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Anesth Analg 2000;90:1184-1190
© 2000 International Anesthesia Research Society

GENERAL ARTICLES

Dual Action of Isoflurane on the {gamma}-aminobutyric Acid (GABA)-Mediated Currents Through Recombinant {alpha}1ß2{gamma}2L-GABAA-Receptor Channels

Susanne Neumahr, MD*, Gerhard Hapfelmeier, MD*, Michaela Scheller, MD*, Hajo Schneck, MD*, Christian Franke, PhD{dagger}, and Eberhard Kochs, MD*

Departments of *Anesthesiology and {dagger}Neurology, Klinikum rechts der Isar, Technische Universität, Munich, Germany

Address correspondence and reprint requests to Susanne Neumahr, MD, Institut für Anaesthesiologie der Technischen Universität München, Klinikum rechts der Isar, Ismaninger Straße 22, D-81675 München, Germany.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Isoflurane (ISO) increased the agonist-induced chloride flux through the {gamma}-aminobutyric acid A receptor (GABAAR). This may reflect an anesthetic-induced increase in the apparent agonist affinity. A dual effect of anesthetics was postulated for both the nicotinic acetylcholine receptor (nAChR) and the GABAAR. We tested the hypothesis that, in addition to a blocking effect, ISO increases {gamma}-aminobutyric acid (GABA)-gated currents through recombinant GABAAR channels. HEK293 cells were transfected with rat cDNA for {alpha}1,ß2,{gamma}2L subunits. Currents elicited by 1 mM or 0.01 mM GABA, respectively, alone, or with increasing concentrations of ISO, were recorded by using standard patch clamp techniques. ISO reduced the peak current elicited by 1 mM GABA. Currents induced by 0.01 mM GABA were potentiated by small ISO (twofold at 0.5 mM ISO) and inhibited by larger concentrations. Withdrawal of ISO and GABA induced rebound currents, suggesting an open-channel block by ISO. These currents increased with increasing concentrations of ISO. At large concentrations of ISO, the inhibitory effect predominated and was caused by, at least partly, an open-channel block. At small concentrations of ISO, potentiation of the GABA-gated currents was more prominent. This dual action of ISO indicates different binding sites at the GABAAR. The balance between potentiation and block depends on the concentrations of both ISO and GABA.

Implications: Isoflurane (ISO) interacts with the inhibitory {gamma}-aminobutyric acid (GABA) receptor. This patch clamp study demonstrates that it may block or potentiate the type A of GABA receptor studied, depending on the concentrations of ISO and of GABA used. At clinically relevant concentrations, ISO considerably potentiates this receptor. This may partly explain its clinical effect.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The {gamma}-aminobutyric acid type A receptor (GABAAR), which is represented at almost one-third of all neurons (1,2) in the mammalian brain, is a heteromeric complex formed by different glycoprotein subunits ({alpha}1–6, ß1–4, {gamma}1–4, {delta}, {epsilon}, and {rho}1–2) that co-assemble to form a chloride channel (1,3). Most GABAAR in vivo consist of pentameric complexes of {alpha}, ß, and {gamma} subunits with a stoichiometry of {alpha}{alpha}ßß{gamma} (4). In mammalian central neurons, {alpha}1ß2{gamma}2 appears to be the predominant subunit combination (1). The GABAAR channel is sensitive for IV and volatile anesthetics (5). Several studies demonstrate a potentiating effect of volatile anesthetics on GABAAR channels, i.e., potentiation of the GABA-mediated chloride flux (6–8). In contrast, some studies report blocking effects of volatile anesthetics at ligand-gated channels, e.g., at the GABAAR (9,10), or the nicotinic acetylcholine receptor (nAChR) (11). The block of Drosophila GABAAR channels by isoflurane (ISO) apparently requires a binding site for picrotoxinin (9), a convulsant drug that binds in the channel lumen of GABAAR (12).

Recently, a dual effect of ISO on the nAChR was reported, determined by stopped-flow spectrofluorometry (13). In addition to a block of the channels, an increase in the apparent agonist affinity was observed. Also, at a recombinant {alpha}1ß1{gamma}2 GABAAR, expressed in Xenopus laevis oocytes, ISO increased the affinity of GABA for its receptor; however, it depressed the maximum GABA response (14). A similar dual effect of volatile anesthetics exists for GABA-mediated inhibitory postsynaptic currents, reflected by a dissociation of blocking and prolonging effects. GABAA miniature inhibitory postsynaptic currents were recorded from CA1 rat pyramidal neurones. Volatile anesthetics reduced the amplitude; however, they prolonged the decay of the IPSPs dose-dependently (15). In dissociated rat pyramidal neurones, sevoflurane induced potentiation or block of GABA-mediated chloride currents, depending on the concentration of GABA used (16). Altogether, the GABA-ergic system, without doubt, interacts with many, if not all, anesthetic substances. Different or even contradictory findings may result from differences in the experimental techniques, in the type of cells used, in the subunit composition of the receptors, or in the concentration of GABA applied. In this study, we used a well defined recombinant receptor and investigated the effects of several concentrations of ISO on the kinetics of this receptor on activation by a large (1 mM), saturating, and desensitizing concentration, and by a small (0.01 mM), nondesensitizing concentration of GABA.

The kinetics of recombinant {alpha}1ß2{gamma}2 GABAAR, transiently expressed in HEK293 cells, have been investigated (17). The application of GABA elicited currents corresponding to the equilibrium of Cl-. Currents were blocked by the competitive antagonist bicuculline. The same receptor proved sensitive for benzodiazepines. Diazepam increased the GABA-mediated currents, apparently by increasing the affinity of GABA to the recombinant {alpha}1ß2{gamma}2 GABAAR, as reflected by a decrease in the rate of unbinding from the receptor (18). We investigated the interactions of ISO with a recombinant {alpha}1ß2{gamma}2 GABAAR, transiently expressed in HEK 293 cells. To match the fast kinetic properties of the channels, we used an ultrafast drug application (19). With this technique, exchange of the solution surrounding the receptors is complete within approximately 1 ms. Thus, currents crossing the membrane can be resolved over time in the same order of magnitude.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Human embryonic kidney cells (HEK293; Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) were cultured in Eagle’s minimal essential medium (GIBCO BRL, Eggenstein, Germany) supplemented with 10% fetal bovine serum (Biochrom, Berlin, Germany), 4 mM/L glutamine (GIBCO), 200 units/mL penicillin/streptomycin, and maintained at 37.0°C in a 5% CO2 air incubator. For transfection of the HEK cells, an electroporation system (Biotechnologies and Experimental Research, Inc., San Diego, CA) was used. The cells were suspended in electroporation buffer containing 50 mM K2HPO4x 3 H2O, 20 mM K-acetate (titrated to pH 7.35 with C2H4O2), and 0.25 mM Mg2SO2 x 7 H2O, and were co-transfected with rat {alpha}1,ß2,{gamma}2L cDNAs and green fluorescent protein by using the pRK5 expression vector. For best expression, the final concentrations of plasmids were 3.3 µg {alpha}1, 16.5 µg ß2, and 3.3 µg {gamma}2. Green fluorescent protein (2-µg final concentration) was used to select the fluorescent transfected cells by means of a fluorescent microscopy (HBO 50; Zeiss, Göttingen, Germany). Cells were replanted in mini- mal essential medium containing fetal calf serum, L-glutamine, and penicillin/streptomycin and incubated for 6–8 h until expression of GABAARs. For electrophysiological experiments, the culture medium was replaced by an extracellular solution with (in mM) 162 NaCl, 5.3 KCl, 0.67 NaHPO4, 0.22 KH2PO4, 15 HEPES, 5.6 glucose, and 2 CaCl2, adjusted to a pH of 7.30 with NaOH. Recording patch pipettes were fabricated from borosilicate glass by using a two-step vertical puller (Zeitz Instruments, Augsburg, Germany). Pipette-to-bath resistances were 5–8 MOhm. Pipettes were filled with intracellular solution containing (in mM) 140 KCl, 2 MgCl2, 11 EGTA, 10 HEPES and 10 glucose, adjusted to a pH 7.30 with KOH and to an osmolarity of 340 mosm with mannitol. Currents recorded from the whole-cell patches reversed at 0 mV, corresponding to the chloride current under these experimental conditions.

Well defined concentrations of 1 mM and of 0.01 mM GABA (Sigma, Deisenhofen, Germany) and of 15 mM to 0.15 mM ISO (ForeneTM; Deutsche Abbott GmbH, Wiesbaden, Germany) were prepared shortly before each experiment by diluting the saturated solutions. A saturated solution of ISO was prepared by adding a surplus of the anesthetic to the extracellular solution and by stirring in an airtight glass bottle for at least 3 h. Maximum solubility of ISO in saline at room temperature was 15 mM as determined by gas chromatography (11). Based on a Bunsen water/gas partition coefficient of 1.08 at 25°C, the concentration of ISO of 0.5 mM corresponds to approximately 1 vol% (20).

Electrophysiological recordings were performed at 20°-23°C by using standard whole-cell patch clamp techniques. The cells were voltage-clamped to -30 mV during recordings, and series resistance was compensated for by the patch clamp amplifier.

A piezo-driven application system (19) was used to apply GABA alone, or combined with ISO to the patches. This system accounts for the rapid kinetics of the GABAAR channel (exchange of solutions was completed in <1 ms) and allows for precise determination of the current onset and rise time. The substances are applied by a liquid filament released by a tube connected to a piezo crystal. The whole-cell patches are placed into the interface between this filament and the rapidly flowing bath solution (extracellular solution alone or combined with ISO) within the recording chamber. On voltage activation of the piezo, the liquid filament containing GABA ± ISO shifts upwards by 20 µm and hits the patch for a defined period of time, thus activating the GABAAR channels. The piezo crystal is driven by protocols in the acquisition program pCLAMPTM 6.0 (Axon Instruments, Foster City, CA). Data were recorded with an Axopatch 200B patch-clamp amplifier, a Digidata 1200 interface, and pCLAMPTM 6.0 software (all Axon Instruments). Currents were low-pass filtered at 5 kHz and stored on a personal computer with a sampling frequency of 30 kHz.

The study protocol consisted of three parts. In the first part, interactions of different concentrations of ISO with large, saturating concentrations of GABA (1 mM) were investigated. Standard protocols repetitively applied 1 mM GABA alone, or combined with varying concentrations of ISO as single pulses lasting 2.5 s. A 10-s interval between the pulses guaranteed full recovery of the channels from desensitization (17). To further investigate the molecular mechanism(s) of this interaction, pulses of 1 mM GABA combined with 15 mM ISO were then varied as to their duration (10–2500 ms).

In the second part, interactions of ISO with small concentrations of GABA (0.01 mM corresponding to an effective concentration of approximately 20%) were studied by using the same application protocols (pulse duration 2.5 s, interval 10 s, variation of pulse duration). Control measurements with the respective concentration of GABA alone preceded and followed every experiment with ISO, and experimental results were used only when control pulses disclosed no rundown. In these parts of the study, the bath solution did not contain ISO.

In a third part, experiments were performed with preexposure of the whole cells to ISO for a period of approximately 1 min by adding ISO to the bath solution. Afterward, GABA, together with the same concentration of ISO as contained in the bath solution, was applied repetitively according to the protocols described for part one.

The tubes of the application system were rinsed carefully after every application of ISO. All experiments began with the lowest concentration of ISO to prevent any artifacts from ISO being retained in the system. Experimental data were expressed as mean ± SD.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
In part one of the study, the whole-cell patches were first exposed to 2.5-s pulses of saturating concentrations of 1 mM GABA. Stepwise, increasing concentrations of ISO (0.15 to 15 mM) were added to the GABA pulses. Repetitively, three to four single pulses were applied, and the currents were averaged.

Figure 1 A shows original current traces from a single patch. The top trace demonstrates a typical current activated by 1 mM GABA alone. The holding potential was -30 mV. The current rapidly rose to a peak amplitude of -1263 pA with a 10% to 90% increase time of 2.3 ms. The current then declined biexponentially with a fast (46 ms) and a slow (1350 ms) time constant caused by desensitization (semilogarithmic plot in Fig. 1B, top trace) (17). At discontinuation of GABA, the current returned to the zero level. When 0.15 mM, 0.5 mM, or 1.5 mM ISO were added to GABA, the peak amplitude remained unchanged. With 15 mM ISO, however, a reduction of the peak amplitude occurred (Fig. 1A, bottom trace). The increase times were not altered by ISO (data not shown), whereas a concentration-dependent reduction of the fast time constant of current decay was observed (Fig. 1B, averaged data in Fig. 2A). The slow time constant, however, was hardly influenced (Fig. 1B, averaged data in Fig. 2B). At discontinuation of both GABA and ISO, rebound currents appeared at concentrations of ISO >=0.5 mM. The amplitude of these reopenings rose with increasing concentrations of ISO (Fig. 1A, bottom traces). The reduction of the fast time constant of current decay, together with the reopenings, suggest an open-channel block mechanism (11,21). The concentration of 1.5 mM ISO without GABA had no intrinsic effect on the GABAAR.



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  		Figure 1. A, Average currents (3–4 single recordings) from one whole-cell patch. The currents were elicited by 2.5 s pulses of GABA alone, or combined with increasing concentrations of ISO as indicated. The holding potential was -30 mV. The peak amplitudes were -1263 pA, -1302 pA, -1318 pA, -1326 pA, and -355 pA for 1 mM GABA alone and together with 0.15 mM, 0.5 mM, 1.5 mM, and 15 mM Iso, respectively. The reopening peak amplitudes were -55 pA, -312 pA, and -396 pA at removal of 1 mM GABA together with 0.5 mM, 1.5 mM, and 15 mM ISO, respectively. B, Semilogarithmic plot of the current decay. The decay was fitted biexponentially with two time constants as indicated. Note the different scales. GABA = {gamma}-aminobutyric acid, ISO = isoflurane.

 


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  		Figure 2. The fast, A, and slow, B, time constant of current decay (1 mM GABA alone, or with increasing concentrations of ISO as indicated) averaged from four separate experiments. C, Concentration-response curve of the relative charge Q [pAs] of the current elicited by 1 mM GABA (Q at a concentration of 1 mM GABA alone = 1) alone, or with increasing concentrations of ISO as indicated (four patches, 3–5 recordings each). The relative Q was 0.93 ± 0.17, 0.78 ± 0.21, 0.25 ± 0.15, and 0.05 ± 0.08 for 1 mM GABA combined with 0.15 mM, 0.5 mM, 1.5 mM, and 15 mM ISO, respectively. GABA = {gamma}-aminobutyric acid, ISO = isoflurane.

 
The block can be quantified by integrating the area under the current curves (integral of the charge Q) after addition of ISO to 1 mM GABA. The value Q is described as the summarized ion flux through all channels during the pulse. With increasing concentrations of ISO, the integral decreased (Fig. 2C), corresponding to an increase in the amount of blocked channels.

Additional experiments were performed to evaluate whether the peak of the reopening current depends on the pulse duration and to provide more information on the existence and impact of additional conformational states during the block. We applied 1 mM GABA combined with 15 mM ISO in pulses for 10 to 2500 ms. Peaks of the reopening currents decreased with increasing pulse duration (Fig. 3). In addition to the block by ISO, a desensitized state is induced by GABA—increasing the pulse duration results in an increasing number of channels that desensitize in the blocked state and cannot reopen after discontinuation of ISO.



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  		Figure 3. One representative example showing the dependency of the peak current of the reopenings at discontinuation of ISO and GABA on the pulse length. Original current traces elicited by 1 mM GABA combined with 15 mM ISO. GABA = {gamma}-aminobutyric acid, ISO = isoflurane.

 
In the second part of the study, the effects of ISO on currents elicited by small concentrations of GABA (0.01 mM = EC10–20) were evaluated. Pulses, 2.5 s, of 0.01 mM GABA combined with increasing concentrations of ISO were applied repetitively. Figure 4A, upper trace, shows the typical current elicited by 0.01 mM GABA alone (17). The current increases to a small peak of -384 pA with a slow increase time of 128 ms. No desensitization occurred. By adding 0.15 mM to 0.5 mM ISO to the pulses, the peak amplitude increased concentration dependently, indicating potentiation by ISO (Fig. 4A and B). At larger concentrations of ISO (>=1.5 mM), however, a blocking effect overrode this potentiation, resulting in a decrease in peak current amplitude (Fig. 4A, bottom traces, Fig. 4B). At >=0.5 mM ISO, reopenings occurred on removal of GABA and of ISO. Again, the reopenings increased in amplitude with increasing concentrations of ISO (Fig. 4A).



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  		Figure 4. A, Average currents (3–4 single recordings) from a single whole-cell patch. Currents were elicited by 2.5 s pulses of 0.01 mM GABA alone, or combined with ISO as indicated. Holding potential was -30 mV. The peak currents were -384 pA, -447 pA, -521 pA, -354 pA, and -49 pA for 0.01 mM GABA alone, or combined with 0.15 mM, 0.5 mM, 1.5 mM, and 15 mM ISO, respectively. The amplitudes of the reopenings were -27 pA, -252 pA, and -622 pA after removal of 0.01 mM GABA combined with 0.5 mM, 1.5 mM, and 15 mM ISO, respectively. B, Concentration-response curve of the relative current amplitude elicited by 0.01 mM GABA combined with ISO, as indicated (relative peak amplitude at 0.01 mM GABA = 1; 13 patches). GABA = {gamma}-aminobutyric acid, ISO = isoflurane.

 
The dependency of the peak current amplitude of the reopenings on the pulse duration was also evaluated at 0.01 mM GABA combined with 15 mM ISO co-applied in single pulses of 10–2500 ms. The interval between the pulses was constant (10 s). At a pulse duration of 10 ms, neither activation nor reopenings could be observed (assuming an open-channel block, the channels must open before they can be blocked). Increasing the pulse duration increased the peak amplitudes of the reopening currents (Fig. 5). This is in clear contrast to the results described previously with 1 mM GABA combined with 15 mM ISO. At small concentrations of GABA, the longer the pulses, the more channels are opened by GABA and are subsequently blocked by ISO.



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  		Figure 5. One representative example showing the dependency of the reopening currents at discontinuation of ISO and of GABA on the pulse length (10–2500 ms). Original current traces elicited by 0.01 mM GABA combined with 15 mM ISO. As expected, the increase in the peak current amplitudes with increasing pulse duration approximately corresponds to the rise time of the current elicited by 0.01 mM GABA alone. GABA = {gamma}-aminobutyric acid, ISO = isoflurane.

 
Finally, effects of ISO on the closed, unliganded state of the GABAAR channel were investigated. Patches were preincubated with 0.3 mM ISO for a period of approximately 1 min before activation with 0.01 mM GABA. ISO was added to the bath solution and was still present during and after exposure of the cells to GABA. GABA was combined with the identical concentration of ISO present in the bath solution. Figure 6 shows typical current traces from one patch. The top trace (Fig. 6A) represents the reference current with 0.01 mM GABA alone (peak current -270 pA; time constant of deactivation, {tau}, 150 ms, best fitted monoexponentially; averaged from three patches with three to five recordings each 160 ± 50 ms). This constant represents the dissociation of GABA from the receptors. Then, 0.3 mM ISO was co-applied (Fig. 6B), identically to the experiment shown in Figure 4A, which resulted in an increase in {tau} to 190 ms (peak current -373 pA). After preincubation with 0.3 mM ISO and co-application of 0.01 mM GABA, {tau} further increased to 260 ms (peak current -339 pA; {tau} averaged 310 ± 120 ms) in the continued presence of ISO (Fig. 6C). This finding points to an increase in the affinity of GABA to its receptor by ISO. The reopening current (as seen with larger concentrations of ISO, Fig. 4A) disappeared in the presence of ISO in the bath solution after removal of GABA. This finding clearly demonstrates that the reopenings are caused by dissociation of ISO from the blocked channels. To test for reversibility, the bath solution was cleared of ISO, and control pulses with 0.01 mM GABA alone, were applied to the same patch after a 2 min washout period (Fig. 6D). The control pulse did not markedly differ from the original control pulse (peak current -245 pA, {tau} 160 ms).



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  		Figure 6. Experiments with 0.3 mM ISO in the background solution. A, control pulse (2.5 s) with 0.01 mM GABA alone in the pulse solution, and extracellular solution in the background solution. B, 0.01 mM GABA, plus 0.3 mM ISO in the pulse solution, and extracellular solution in the background solution. A rebound current appeared after the end of the application pulse. C, 0.01 mM GABA, plus 0.3 mM ISO in the pulse solution, and 0.3 mM ISO in the background solution. The presence of ISO in the background solution prevented the unbinding of ISO from the receptor channels, and thus, no rebound current appeared. D, control pulse with 0.01 mM GABA alone in the pulse solution. The time constant of current decay after the end of the GABA pulse significantly rose from 160 ± 50 ms (A, n = 6) to 220 ± 70 ms (B, n = 6) and to 310 ± 120 ms (C, n = 3) on average. GABA = {gamma}-aminobutyric acid, ISO = isoflurane.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our results provide evidence that ISO has a blocking, as well as a potentiating, effect on the GABA-mediated current. The balance between these opposing effects depends on both the concentration of GABA and ISO. In agreement with other findings (8,9), a potentiating effect of ISO was observed at small concentrations of ISO (0.15–0.3 mM) together with small concentrations of GABA (0.01 mM). A blocking effect predominates at larger concentrations of ISO (15 mM) and may reflect an open channel block. Such an open channel block has been described for ISO at the GABAAR (10,15) and at the nAChR (11). An additional allosteric block by ISO, however, cannot be ruled out by our results.

In the first part of the study, experiments were performed with 1 mM GABA, a saturating transmitter concentration, alone and with increasing concentrations of ISO (Fig. 1A). As expected, the current elicited by GABA plus ISO was not potentiated. The blocking effect of ISO, however, was obvious. First, the peak current amplitude decreased with ISO at concentrations of >=0.5 mM. Second, the current decay could be fitted with two exponentials. The initial faster component reflects the block of the open channels, as ISO binds to the channels opened by GABA. Concentration-dependency of this open-channel block is reflected by the decrease in the fast time constant of current decay with increasing concentration of ISO (Fig. 1B, Fig. 2A) (11). The slow component of current decay expresses the equilibrium between the blocked and the unblocked state (11,21), reflecting desensitization in the continued presence of 1 mM GABA. This slow time constant of current decay was not affected by ISO (Fig. 2B). Thus, desensitization of the receptor channels was not influenced by ISO. Third, on simultaneous discontinuation of ISO and of GABA, rebound currents appear. These reopenings reflect the transition from the blocked state through the open state back to the resting, unliganded state (Fig. 1A). Similar results have been reported for ISO (11) and for the open-channel blockers physostigmine and procaine (21) at the nAChR. From their studies with picrotoxinin that binds inside the channel, Edwards and Lees (9) proposed a channel block by ISO via an interaction with the picrotoxinin recognition site within the lumen of the open GABAAR channel.

To study the mechanism of the block in more detail, we investigated the effect of the duration of the pulse containing GABA and ISO on the reopening currents (Fig. 3). In our experiments with 1 mM GABA, the reopening current amplitude decreased as pulse duration increased. These results strongly suggest the induction of at least one additional conformational state of the channel, which is unable to reopen on removal of the blocker; this state may be called "open-blocked-desensitized" (22). The number of channels in this conformational state would increase over time in the presence of ISO and GABA, and the channels would not contribute to the reopening current on removal of both substances. The induction of a blocked-desensitized state by ISO and GABA has been suggested by Adelsberger et al. (10) from their results on crayfish GABAAR and computer simulations of the respective reaction scheme.

In the second part of the study, experiments were performed with 0.01 mM GABA, a nonsaturating and nondesensitizing concentration of the transmitter eliciting approximately 20% to 30% of the maximum current by 1 mM GABA alone, and combined with increasing concentrations of ISO. In these protocols, a potentiating effect of ISO was observed, i.e., the peak current amplitude increased at concentrations of ISO up to 0.5 mM (Fig. 4A). This suggests an increase in the affinity of the agonist to its receptor, as shown for Drosophila GABAAR (9) and for the nAChR (13). Banks and Pearce (15) reported both a blocking and a prolonging effect of volatile anesthetics on GABAA IPSCs. Taking into account that we used a very fast application system, this latter finding may well be explained by the summation of peak current and reopening current that cannot be resolved with the experimental approach used in their study (15). Further experiments were performed to determine the impact of pulse duration on the reopenings. Experiments were performed with 15 mM ISO to induce a strong effect. In the presence of small concentrations of GABA, no desensitization was expected. Thus, no open-blocked desensitized state would be induced and the amplitude of the reopenings should, therefore, depend only on the number of channels blocked by ISO. Longer exposure to ISO was expected to block a higher number of channels and more channels were expected to reopen at the end of the pulse. The results shown in Figure 5 were in full accordance with these hypotheses.

Finally, assuming that ISO increases the affinity of GABA to its receptor, we studied the influence of ISO on the closed unliganded GABAAR channel. An increase in affinity would result in a slower dissociation of GABA from the receptor in the presence of ISO. The patches were preincubated with small concentrations of ISO before, during, and after exposure to GABA. The time constant of deactivation after discontinuation of GABA (which reflects the dissociation of GABA from the receptor) increased from 160 ± 50 ms (control, Fig. 6A) to 310 ± 120 ms (dissociation of GABA in the presence of ISO, Fig. 6C) on average. An increase in transmitter affinity, as suggested by these findings for ISO, has been postulated at the same receptor for halothane (23) and for ISO at a different type of GABAAR (9). An identical mechanism was shown at the nAChR (13). Because both the GABAAR and the nAChR are closely related ontogenetically and exhibit a striking amino-acid homology, a common mechanism or even site of action may be assumed for the effect of volatile anesthetics on these ligand-gated receptors. Volatile anesthetics, however, have also been shown to block agonist-induced ion flux through GABAAR (24–26). An inhibitory effect, which was also obvious in our findings, occurred only at large concentrations of the respective anesthetic. In this study, we observed that, similar to the nAChR (27), the GABAAR is simultaneously potentiated and blocked by ISO and the resulting effect depends on the concentration of ISO. In the clinically relevant range, the potentiating effect of ISO predominates over its blocking effect.


   Acknowledgments
 
Supported, in part, by the Deutsche Forschungsgemeinschaft Grant Schn-514/2–2 and by the Dr.-Ing. Leonhard Lorenz-Stiftung, Munich, Az. 376/97.

We thank Mrs. Monika Hammel for expert technical assistance and W. Zieglgänsberger, PhD, Max-Planck-Institut für Psychiatrie, Munich, Germany, for providing the cDNAs. We greatly appreciate the helpful comments by J. P. Dilger, PhD, New York State University, Stony Brook, New York.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication January 11, 2000.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press