Direct activation of GABAA receptors by barbiturates in cultured rat ...

Journal of Phy8iology (1996), 497.2, pp.509-522

Direct activation of GABAA receptors by barbituratesin cultured rat hippocampal neurons

Jong M. Rho, Sean D. Donevan and Michael A. Rogawski *

Neuronal Excitability Section, Epilepsy Research Branch, National Institute of NeurologicalDisorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA

1. The direct activation of the GABAA receptor by pentobarbitone (PB) and phenobarbitone(PHB) was characterized in cultured rat hippocampal neurons using whole-cell voltage clampand single channel recording techniques.

2. In whole-cell recordings, PB and PHB produced a concentration-dependent activation of Cl-current (EC50 values, 0 33 and 3 0 mm, respectively). The response to the barbiturates wassimilar to that produced by GABA, although GABA was more potent (EC50, 5.5 /UM). PB andPHB were substantially more potent in enhancing the response to 1 ,UM GABA (EC50 values,94 uM and 0'89 mm, respectively). The maximal magnitude of the responses to PB wassimilar to that of the maximal response to GABA or GABA + PB. PHB appeared to bemodestly less efficacious.

3. The mean deactivation time constant for whole-cell Cl- currents evoked by 1 mm PB + 1 /SMGABA was significantly longer (480 + 34 ms) than for 1 mm PB (170 + 9 ms) or 1 /M GABA(180 + 14 ms) alone.

4. Whole-cell currents directly activated by 300 /M PB and 1 FM GABA were blocked by theGABA receptor antagonists bicuculline and picrotoxin.

5. Unitary GABAA receptor channel currents evoked by 300 FM PB had similar mainconductance, mean open time and mean burst duration as those activated by 2 FM GABAalone. Single channel openings and bursts were of shorter mean duration when 100 and300 FM PHB were used.

6. High concentrations of PB (1-3 mM) and PHB (3-10 mM) produced a rapid block of currentsactivated by the barbiturate alone or by the barbiturate in the presence of 1 FM GABA. Theestimated IC50 values for block of PB- and PHB-potentiated GABA currents were 2-8 and12'9 mm, respectively.

7. Single channel currents activated by high concentrations of PB and PHB alone or in thepresence of GABA demonstrated flickering, probably reflecting fast channel block.

8. We conclude that the gating of the GABAA receptor channel by PHB and PB is functionallysimilar to that produced by the natural agonist GABA alone, but distinct from that obtainedwhen barbiturates modulate the response to GABA. At high concentrations, the barbituratesproduce a channel blocking action that limits the maximum total current conducted by thechannel.

Hypnotic-anaesthetic barbiturates such as pentobarbitone GABAA receptor-modulating barbiturates, such as PB and(PB) and the anticonvulsant barbiturate phenobarbitone PHB, exert at least two separate actions on GABAA(PHB) are well known to interact with the GABAA receptor- receptors: they enhance the activation of the receptor byCl- channel complex. Although barbiturates have been GABA recognition-site agonists (Ransom & Barker, 1976;reported to inhibit excitatory amino acid receptors Macdonald & Barker, 1979), and, at high concentrations,(Marszalec & Narahashi, 1993) and to block voltage- they directly activate the receptor in the absence of suchactivated Ca2P currents (ifrench-Mullen, Barker & Rogawski, agonists (Mathers & Barker, 1980; Nicoll & Wojtowicz,1993), a major action of barbiturates is allosteric modulation 1980; Suzdak, Schwartz, Skolnick & Paul, 1986; Yang &of the GABAA receptor complex (Macdonald & Olsen, 1994). Olsen, 1987; Robertson, 1989).

* To whom correspondence should be addressed.

5740 509

J M. Rho, S. D. Donevan and M. A. Rogawski

The potentiation of GABA-activated Cl- currents by PB hasbeen studied using current fluctuation analysis (Barker &McBurney, 1979; Study & Barker, 1981) and more recentlywith single channel recording techniques. Barbituratesincreased the mean open time of GABA-activated singlechannel currents without affecting the open frequency or theconductance of the main open state (Macdonald, Rogers &Twyman, 1989b). Single channel recordings have indicatedthat there are at least three kinetically distinct open statesof the GABAA receptor. PB and PHB, at clinically relevantconcentrations, did not alter the kinetic parameters of theprincipal open states; rather, they increased the relativeproportion of longer openings, thereby increasing theoverall mean open duration (Macdonald et al. 1989 b).

The direct actions of barbiturates on GABAA receptors inthe absence of GABA recognition-site agonists have beenless well characterized. Mathers & Barker (1980) firstdescribed the direct activation of Cl- current by (-)-PB instudies of voltage-clamped mouse spinal neurons in culture.Current fluctuation analysis suggested that both GABA and(-)-PB activated ion channels of similar conductance;however, PB-activated channels remained open longer thanthose activated by GABA. Several additional studies haveconfirmed that barbiturates can directly activate GABAAreceptors. In experiments with isolated membrane vesiclesfrom rat cerebral cortex, Suzdak et al. (1986) reported thatPB (0-2 mM) directly stimulated the uptake of 36CF. Later,Yang & Olsen (1987) found that barbiturates directlystimulated the efflux of 36CF- in mouse cortical slicespreloaded with the isotope (EC50, 0 3 mM). Finally, PB wasobserved to directly activate Cl- currents in Xenopusoocytes injected with rat or chick brain poly(A)+ mRNA(Parker, Gunderson & Miledi, 1986). Recently, amodification in the fl-subunit of the GABAA receptor hasbeen described that eliminates responsiveness to GABA butnot to PB (Amin & Weiss, 1993). This provides compellingevidence that barbiturate activation of the GABAA receptoroccurs independently of the GABA recognition site.

Despite previous evidence that barbiturates can directlyevoke a Cl-current in the absence of GABA, the directactivation of GABAA receptors by barbiturates has not beenstudied in detail. Therefore, in the present study, weutilized whole-cell and single channel recording techniquesto characterize the kinetic properties of channel activationby PB and PHB, and to compare the direct actions of thesebarbiturates with those of GABA, the endogenous ligand.

METHODSCell cultureHippocampal neurons were grown in monolayer culture asdescribed previously (Segal, 1983; Donevan, Jones & Rogawski,1992). In brief, timed pregnant Sprague-Dawley rats with 19-day-old embryos were anaesthetized by CO2 narcosis, the fetuses wereremoved and killed by decapitation, and the mothers weresubsequently killed by cervical dislocation in accordance with the

Animals (Scientific Procedures) Act 1986. (All animal procedureswere carried out in strict compliance with the National Institutes ofHealth (NIH) Guide for the Care and Use of Laboratory Animals,under a protocol approved by the NIH Animal Use Committee.) Thefetal hippocampi were dissected, triturated in modified minimalessential medium with Earle's salt (Advanced Biotechnologies,Columbia, MD, USA), and plated on 35 mm polystyrene Petridishes (Falcon 3001; Becton Dickinson Labware, Oxnard, CA,USA) precoated with Matrigel (Collaborative Biomedical Products,Bedford, MA, USA). The plating medium was supplemented withN3 (composed of transferrin, putrescine, sodium selenite, tri-iodothyronine, insulin, progesterone and corticosterone), horseserum (Gibco), fetal calf serum, and glutamine (Bottenstein, 1985;Guthrie, Brenneman & Neale, 1987). Cell cultures were placed in ahumidified atmosphere containing 10% CO2 at 37 °C for 6-12 daysprior to use. Fresh growth medium (without fetal calf serum andN3) was added after 6 days in culture.

SolutionsPrior to each recording session, the culture medium was replacedby bathing solution containing (mM, except as noted): 145 NaCl,10 Hepes, 2-5 KCl, 0-1 CaCl2, 10 glucose, 1 ,CM tetrodotoxin (toblock voltage-activated Na+ channels), and 1 /SM strychnine (to blockglycine-activated Cl- currents). The bath solution was adjusted toan osmolality of 315-325 mosmol kg' with sucrose and to a pH of7-4 with NaOH. For experiments in which the extracellularconcentration of Cl- was varied, NaCl was replaced with sodiumgluconate.

The micropipette filling solution contained (mM): 145 CsCl, 0-1CaCl2, 10 Hepes, and 1 EGTA (adjusted to an osmolality of305-310 mosmol kg-' and a pH of 7 2). In excised patchrecordings, Mg-ATP (2 mM) was included in the intracellularsolution to retard run-down.

All drugs and chemicals, except where otherwise noted, wereobtained from Sigma or Aldrich.

ElectrophysiologyPatch pipettes (4-8 MQI) were prepared from filament-containingthin-walled glass capillary tubes (1-5 mm outer diameter; WorldPrecision Instruments, Sarasota, FL, USA) using a four-stagehorizontal pipette puller (Model P-80/PC Flaming Brown, SutterInstrument Co., Novato, CA, USA). Micropipette tips wereroutinely fire polished, and, for single channel recordings, werecoated with Sylgard (Dow Corning).

All electrophysiological recordings were conducted on the stage of aNikon Diaphot inverted phase-contrast microscope at roomtemperature (23-25 °C). Currents were monitored with either anAxopatch 1B or 200A patch-clamp amplifier (Axon Instruments).Voltages corresponding to the currents were acquired with a high-speed chart recorder (Gould), and digitized for off-line analysis. Theholding potential for whole-cell recordings was -60 mV unlessotherwise noted.

Unitary GABAA receptor currents were recorded in excised outside-out membrane patches. The currents were filtered at 1 kHz (-3 dB;four-pole, low-pass Bessel filter) and digitally sampled at 10 kHz.The holding potential was -80 mV. Drugs were applied for 20-60 stime periods, separated by 30-60 s washes with bathing solution.

Drug applicationDrugs were dissolved in bathing solution on the day of use andapplied via a rapid perfusion system consisting of a four- or nine-barrel array in which all barrels (0-32 mm outer diameter; J & WScientific, Folsom, CA, USA) emptied via a common tip positioned

510 J. Phy8iol.497.2

Barbiturate activation of GABAA receptors

-200 #um from the cell under study. Flow through each barrel wasgravity fed and regulated by high-speed solenoid microvalves (LeeCo., Westbrook, CT, USA) operated by a programmablemicroprocessor-based controller. Switching between solutionsoccurred within < 10 ms (see Donevan et al. 1992). One barrelcontained buffer and the others were filled with various drugs aloneand in combination. Only one valve was open at a time, and thebuffer solution was applied continuously between drug applications.In whole-cell recordings, applications of GABA, PHB and PB,either alone or in combination, were separated by a time interval ofat least 1 min to minimize desensitization.

Analysis of data from whole-cell recordingsWhole-cell currents were analysed off-line using the Axotapesoftware package and the Clampfit module of the pCLAMP softwarepackage (Axon Instruments). Concentration-response data werefitted using a non-linear least-squares program (NFIT, IslandProducts, Galveston, TX, USA) to the logistic equation:

I/Io = Rmax/(1 + (EC50/[D])nH) + C,

where I/Io represents the ratio of potentiated current (I) and thecontrol current (IO) evoked by 1 /uM GABA, Rmax is the maximumratio, EC50 is the concentration producing 50% maximum effect,[D] is the drug concentration, nH is an empirical parameterdescribing the steepness of fit (equivalent to the Hill coefficient),and C is 0 for direct responses or 1 for barbiturate potentiation of1 /uM GABA responses. Concentration-response curves for blockwere fitted to the function 100/(1 + (IC50/[D])UH), where IC50 is theconcentration producing 50% block.

A

B

GABA1 #M

GABA1 /FM

>,

500 pA L

Analysis of data from excised-patch recordingsPatch currents were analysed using the Fetchan and pSTAT modulesof pCLAMP. Single channel openings were determined by detectingcurrent level changes exceeding a 50% threshold criterion. Openingsbriefer than 200 pss (twice the system dead time) were ignored.Only patches demonstrating infrequent multiple openings (no morethan 3 simultaneous openings apparent) were used for analysis;simultaneous channel openings were not included in the dataanalysis. Although GABAA receptor channels exhibit as many asfour conductance states (Hamill, Bormann & Sakmann, 1983;Bormann, Hamill & Sakmann, 1987), only the 30 pS mainconductance state was analysed. Bursts were defined as an openingor a series of closely spaced openings separated by relatively longclosed periods (Colquhoun & Hawkes, 1982). Operationally, burstswere taken to be openings in which closed intervals briefer than5 ms were ignored (Macdonald, Roger & Twyman, 1989a).

Mean open times and mean burst durations were determined fromthe pooled data of at least three separate patches. These pooledopen dwell-time values were displayed in the form oflogarithmically binned histograms, with the ordinate representingthe square root of the normalized counts per bin, similar to themethod of Sigworth & Sine (1987). The histogram data were fittedwith a second-order Gaussian function, yielding two timeconstants. Closed duration dwell times were displayed in linearlybinned frequency histograms, with bin sizes of 2 and 4 ms for PBand PHB, respectively. The closed duration histograms were fittedwith the sum of two exponential functions. Single channel data arepresented as means + S.E.M., unless otherwise indicated; n denotesthe total number of patches examined.

PHB300 FLM-M

PB PB200 /M 500 ,M

E-s_ e

PHB1 mM

PB1 mMm

PHB3 mM

Fig. 1. Phenobarbitone (PHB) and pentobarbitone (PB) directly activate inward currents incultured hippocampal neuronsNo GABA was present in the perfusion solution during the barbiturate applications. For comparison,responses to 1 ,UM GABA are also shown. A and B represent 2 separate cells. Holding potential, -60 mV.

J Physiol.497.2 511

B25 -

20 -

15 -

10 -

5-

10-7 10-6 10-5 10-4 10-3

[Drug] (M)10-2 10-1

° - I"I""II'II ""'I """'III IIII ' ""'I """'1I I"""'I10-7 10-6 10-5 10-4 10-3 10-2 10-1

[Drug] (M)

Figure 2. Concentration-response curvesActivation of whole-cell currents by GABA (O), PB (0) or PHB (a) alone (A), and PB (a) and PHB (0) in thepresence of 1 ,tM GABA (B). Currents were normalized to the control current evoked by 1 ,CM GABA. Errorbars not shown were smaller than the size of the symbols. Each point represents the mean + S.E.M. of datavalues from 3-17 cells.

APTX

PB

10

500 ,

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PTX -

GABA

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pA 250 pA

GABA

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0

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Figure 3. Currents evoked directly by PB and GABA are blocked by the GABAA receptor

antagonists bicuculline (BIC) and picrotoxin (PTX)A, representative whole-cell current traces demonstrating block by 10 M PTX of currents activated by300 /M PB (left) and 1 uM GABA (right). B, steady-state block of whole-cell currents by 10 and 100 uM BICand PTX. Data are normalized to the control currents evoked by 300 /SM PB and 1 /SM GABA. Each barrepresents the mean + S.E.M. of 3-8 cells studied. Mean amplitudes of PB and GABA-evoked currents were

1300 + 640 pA (n = 5) and 210 + 70 pA (n = 5), respectively. Holding potential, -60 mV.

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During the course of excised outside-out patch recordings there wasa high degree of variability in the channel open and burstfrequency, as observed previously (Twyman & Macdonald, 1992;Porter, Angelotti, Twyman & Macdonald, 1992). At present, theprecise factors that produce fluctuations in the open probability ofthe GABAA receptor are obscure; however, receptor desensitizationand run-down may play a role. Nevertheless, even with the additionof 2 mm Mg-ATP to the intracellular solution (which has previouslybeen shown to reduce run-down; Horn & Korn, 1992), suchvariability was still observed. Consequently, no attempt was madeto compare channel open probabilities among drug treatments.

RESULTSPHB- and PB-activated whole-cell currentsAs illustrated in Fig. 1, perfusion of hippocampal neuronswith PHB and PB (in the absence of GABA) resulted in the

activation of robust inward current responses. The currentdirectly activated by barbiturates was similar to thatactivated by 1IuM GABA except that the barbiturateresponse had a somewhat slower onset. Concentration-response curves for the peak current activated by GABAand the barbiturates alone and in combination are presentedin Fig. 2A and B. The current values are normalized withrespect to the peak 1 uM GABA response obtained in thesame cell. The maximum magnitudes of the barbiturateresponses nearly reached that obtained with GABA. TheEC50 values (and nH values), obtained from the best fits tothe concentration-response data according to the logisticfunction given in the Methods, were: GABA, 5-5 m (1P5);PB+ 1/UM GABA, 94 sM (1-6); PB, 033mM (2-3);PHB + 1 ,UM GABA, 089 mm (1P5); and PHB, 3 0 mm (2 2).

n.s.

Control

c

I I0-0 0-2 0-4

Time constant (s)

**

0-6

Figure 4. Deactivation rates for whole-cell currents evoked by PB, GABA alone, and PB in thepresence of GABAThe duration of drug perfusions varied from 3 to 5 s. A, deactivation rates derived from the best single-exponential fits to whole-cell current relaxations occurring at the end of drug perfusion. Fits excluded theinitial rebound. Each bar represents the mean time constant + S.E.M. for 4-10 cells. **P < 0-01 (Student's ttest, grouped data); n.s., not significant. B, representative whole-cell current traces after termination ofdrug perfusion (vertical line). Traces a, b and c represent deactivation of currents evoked by 300 uM PB,10/M GABA, and 300 /M PB + 1 /M GABA, respectively. The inset at the bottom of panel B shows thedeactivation currents scaled to the same amplitude to allow a better comparison of their time courses.

Holding potential, -60 mV.

A B

300 /FM PB

1 mM PB

1 uM GABA

3 FM GABA

10 uM GABA

300 uM PB +1 FM GABA

1 mM PB +1 FM GABA

513J Physiol.497.2

514 J M. Rho, S. D. Done

Currents directly activated by PB are carried by Cl-ionsThe similarity between the responses elicited by GABA andthe barbiturates raised the possibility that the barbiturate-induced current could be carried by Cl-, as is the GABAcurrent (Macdonald & Olsen, 1994). This possibility was

evaluated by determining the reversal potential of the300 /M PB response in the presence of three differentextracellular Cl- concentrations. With external Cl-concentrations of 33, 93 and 148 mm, the mean reversalpotential values were (mV): 37-5 + 0 3 (n = 4), 13-5 + 0 5(n =4) and 5-7 + 0-6 (n = 3), respectively. These valuesclosely match the theoretical values predicted by the Nernstequation.

?va?n and M. A. Rogawski J Phy8iol.497.2

Currents directly activated by PB are blocked byGABAA receptor antagonistsTo further investigate whether Cl- currents activated by PBare mediated by GABAA receptors, we examined whetherthe currents could be antagonized by the GABAA receptorantagonists bicuculline and picrotoxin. At a concentrationof 10 /SM, picrotoxin reduced the current activated by300 /SM PB (Fig. 3A, left) and at 100 /UM suppressed thecurrent completely (Fig. 3B, left). Similarly, 10 and 100 ,UMbicuculline markedly reduced the current, but even at thehigher concentration, the block was not complete. Forcomparison, we also determined the blocking effects ofpicrotoxin and bicuculline on currents activated by 1 ,lMGABA. As illustrated in Fig. 3A and B (right) both

2/SM GABA

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300 /FM PB

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Figure 5. Single-channel currents in three separate outside-out patches evoked by GABA, PBand PHBBurst openings marked by boxes are shown on an expanded scale to the right. Holding potential, -80 mV.

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Table 1. Mean open time and burst durations for unitary currents activated by PB, PHB and GABA

Agonist Concentration Mean open time Mean burst duration(/SM) (ms) (ms)

PB 30 10+0 05 2-7+00233(2424) 3(2168)

100 2-0+0 11 4-3+00563(2623) 3(1825)

300 2-4+0 15 8-0+103(1409) 3 (361)

PHB 100 1-0 + 0.16** 1.7 + 0.23*3(1153) 3(996)

300 1-0 + 0 07** 1.9 + 0.31**5(3294) 5(2210)

GABA 0-3 1P6+020 2-9+00353(1898) 3(1115)

1 2-0 + 0-41 8.0 + 0-915(2708) 5(2365)

2 2-4 + 0-43 8-3 + 0-935 (1290) 5(1485)

GABA+PB 1,30 3-9 + 0.46** 118+ 0.67*3(1842) 3(1641)

Numerical values indicate the means + S.E.M. of data from individual patches analysed separately. Thetotal number of patches are shown below each mean and the number of events is given in parentheses.Statistically significant differences in the mean open times and mean burst durations between comparableconcentrations of pentobarbitone (PB) and phenobarbitone (PHB) are indicated by asterisks, as aredifferences between 1 /iM GABA and 1 UM GABA + 30 uM PB. * P < 0.05 and ** P < 0.01 (Student'st test, grouped data). In addition, there were significant differences (P < 0 05) between the mean burstdurations, but not mean open times, of minimally effective concentrations of PHB (100 and 300 uM) and aminimally effective concentration of PB (30 uM).

antagonists produced a concentration-dependent depressionof the current with complete block at the higherconcentration.

PB- and GABA-activated currents exhibit similardeactivation kineticsThe time constants for current deactivation were determinedin experiments where the barbiturates and GABA, eitheralone or in combination, were applied to activate Cl- currentresponses and then abruptly terminated. Off-rates wereestimated by fitting the current decay at the termination ofthe perfusion to a single exponential function. The meandeactivation time constants for currents evoked by differentconcentrations of GABA (1, 3, and 10 /tM) or PB (300 /SMand 1 mM) alone were very similar, ranging between 110and 180 ms (Fig. 4A). However, the deactivation timeconstants for currents evoked by 300,uM or 1 mMPB + 1 ,UM GABA were significantly longer. Figure 4Bshows representative deactivation currents, in the same cell,for 300 /SM PB (a), 10 M GABA (b), and 300 /M PB + 1 uMGABA (c). The combined application of PB and GABAproduces a current of substantially greater amplitude andslower decay.

Single channel currents activated by PB, PHB andGABAUnitary currents induced by PHB (100-300 uM), PB(30-300 uM) or GABA (0-3-2 uM) in outside-out patchrecordings exhibited a principal conductance level of 30 pS(see Fig. 5 for sample records). Mean open time and meanburst duration values for the single channel currentsactivated by various concentrations of PB, PHB and GABAare given in Table 1. Except in the case of PHB (where itwas only possible to obtain reliable single channel data withminimally effective concentrations), longer mean open timesand burst durations occurred at the higher agonistconcentrations (P < 0'05; one-way analysis of variance). Themean open times and burst durations of currents activatedby PHB were briefer than those activated by comparableconcentrations of PB or GABA. Since PHB and PB havemarkedly different potencies, it is also noteworthy thatthere were significant differences between the mean burstdurations (but not mean open times) of minimally effectiveconcentrations of PHB (100 and 300 #M) and a minimallyeffective concentration ofPB (30 /M). The distinctive kineticproperties of the unitary currents activated by PHB arefurther demonstrated in the frequency distribution

J Physiol.497.2 515

J M. Rho, S. D. Donevan and M. A. Rogawski J Physiol.497.2

Table 2. Parameters of fits to open time and burst duration distributions of unitary currentsactivated by PB, PHB and GABA

Agonist Concentration Open time Burst durationCUM) (ms) (ms)

Ti T2 71 T2

PB 30 0-20 1P2 0-21 3-2100 0.19 2-4 0 30 6'1300 0-20 2'1 0-64 9-7

PHB 100 0-21 0-8 0-23 2-5300 0-18 1.1 0-21 3-8

GABA 0 3 0X19 1X4 0X23 5-11 0-21 19 0-25 5-32 0.19 2-4 0-18 6-1

GABA + PB 1,30 0-22 3-3 0-27 11P3

Summary of time constants derived from the best second-order Gaussian fits to logarithmically binnedfrequency distribution histograms as illustrated in Fig. 6. The two time constants (r, and 12) are similar foropenings and bursts evoked by GABA, PB and PHB, but the slow time constant value (12) is prolongedwhen 30 /M PB potentiates GABA-activated single channel currents.

A04

0Ixco

08 0.20)N

E 0-10z

0-0

BPB PB

100 101 102

D0-4

03

02

0*1

0oo100 10'

Time (ms)102

100 101 102 103

100 101Time (ms)

102 103

Figure 6. Open and burst dwell-time distributions for unitary currents activated by PB and PHBLogarithmically binned open time (A and C) and burst duration (B and D) frequency distributions forunitary currents evoked by 1 ,UM GABA (dotted lines) and two concentrations (100 #M, thin lines; 300 #M,thick lines) of PB and PHB. The histograms were fitted to second-order Gaussian functions withparameters as presented in Table 2. Each histogram is derived from pooled data obtained in 3-5 outside-out patch recordings.

516

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histograms of Fig. 6 where the open time and burstdurations for PHB-evoked unitary currents are shifted toshorter times compared with those of GABA-evoked currents.This is predominantly due to smaller long open and burstduration time constants (T2; Table 2). In contrast, thedistributions for PB and GABA are similar (see Fig. 6 andTable 2). Single channel currents recorded in the presence of1 uCM GABA + 30 juM PB exhibited distinctly longer opentimes than any concentration of the agonists alone.

Closed dwell-time distributions vary with barbiturateconcentrationClosed dwell-time distributions for unitary currentsactivated by PB and PHB are shown in Fig. 7. There is anoverall reduction in the closed times as the concentration ofPB is increased from 30 to 300 ,CM (Fig. 7A) and theconcentration of PHB is increased from 100 to 300 /IM(Fig. 7B). There is a dramatic increase in the proportion ofbriefer closings as the barbiturate concentration is increased(see legend to Fig. 7). There are also modest concentration-dependent reductions in the values of the fast and slow timeconstants (except in the T2 values for PB). Overall, theconcentration-dependent changes observed reflect theincreased frequency of opening and the shift frominterburst to intraburst events.

High concentrations of barbiturates cause channelblockWhole-cell currents activated by high concentrations ofPHB (3-10 mM) and PB (1-3 mM), either alone or in

combination with GABA (1 uSM), exhibited a transientrebound upon discontinuation of the agonist application(Fig. 8). This 'off-effect' phenomenon suggests that thebarbiturates have a channel-blocking action in addition totheir direct agonist and GABA-potentiating activities. Thus,the current rebound probably reflects rapid unblockingsuperimposed upon the slower decay of the potentiatedcurrent. The blocking potencies of the barbiturates wereestimated by comparing the amplitude of the peak currentimmediately prior to termination of the agonist perfusionwith the peak of the current transient occurringimmediately after the perfusion. As shown in Fig. 8(bottom), for both PB and PHB, there was a concentration-dependent increase in the degree of block. Using thepercentage block values from Fig. 8, IC50 values for PB- andPHB-potentiated GABA currents (derived from a logistic fitwith n. constrained to 1, i.e. assuming that one moleculecan block the open receptor channel and that full block couldbe achieved) were 3-2 and 19-3 mm, respectively. Forcurrents activated directly by PB and PHB, the estimatedIC50 values were 5*0 and 151 mm, respectively.

To examine the basis of the putative blocking phenomenonin more detail, recordings were conducted of unitarycurrents activated by various barbiturate concentrations,including the high concentrations producing apparentchannel block. As described above, increasing concentrationsof PB (30-1000 /M) produced a concentration-dependentprolongation in the open time and burst duration. However,at a concentration of 300,M, and more prominently at

BPB 1 -0 m, PHB

08

0-6

0-4

0-2

0 20 40 60Time (ms)

80 100 0

c

50 100 150Time (ms)

Figure 7. Closed dwell-time distributions for unitary currents activated by PB and PHBA, normalized distributions of dwell times for currents in patches exposed to 30 uM (a) and 300 UM (b) PB.B, distributions for 100 /M (c) and 300 uM (d) PHB. The histograms were fitted to the bi-exponentialfunction Ylexp(-t/71) + Y2exp(- t/r2), where t is time and T, and T2 are fast and slow time constants,respectively (smooth curves). Parameter values Y1, Y2, T, and T2 for the fits to a and b are 470, 290, 2-5 msand 18 8 ms, and 2300, 48, 1P5 ms and 18-9 ms, respectively; and for the fits to c and d are 110, 80, 7 0 msand 49-6 ms, and 1000, 350, 4 9 ms and 29-8 ms, respectively. A fit to data from an additional series ofexperiments with 100 /SM PB (not shown for clarity) gave parameter values of 1100, 130, 2-1 ms and18-8 ms. The fit to the normalized closed-time distribution of unitary currents activated by 2 /M GABA isrepresented by the dashed lines (parameter values, 2100, 40, 1P1 ms and 18-1 ms). Each histogram isderived from pooled data obtained in 3-6 outside-out patch recordings.

A

N

C00r0

0

0z 0-2

0-0200

J Physiol.497.2 517


1 mM, PB induced flickering of the unitary currents,reflecting fast channel block. This is manifest as a reversalin the trend toward longer open times with increasing PBconcentration (Table 1). Thus, in five patches exposed to1 mM PB the mean open time was 1P0 + 0X06 ms (2644events), a statistically significant reduction from the meanopen time with 300 ,UM PB (P < 0 01; t test).

DISCUSSIONIn this study we have confirmed that PB and PHB can, inthe absence of GABA recognition-site agonists, produce a

A GABA300 /FM

B

1 mM

GABA O

60-

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0

nA

PB

3 mM

0

concentration-dependent activation of GABAA receptors.Furthermore, we have demonstrated that GABAA receptorcurrents directly activated by barbiturates have certaindistinct properties from the currents evoked by barbituratesin the presence of GABA.

Whole-cell currentsIn whole-cell recordings, maximal concentrations of PBalone activated currents that were of equivalent magnitudeto the maximal GABA responses or the responses obtainedwith maximal PB + 1 /M GABA. Thus, PB by itself isequally efficacious to GABA in activating GABAA receptors,

PB3 mM

PHB10 mM

PHB

3 mM 10 mMm m

03 03

Figure 8. High concentrations of PB and PHB block whole-cell currents activated by thebarbiturates alone (A) or by the barbiturates + 1 j/m GABA (B)The block is reflected by transient increases in the current immediately after termination of the drugperfusion; this rebound is not seen with a high (supramaximal) concentration of GABA. The concentration-dependent increase in the degree of block is indicated in the bar chart. Percentage block was calculatedfrom the minimum current level immediately before termination of the perfusion and the peak currentlevel after termination of the perfusion.

J. Physiol.497.2518


although it is substantially less potent. The EC50 obtained inthe present study for direct activation of Cl- currentresponses by PB (0 33 mM) was similar to that previouslyreported in 36C1- flux studies (Yang & Olsen, 1987). In thepresence of 1 /M GABA, there was a shift to the left in theconcentration-response relationship for PB. Thus, GABAappears to enhance the binding affinity of PB. Thisphenomenon is complementary to the well recognizedability of PB to enhance the affinity of GABAA receptors forGABA (Willow & Johnston, 1980; Nicoll & Wojtowicz,1980; Olsen & Snowman, 1982). However, the maximalcurrent is similar whether GABA, PB or GABA + PB isused. Theoretically, the response in each case is limited bythe current-carrying capacity of channels in the fully openstate. However, whether such a fully open state is achievedis not known since, in single channel recordings, receptordesensitization was a confounding factor (see below).

Our finding that PB can induce currents comparable inamplitude to those evoked by GABA is in contrast to aprevious report that the maximal PB-induced current(estimated EC50, 0'6 mM) was only half that of GABA(Akaike, Shirasaki & Yakushiji, 1991). The reason for thediscrepancy is not apparent.

The maximal response to PHB appeared to be somewhatreduced from that of PB, so that PHB appeared to be apartial agonist (see also, ifrench-Mullen et al. 1993). PHBalso enhanced currents evoked by 1 /IM GABA. Here again,the maximal response was modestly less than that obtainedwith PB + 1 /uM GABA. The apparent partial agonism couldreflect reduced intrinsic efficacy of PHB in relation to PB,but more probably is due to channel block by PHB(discussed below).

Reversal potential measurements confirmed that thecurrent activated by PB is carried by Cl-. This is consistentwith the possibility, but does not prove, that PB opensGABAA receptors. The current activated by PB is not due toan action on glycine receptors since 1 uM strychnine wasused in the perfusion buffer and in all drug solutions.

Further evidence that barbiturates can act directly onGABAA receptors is provided by our finding that theGABAA receptor antagonists bicuculline and picrotoxinproduced a similar block of whole-cell currents evoked by300,uM PB and 1 uM GABA (Fig. 3). Interestingly,picrotoxin was more potent than bicuculline in blocking PB,whereas the reverse situation applied for GABA. Picrotoxinis an allosteric inhibitor of the GABAA receptor that acts ata distinct, but poorly defined site on the receptor-Cl-channel complex (Olsen, 1981; Yoon, Covey & Rothman,1993). It has been proposed that barbiturates and picrotoxinact in a functionally reciprocal fashion, with barbituratesprolonging the time spent in a long duration open state andpicrotoxin having the opposite effect (Twyman, Rogers &Macdonald, 1989). Whether this functional interactionreflects a direct interaction at a common site on the GABAAreceptor is not clear. However, as illustrated in Fig. 3A,

picrotoxin block of PB-activated current occurred slowlycompared with the rapid block of GABA-activated current(Fig. 3B). This may indicate a requirement for PBunbinding in order for picrotoxin block to occur, and wouldbe consistent with binding of PB and picrotoxin to the sameor adjacent sites.

Bicuculline, an antagonist at the GABA recognition site,also partially inhibited the current activated by PB. SincePB is not believed to interact with the GABA recognition site,the block presumably occurs by an allosteric mechanism.(Bicuculline has previously been shown to antagonize directPB-induced hyperpolarizations of frog motoneurons; Nicoll& Wojtowicz, 1980.) As noted above, agonist binding to theGABA recognition site enhances binding to the barbituratesite (i.e. reduces the barbiturate EC50). Thus, there appearsto be a reciprocal relationship such that agonists at theGABA recognition site allosterically promote barbituratebinding and antagonists inhibit such binding.

Amin & Weiss (1993) recently demonstrated that asubstitution of tyrosine and threonine in each of two /2 -subunits of a cloned rat GABA receptor (Lzfl2y2) results in asubstantial decrease in sensitivity to GABA and muscimol,but no change in the ability of PB to directly activate thechannel. This finding supports the view that GABA and PBactivate GABAA receptors by binding at separate sites.

Barbiturates may, in fact, interact with the GABAA receptorvia two distinct sites: the well-recognized allostericregulatory site, where barbiturates modulate the action ofGABA (and perhaps also induce direct activation of thechannel), and a low-affinity channel-blocking site. Wepropose that block of the channel at this latter site limits theextent to which barbiturates can enhance and directlyactivate Cl- current responses. Data from our study supportthe existence of this channel-blocking site. Thus, weobserved transient increases ('rebound') in barbiturate-evoked or barbiturate-potentiated GABA currents uponabrupt termination of the barbiturate perfusion. Weinterpret this rebound as the relief of channel blockoccurring as a result of the rapid dissociation of thebarbiturate from the low-affinity channel blocking site inthe face of a relatively slow relaxation of the agonist effect.

Characterization of the deactivation of whole-cell currentsinduced by PB, GABA and PB + GABA provided furtherinsight into the interaction between the barbiturate andGABA binding sites. Thus, the whole-cell currents activatedby PB and GABA alone deactivated in a roughly single-exponential fashion with time constants in the range of100-150 ms (Fig. 4). In contrast, there was a dramaticslowing in the deactivation rate of PB-potentiated GABAcurrents, so that the deactivation time constants were two-to threefold longer. This implies that, when GABA and PBboth bind to the GABAA receptor, there may be mutualstabilization of the binding of these ligands to theirrespective recognition sites. Moreover, the observation thatthe PB current has distinctly different deactivation

J Physiol.497.2 519


properties from the current induced by GABA + PBunequivocally eliminates the possibility that there aresignificant trace levels of GABA present in the perfusionswith PB alone.

GABA and barbiturate-activated whole-cell currents differin another respect. At millimolar concentrations, both PBand PHB exhibit a blocking action (Fig. 8) when they eitherdirectly evoke currents or when they potentiate GABAresponses. However, there is no blocking action whencurrents are evoked by supramaximal concentrations ofGABA alone.

The mechanism by which barbiturates mediate this blockingaction is not fully defined, although open channel block is alikely possibility. We derived crude estimates of theblocking potencies of the whole-cell block seen withbarbiturate-potentiated GABA currents, and it is clear thatPB is a more potent blocker than PHB (IC50 values, 2-75 vs.12-9 mm, respectively). Single channel recordings showedfrequent but very brief openings, often referred to as'flickery' block.

Single channel currentsThe principal conductance state of unitary currentsactivated by PB and PHB was approximately 30 pS. This issimilar to previously reported values for the GABAAreceptor (Bormann et al. 1987; Macdonald et al. 1989b),supporting the view that the currents directly activated bythe barbiturates are carried by GABAA receptors. The meanopen times and burst durations of the unitary currentsactivated by PB and GABA were similar (Table 1). For bothagonists, the mean open and burst dwell times increased ina concentration-dependent fashion.

GABAA receptors activated by GABA exhibit severaldifferent open states. It has been previously observed thatthe mean open dwell time increases with increasing GABAconcentration due to a shift toward longer openings(Macdonald & Olsen, 1994). There are believed to be twobinding sites for GABA on each GABAA receptor complex(Macdonald & Olsen, 1994). Presumably, occupation of one ofthese sites, as would occur with low GABA concentrations,induces a transition of the channel to the first open statefrom which closing is rapid. At higher GABA concentrations,in which case a greater number of receptors have both GABAbinding sites occupied, the channel is more easily able toenter open states from which closing occurs more slowly. Ourresults, as summarized in Tables 1 and 2, confirm priorstudies showing a shift in the relative frequency of longeropenings with higher GABA concentrations. PB induced asimilar concentration-dependent shift in opening frequency,so that the mean open and burst dwell times increased withincreasing PB concentration. This implies that there may betwo (or more) binding sites for PB on the GABAA receptor.In fact, the Hill slope of 2-3 for PB in the isotherm ofFig. 2A is compatible with the presence of more than one PBbinding site.

In contrast to the situation with PB, unitary currentsactivated by PHB were generally briefer than thoseactivated by GABA. Thus, the apparent partial agonism ofPHB may be due to its inability to activate the channelbeyond the initial first (rapidly closing) open state. Thisprobably results from a difference in the intrinsic activity ofPHB. (Although it is theoretically possible that the GABAAreceptor is capable of binding only one molecule of PHB,this is unlikely given the Hill slope of the concentration-response curve for PHB activation of GABA receptorcurrent (n = 2-2; Fig. 2A).) The channel blocking activityof PHB mainly occurs at higher concentrations and wouldnot be expected to be a major factor in the kinetic propertiesof the unitary currents induced by 100 and 300 uM PHB.

The combination of GABA + PB resulted in substantiallylonger openings and bursts than with GABA or PB alone.Thus, openings induced by PB alone are not due topotentiation of contaminating GABA; rather, at the singlechannel level, PB potentiation of GABA is clearlydistinguishable from direct activation by PB.

It has previously been observed that barbiturates increasethe mean channel open duration of GABAA receptors(Barker & McBurney, 1979; Study & Barker, 1981;Macdonald et al. 1989 b). This has been reported to occur asa result of a shift in the distribution of openings to favourthose with longer duration. If we assume that the opentimes for GABA represented by T, and r2 in Table 2 reflectthe two fastest decaying open states, then in the case ofGABA + PB, T2 (3 3 ms) may reflect a third, slowerdecaying open state. Consequently, our data withGABA + PB are in agreement with the results of previousstudies examining the effects of PB on GABAA receptorchannel kinetic properties.

In contrast to our results, Mathers & Barker (1980), in astudy using fluctuation analysis, observed that PB-activatedchannels had longer open times than those activated byGABA. However, the microperfusion method used in thisearlier investigation would not have been expected to aseffectively wash away endogenous GABA as the rapidperfusion system of the present study. Consequently, it ispossible that there was to some extent contamination bylong openings induced by GABA + PB.

Prior kinetic studies of single GABAA receptor channelshave indicated the existence of multiple (as many as 10)closed states (Macdonald et al. 1989a; Twyman et al. 1989;Twyman & Macdonald, 1992). We observed that closed-duration histograms were adequately fitted to second-orderexponential functions. The two time constants derived fromthese fits largely represent the closings within and betweenbursts. No attempt was made to fit the data to morecomplex kinetic schemes. As is apparent from the data inFig.7 (legend), PB and PHB produce a dramaticconcentration-dependent increase in the relative frequencyof short closings compatible with an increase in the numberand duration of burst events.

520 J Phy8iol.497.2

J Physiol.497.2 Barbiturate activation of GABAA receptors 521

Clinical relevanceThe direct activation of CF- current responses by PB andPHB may contribute to the central nervous systemdepressant effects of the barbiturates. However, atconcentrations severalfold higher than those which activatethe channel, the barbiturates produce channel block. Thiseffect serves to limit the magnitude of the barbiturateresponse. Channel block may be of particular pharmaco-logical significance in the case of PHB, where theconcentrations producing channel block and GABA receptorpotentiation are within the same range. Channel block maylimit the extent to which PHB can potentiate GABAAreceptor responses, so that it appears to act as a 'partialagonist.' In addition, PHB may be less efficacious inactivating GABAA receptors because it only induces entryinto a rapidly closing open state. Whatever the mechanism,the partial agonist-like character of PHB may, in part,account for the reduced propensity of this barbiturate toinduce central nervous system depression at anticonvulsantdoses. This property allows PHB to be used clinically in thetreatment of seizure disorders.

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AcknowledgementsThe assistance of Ms Karen Wayns with the cell cultures isgratefully acknowledged.

Authors' present addressesS. D. Donevan: Department of Neurology, University of UtahSchool of Medicine, Salt Lake City, UT 84112, USA.

J. M. Rho: Department of Neurology, Children's Hospital andMedical Center, University of Washington School of Medicine,Seattle, WA 98105, USA.

Author's email addressM. A. Rogawski: [email protected]

Received 13 May 1996; accepted 30 August 1996.

522 J Physiol. 497.2

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