gamma - aminobutyric acid ( gaba ) receptora ( gabaa ) receptors are members of the large  cys - loop super - family of evolutionarily related and structurally similar ligand - gated ion channels that also includes the nicotinic acetylcholine , glycine , and 5-ht3 receptors . the gaba receptor is predominantly expressed in the central nervous system , and forms a chloride - selective transmembrane channel in the post - synaptic sites of nerve terminals . for example , kimura et al .   showed that ginsenosides differentially regulate [ h]-f1unitrazepam or [ h]-muscimol binding to the gabaa receptor in a rat brain membrane fraction . kim et al .   reported that prolonged infusion with ginsenoside rc , but not ginsenoside rg1 , into rat brain elevates [ h]-muscimol binding to the gabaa receptor in a brain region - specific manner . thus , ginsenosides may regulate the gabaa receptor by affecting ligand affinity for its receptor , but there is no direct evidence on the regulation of gabaa receptor channel activity by ginsenosides . on the other hand , choi et al .   showed that ginsenoside rc enhances gaba - mediated ion currents in oocytes expressing the gabaa receptor . it is known that protopanaxadiol and protopanaxatriol ginsenosides are metabolized into protopanaxadiol ( ppd ) and m4 , respectively , by gastric juice and intestinal microorganisms after ginseng intake . however , little is known about whether ppd or m4 regulates gabaa receptor channel activity . in the present study we injected neuronal human gabaa ( 112s ) receptor crnas into xenopus oocytes and examined the effect of ginsenoside metabolites on the gaba - elicited inward peak currents ( igaba ) . treatment with ginsenoside metabolites inhibited igaba in a reversible , dose - dependent and non - competitive manner . the ginsenoside metabolites m4 and ppd were provided by the ambo institute ( seoul , korea ) ( fig . all other reagents were purchased from sigma - aldrich ( st . louis , mo , usa ) . xenopus laevis frogs were purchased from xenopus i ( ann arbor , mi , usa ) . their care and handling were in accordance with the highest standards of institutional guidelines of konkuk university . for isolation of oocytes , frogs were anesthetized with an aerated solution of 3-amino benzoic acid ethyl ester followed by removal of ovarian follicles . the oocytes were treated with collagenase and then agitated for 2 h in a ca - free medium containing 82.5 mm nacl , 2 mm kcl , 1 mm mgcl2 , 5 mm hepes , 2.5 mm sodium pyruvate , 100 units / ml penicillin , and 100 g / ml streptomycin . stage v - vi oocytes were collected and stored in nd96 medium ( in mm : 96 nacl , 2 kcl , 1 mgcl2 , 1.8 cacl2 , and 5 hepes , ph 7.5 ) supplemented with 50 g / ml gentamicin . the oocyte - containing solution was maintained at 18 with continuous gentle shaking and renewed daily . electrophysiological experiments were performed within 5 to 6 d of oocyte isolation , with ginsenoside metabolites added to the bath . for gabaa receptor activity experiments , each gabaa receptor subunit - encoding crna ( 40 nl ) was injected into the animal or vegetal pole of each oocyte one day after isolation , using a 10 l microdispenser ( vwr scientific , san francisco , ca , usa ) fitted with a tapered glass pipette tip 15 to 20 m in diameter . a custom - made plexiglas net chamber the oocytes were impaled with two microelectrodes filled with 3 m kcl ( 0.2 - 0.7 m ) , and electrophysiological experiments were carried out at room temperature using an oocyte clamp ( oc-725c ; warner instruments , hamsden , ct , usa ) . stimulation and data acquisition were controlled with a pclamp 8 ( axon instruments , union city , ca , usa ) . for most electrophysiological experiments , oocytes were perfused initially with nd96 solution ( in mm : 96 nacl , 3 kcl , 2 cacl2 , and 5 hepes ; ph 7.4 with naoh ) and control current recordings were obtained . for most electrophysiological data , the oocytes were clamped at a holding potential of 80 mv . for current and voltage ( i - v ) relationship , voltage ramps were applied from 100 to + 40 mv for 300 ms . in the different membrane - holding potential experiments , linear leak and capacitance currents were corrected by means of the leak subtraction procedure . to obtain the concentration - response curve for the effect of ginsenoside metabolites on the inward peak igaba mediated by the gabaa receptor , the igaba peak was plotted at different concentrations of gaba and origin software ( origin , northampton , ma , usa ) was used to fit the plot to the hill equation : y / ymax=[a]/([a ] + [ ic50 ] ) , where y is the peak current at a given concentration of ginsenoside metabolites , ymax is the maximal peak current , half - inhibitory concentration ( ic50 ) is the concentration of ginsenoside metabolites producing a half - maximal effect , [ a ] is the concentration of ginsenoside metabolites , and nh is the hill coefficient . the significance of differences between the mean control and treatment values was determined using student s t - test the ginsenoside metabolites m4 and ppd were provided by the ambo institute ( seoul , korea ) ( fig . all other reagents were purchased from sigma - aldrich ( st . louis , mo , usa ) . xenopus laevis frogs were purchased from xenopus i ( ann arbor , mi , usa ) . their care and handling were in accordance with the highest standards of institutional guidelines of konkuk university . for isolation of oocytes , frogs were anesthetized with an aerated solution of 3-amino benzoic acid ethyl ester followed by removal of ovarian follicles . the oocytes were treated with collagenase and then agitated for 2 h in a ca - free medium containing 82.5 mm nacl , 2 mm kcl , 1 mm mgcl2 , 5 mm hepes , 2.5 mm sodium pyruvate , 100 units / ml penicillin , and 100 g / ml streptomycin . stage v - vi oocytes were collected and stored in nd96 medium ( in mm : 96 nacl , 2 kcl , 1 mgcl2 , 1.8 cacl2 , and 5 hepes , ph 7.5 ) supplemented with 50 g / ml gentamicin . the oocyte - containing solution was maintained at 18 with continuous gentle shaking and renewed daily . electrophysiological experiments were performed within 5 to 6 d of oocyte isolation , with ginsenoside metabolites added to the bath . for gabaa receptor activity experiments , each gabaa receptor subunit - encoding crna ( 40 nl ) was injected into the animal or vegetal pole of each oocyte one day after isolation , using a 10 l microdispenser ( vwr scientific , san francisco , ca , usa ) fitted with a tapered glass pipette tip 15 to 20 m in diameter . a custom - made plexiglas net chamber was used for two - electrode voltage - clamp recordings as previously reported . the oocytes were impaled with two microelectrodes filled with 3 m kcl ( 0.2 - 0.7 m ) , and electrophysiological experiments were carried out at room temperature using an oocyte clamp ( oc-725c ; warner instruments , hamsden , ct , usa ) . stimulation and data acquisition were controlled with a pclamp 8 ( axon instruments , union city , ca , usa ) . for most electrophysiological experiments , oocytes were perfused initially with nd96 solution ( in mm : 96 nacl , 3 kcl , 2 cacl2 , and 5 hepes ; ph 7.4 with naoh ) and control current recordings were obtained . for most electrophysiological data , the oocytes were clamped at a holding potential of 80 mv . for current and voltage ( i - v ) relationship , voltage ramps were applied from 100 to + 40 mv for 300 ms . in the different membrane - holding potential experiments , to obtain the concentration - response curve for the effect of ginsenoside metabolites on the inward peak igaba mediated by the gabaa receptor , the igaba peak was plotted at different concentrations of gaba and origin software ( origin , northampton , ma , usa ) was used to fit the plot to the hill equation : y / ymax=[a]/([a ] + [ ic50 ] ) , where y is the peak current at a given concentration of ginsenoside metabolites , ymax is the maximal peak current , half - inhibitory concentration ( ic50 ) is the concentration of ginsenoside metabolites producing a half - maximal effect , [ a ] is the concentration of ginsenoside metabolites , and nh is the hill coefficient . the significance of differences between the mean control and treatment values was determined using student s t - test the addition of gaba ( 10 m ) to the bathing medium induced a large inward current ( igaba ) in oocytes injected with gabaa receptor subunits crnas , indicating that the gabaa receptor was functionally expressed ( fig . expressing gabaa receptor , the treatment of ginsenoside metabolites such as m4 or ppd had no effect . moreover , co- or pre - treatment with ginsenoside metabolites for 30 s with gaba induced an inhibition of igaba in a reversible manner ( fig . 2a , b ; n=15 from three different frogs ) . as shown in fig . 2c , m4 more significantly inhibited igaba than ppd . in concentration - response experiments with m4 or ppd , co - treatment with m4 and ppd inhibited igaba in a dose - dependent manner in oocytes expressing the gabaa receptor ( fig . the ic50 of igaba by m4 and ppd was 17.12.2 and 23.18.6 m , respectively , in oocytes expressing the gabaa receptor ( n=9 - 12 from three different frogs at each point ) ( fig . the membrane potential was held at 80 mv and a voltage ramp was applied from 100 to + 40 mv for 300 ms . in the absence of gaba , the inward current at 100 mv was < 0.3 a and the outward current at + 40 mv was 0.3 - 0.5 a . the addition of gaba to the bathing medium resulted in an increase of the inward current at a potential more negative than 20 mv . in contrast , at a potential more positive than 20 mv , gaba caused a large increase in the outward current . co - treatment of gaba with m4 or ppd inhibited both inward and outward currents as compared with those induced by gaba treatment alone . the reversal potential was near 20 mv in gaba alone and in gaba + m4 or ppd , which indicates that gaba induces the ci current . also , co - treatment of gaba with m4 or ppd did not affect the channel property of the gabaa receptor ( fig . , our results further revealed that the inhibitory effects of m4 and ppd on igaba in oocytes expressing human gabaa receptors were independent of the membrane holding potential ( fig . holding potentials of 100 , 80 , 60 , 40 , and 20 mv , m4 inhibited igaba by 53.82.5% , 52.44.0% , 52.81.5% , 54.11.7% and 53.92.9% , respectively ( n=9 - 12 , from three different frogs ) . ppd inhibited igaba by 20.91.9% , 23.31.4% , 22.13.8% , 20.71.3% and 21.63.0% , same respective order ( n=9 - 12 , from three different frogs ) . to begin studying the mechanism by which m4 or ppd inhibit igaba in oocytes expressing human gabaa receptors , we analyzed the effect of 100 m m4 or ppd on the igaba evoked by different gaba concentrations ( fig . co - treatment of oocytes expressing human gabaa receptors with 100 m m4 or ppd plus different concentrations of gaba did not significantly shift the dose - response curve of gaba to the right . the ec50 values were 15.82.5 , 18.32.4 and 15.42.6 m for gaba alone , gaba+m4 and gaba+ppd , respectively , and the hill coefficients were 1.6 , 1.5 and 1.7 , in the same respective order . thus , m4 and ppd significantly inhibited the igaba elicited by 10 , 30 , and 100 m of gaba , independent of the gaba concentration ( n=6 - 8 from three different frogs ) ( fig . aglycone is the backbone of the ginsenoside , with a hydrophobic four - ring steroid - like structure that may be non - polar , whereas the carbohydrates on carbons 3 , 6 , and 20 of the backbone are polar ( fig . have shown that ginsenosides administered orally are metabolized and finally become an agylcone such as m4 and ppd . these ginsenoside metabolites might also induce apoptosis of cancer cells and play a role as anti - cancer agents , suggesting that ginsenosides are pro - drugs of these metabolites . however , relatively little is known regarding how ginsenoside metabolites regulate ion channel or receptor activity , especially on gabaa receptor . in the present study , we investigated the effects of ginsenoside metabolites on human gabaa receptors heterologously expressed in xenopus oocytes . we found that co- or pre - treatment with m4 rather than ppd induced a large inhibition of igaba in reversible manners , treatment of m4 and ppd inhibited igaba in oocytes expressing gabaa receptors in concentration - dependent manners , and inhibition of igaba by treatment of m4 or ppd occurred in a voltage - independent and non - competitive manner in oocytes expressing gabaa receptors . these results indicate that the ginsenoside metabolites m4 and ppd might be novel gabaa receptor regulators . gaba is one of major inhibitory neurotransmitters in the mammalian brain and it has long been known that many neuroactive drugs , such as the benzodiazepines , nonbenzodiazepines , barbiturates , ethanol , neuroactive steroids , anaethetics , and picrotoxin interact with gabaa receptors by binding to modulatory sites of the receptor . thus , gabaa receptor regulators are clinically important for treatment of various mental dysfunctions . for example , benzodiazepines , which are known to potentiate igaba , have been used for their anxiolytic , sedative , and myorelaxant effects . however , many unwanted side effects such as amnesic - like effects , ataxia , ethanol and barbiturate potentiation , and tolerance and dependence , appear and result in a reduction in the therapeutic value of benzodiazepines [ 13 - 15 ] . on the other hand , we have shown that ginsenoside rc enhances igaba 
 ; these results are well coupled to the report that some subsets of ginsenosides have anxiolytic - like effects in animal model . presently , m4 and ppd displayed inhibitory effects on igaba rather than enhancing effects , indicating the possibility that ginsenoside metabolites are different from intact ginsenosides in the regulation of gabaa receptor - mediated ion channel regulations . we have previously reported that ginsenoside metabolites such as compound k ( ck ) , m4 , and ppd differentially regulate ion channels and receptors . for example , ck , but not protopanaxatriol ( ppt ) , potently inhibits the voltage - dependent 1g - type ca channel . similarly , we also demonstrated that ck , but not m4 ( ppt ) , inhibits a neuronal na ( nav1.2 ) channel , and that m4 , but not ck , inhibits 5-ht3a receptor - mediated currents . recently , we also reported that , although ppd itself does not affect human herg k channel activity , it inhibits ginsenoside rg3-mediated decelerating effects of herg k channel currents . in the present study , m4 more strongly inhibited gabaa - receptor - mediated ion currents than ppd . the previous and present findings indicate that ginsenoside metabolites as well as ginsenosides have regulatory effects on voltage - dependent ion channel and receptor activities , but they differentially affect the regulation of ion channels or receptors . in summary , we have examined the effects of ginsenoside metabolites such as m4 and ppd on human gabaa receptor channel activities heterologously expressed in xenopus oocytes . human gabaa receptors are mainly involved in the modulations of various physiological and pathophysiological activities in the central nervous system , the inhibitory effects of m4 on igaba could provide a molecular basis for the pharmacological actions of ginsenoside metaobolites in the nervous system .