂γ₂ GABA_A receptors, with a half-maximal potentiation observed at a concentration of about 4 μM and a maximal potentiation of >4000%. Significant potentiation was already evident at a concentration as low as 0.1 μM. Extent of potentiation strongly depends on the type of α subunit, the type of β subunit and the presence of the γ subunit.     

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.     

Co-expression of γ2 Subunits Hinders Processing of N-Linked Glycans Attached to the N104 Glycosylation Sites of GABAA Receptor β2 Subunits

GABA_A receptors, the major mediators of fast inhibitory neuronal transmission, are heteropentameric glycoproteins assembled from a panel of subunits, usually including α and β subunits with or without a γ2 subunit. The α1β2γ2 receptor is the most abundant GABA_A receptor in brain. Co-expression of γ2 with α1 and β2 subunits causes conformational changes, increases GABA_A receptor channel conductance, and prolongs channel open times. We reported previously that glycosylation of the three β2 subunit glycosylation sites, N32, N104 and N173, was important for α1β2 receptor channel gating. Here, we examined the hypothesis that steric effects or conformational changes caused by γ2 subunit co-expression alter the glycosylation of partnering β2 subunits. We found that co-expression of γ2 subunits hindered processing of β2 subunit N104 N-glycans in HEK293T cells. This γ2 subunit-dependent effect was strong enough that a decrease of γ2 subunit expression in heterozygous GABRG2 knockout (γ2^+/?) mice led to appreciable changes in the endoglycosidase H digestion pattern of neuronal β2 subunits. Interestingly, as measured by flow cytometry, γ2 subunit surface levels were decreased by mutating each of the β2 subunit glycosylation sites. The β2 subunit mutation N104Q also decreased GABA potency to evoke macroscopic currents and reduced conductance, mean open time and open probability of single channel currents. Collectively, our data suggested that γ2 subunits interacted with β2 subunit N-glycans and/or subdomains containing the glycosylation sites, and that γ2 subunit co-expression-dependent alterations in the processing of the β2 subunit N104 N-glycans were involved in altering the function of surface GABA_A receptors.     

Inhibitory effects of insulin on GABAA currents modulated by the GABAA alpha subunit

Abstract

Insulin, when co-applied with GABA, can cause an inhibition of the induced current at GABA_A receptors. This study investigated that inhibitory effect of insulin at a variety of receptor isoforms, concentrating on α1, α2 and α4 containing receptors. Various isoforms were expressed in Xenopus oocytes and currents determined using two-electrode voltage clamp. Submaximal GABA currents at all isoforms studied were inhibited by nanomolar concentrations of insulin. At α2 and α4 containing forms, insulin could inhibit maximal GABA currents. The ability to inhibit maximal currents, and the general potency and effects at submaximal currents paralleled the number of potential MAPK sites on the α subunits. The differences in insulin inhibition of GABA currents at different α containing GABA_A receptors could be important in autocrine and paracrine control of hormone secretion in the pancreas, and in control of reward and food intake circuits of the brain.     

Identification of GABA A receptor modulators in Kadsura longipedunculata and assignment of absolute configurations by quantum-chemical ECD calculations

A petroleum ether extract of Kadsura longipedunculata enhanced the GABA-induced chloride current (I_GABA) by 122.5 ± 0.3% (n = 2) when tested at 100 μg/ml in Xenopuslaevis oocytes expressing GABA A receptors (α₁β₂γ_2S subtype) in two-microelectrode voltage clamp measurements. Thirteen compounds were subsequently identified by HPLC-based activity profiling as responsible for GABA A receptor activity and purified in preparative scale. 6-Cinnamoyl-6,7-dihydro-7-myrceneol and 5,6-dihydrocuparenic acid were thereby isolated for the first time. The determination of the absolute stereochemistry of these compounds was achieved by comparison of experimental and calculated ECD spectra. All but one of the 13 isolated compounds from K. longipedunculata potentiated I_GABA through GABA A receptors composed of α₁β₂γ_2S subunits in a concentration-dependent manner. Potencies ranged from 12.8 ± 3.1 to 135.6 ± 85.7 μM, and efficiencies ranged from 129.7 ± 36.8% to 885.8 ± 291.2%. The phytochemical profiles of petroleum ether extracts of Kadsura japonica fruits (114.1 ± 2.6% potentiation of I_GABA at 100 μg/ml, n = 2), and Schisandra chinensis fruits (inactive at 100 μg/ml) were compared by HPLC-PDA-ESIMS with that of K. longipedunculata.