QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: mol.104.003426v1 66/6/1517    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Feng, H.-J. Articles by Macdonald, R. L. Search for Related Content PubMed PubMed Citation Articles by Feng, H.-J. Articles by Macdonald, R. L.

Multiple Actions of Propofol on and GABA_A Receptors

Departments of Neurology (H.-J.F., R.L.M.), Molecular Physiology and Biophysics (R.L.M.), and Pharmacology (R.L.M.), Vanderbilt University Medical Center, Nashville, Tennessee

Received June 1, 2004; accepted August 24, 2004

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
GABA_A receptors are predominantly composed of and isoforms in the brain. It has been proposed that receptors mediate phasic inhibition, whereas receptors mediate tonic inhibition. Propofol (2,6-di-isopropylphenol), a widely used anesthetic drug, exerts its effect primarily by modulating GABA_A receptors; however, the effects of propofol on the kinetic properties of and receptors are uncertain. We transfected human embryonic kidney (HEK293T) cells with cDNAs encoding rat 1, 6, 3, 2L, or subunits and performed whole-cell patch-clamp recordings to explore this issue. Propofol (3 µM) increased GABA concentration-response curve maximal currents similarly for both 132L and 632L receptors, but propofol increased those for 13 and 63 receptors differently, the increase being greater for 13 than for 63 receptors. Propofol (10 µM) produced similar alterations in 132L and 632L receptor currents when using a preapplication protocol; peak currents were not altered, desensitization was reduced, and deactivation was prolonged. Propofol enhanced peak currents for both 13 and 63 receptors, but the enhancement was greater for 13 receptors. Desensitization of these two isoforms was not modified by propofol. Propofol did not alter the deactivation rate of 13 receptor currents but did slow deactivation of 63 receptor currents. The findings that propofol reduced desensitization and prolonged deactivation of 2L subunit-containing receptors and enhanced peak currents or prolonged deactivation of subunit-containing receptors suggest that propofol enhancement of both phasic and tonic inhibition may contribute to its anesthetic effect in the brain.

Several widely used general anesthetic drugs, including propofol (2,6-di-isopropylphenol), exert their effects in the central nervous system mainly by enhancing GABA_A receptor currents (Olsen and Macdonald, 2002). Modulation of receptor current amplitudes by propofol has been substantially explored (Hill-Venning et al., 1997; Uchida et al., 1997; Lam and Reynolds, 1998; Pistis et al., 1999; Carlson et al., 2000; Davies et al., 2001; Krasowski et al., 2001; Williams and Akabas, 2002), but propofol effects on the kinetic properties of recombinant receptors are unclear. Although one study suggested that propofol slightly enhanced the function of 43 receptors (Brown et al., 2002), its effects on current kinetics of other isoforms are unknown. Therefore, modulation of propofol on recombinant and receptors was examined to explore the potential effects of propofol on phasic and tonic GABAergic inhibition. GABA_A receptor 1 subunit mRNA is ubiquitously expressed in the brain, whereas 6 subunit mRNA is restrictively found in the cerebellum (Wisden et al., 1992). In addition, 6 subunits preferably coassemble with subunits, and the 6 receptor is one of the predominant subunit-containing GABA_A receptor isoforms in the brain (Poltl et al., 2003). It is of interest to determine whether propofol has different effects on 1 and 6 subunit-containing GABA_A receptors. Therefore, modulation of recombinant 1, 6, 1, and 6 receptors by propofol was examined to explore the potential subunit-dependent effects of propofol.

In the present study, we demonstrated subunit-specific propofol activation of 2L and subunit-containing GABA_A receptors. Propofol evoked a greater maximal conductance change (G) from 2L than from subunit-containing receptors. Propofol similarly decreased the desensitization and prolonged the deactivation of 132L and 632L receptors without affecting the peak current amplitudes. Although propofol modulation of GABA_A receptor currents was relatively insensitive to the subunit subtype, subtype-specific effects of propofol were observed for receptors. Propofol produced a greater enhancement of peak current amplitudes for 13 than for 63 receptors and prolonged the deactivation of 63 receptor currents without altering deactivation of 13 receptor currents.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Expression of Recombinant GABA_A Receptors in Human Embryonic Kidney Cells. HEK293T cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) in an incubator at 37°C with 5% CO₂ and 95% air. The cells were seeded at a density of 400,000/dish in 60-mm culture dishes (Corning Incorporated, Corning, NY) and transfected the following day with the combinations of cDNAs encoding rat 1, 6, 3, 2L, and GABA_A receptor subunits (2 µg of each subunit in different ternary combinations) along with 2 µg of pHOOK (Invitrogen) using a modified calcium phosphate precipitation method (Fisher and Macdonald, 1997). The cells were incubated at 37°C for 4 h with 3% CO₂ and then shocked for 30 s with 15% glycerol (Sigma-Aldrich, St. Louis, MO). The selection marker pHOOK encoded a cell surface antibody (sFv) that bound to the antigen (phOx) coated on the ferromagnetic beads (Invitrogen). The bead-bound transfected cells were separated from nontransfected cells using a magnetic stand (Greenfield et al., 1997). Electrophysiological recordings were obtained 24 h later. Eighty two percent of cells that bound beads also expressed GABA_A receptors (80% for 132L receptors, 79% for 13 receptors, 86% for 632L receptors, and 87% for 63 receptors).

Whole-Cell Recordings. Whole-cell macroscopic currents were recorded using the patch-clamp technique at room temperature. The recording electrodes were pulled from the thin-wall borosilicate glass tubing (i.d., 1.12 mm; o.d., 1.5 mm) (World Precision Instruments Inc., Sarasota, FL) on a P-2000 Quartz Micropipette Puller (Sutter Instrument Company, Novato, CA). The electrodes were fire-polished on an MF-830 Microforge (Narishige, Tokyo, Japan), and the resistances of the electrodes were 0.9 to 1.6 M when filled with an internal solution (see Chemicals, Solutions, and Drug Application for ionic composition).

Currents were recorded with an Axopatch 200A patch-clamp amplifier (Axon Instruments Inc., Union City, CA) and Digidata 1200 series interface (Axon Instruments Inc.). Series resistance was not compensated given that we previously reported that desensitization rate and extent were not affected by the current size we usually obtained from these recombinant GABA_A receptors (Bianchi and Macdonald, 2002), suggesting that series resistance errors did not significantly affect our interpretations.

Chemicals, Solutions, and Drug Application. All chemicals were purchased from Sigma-Aldrich. The external bath solution was composed of 142 mM NaCl, 1 mM CaCl₂, 6 mM MgCl₂, 8 mM KCl, 10 mM glucose, and 10 mM HEPES (pH 7.4, 328-330 mOsm). The internal micropipette solution consisted of 153 mM KCl, 1 mM MgCl₂, 10 mM HEPES, 2 mM MgATP, and 5 mM EGTA (pH 7.3, 301-309 mOsm). This combination of the external and internal solutions produced an E_CL near 0 mV and an E_K at -75 mV.

GABA was dissolved in water, and propofol was dissolved in dimethyl sulfoxide to make 1 M stock solutions. The working solutions were prepared by diluting the stock solution with external solution on the day of the experiment. The maximal final concentration of dimethyl sulfoxide in working solutions was 0.3%. Drugs were applied by gravity using an ultrafast delivery device consisting of multibarrel tubes connected to a Perfusion Fast-Step system (Warner Instruments Inc., Hamden, CT). The 10 to 90% open electrode tip rise time of solution exchange was approximately 0.4 ms. Consecutive drug applications were separated by an interval of at least 45 s to minimize accumulation of desensitization. The duration of GABA or propofol application was 4 s.

Data Analysis. Whole-cell currents were analyzed offline using Clampfit 8.1 (Axon Instruments). Peak currents were measured manually from the baseline to the transient peak. Potentiation of GABA current by propofol (percentage of GABA current) was determined by dividing the peak current of coapplication of GABA and propofol by the peak current evoked by GABA alone and multiplying by 100. Normalized concentration-response data were fitted using a logistic equation with a variable slope: I = I_max/(1 + 10^{(LogEC₅₀-Logdrug) x nH}). I was the peak current evoked by a given concentration of GABA or GABA and propofol coapplication. I_max was the maximal peak current. EC₅₀ was defined as the GABA concentration at which 50% of maximal response was evoked. Peak conductance change (G) was calculated by dividing the peak current by the holding potential. The extent of desensitization (percent) was calculated by dividing the amount of current loss after 4 s of drug application by peak current and multiplying by 100. The deactivation current phase was analyzed by fitting using the standard exponential Levenberg-Marquardt methods, and the exponential components were expressed in the form of a_nn, where a was the relative amplitude, was the time constant, and n (= 1 or 2) was the number of exponential components. A weighted was used to compare the rates of deactivation: a₁ x ₁/(a₁ + a₂) + a₂ x ₂/(a₁ + a₂), where a₁ and a₂ were the relative amplitudes of the fast and slow exponential components (at time 0), and ₁ and ₂ were the corresponding time constants. Data were reported as mean ± S.E.M. Paired Student's t test was used to compare the changes before and after propofol treatment. Un-paired Student's t test was used to compare the alterations between different treatment groups. The difference was considered to be statistically significant if p was less than 0.05.

	Results

Top Abstract Materials and Methods Results Discussion References

 
GABA Sensitivity of 2L and Subunit-Containing GABA_A Receptors Assembled with Either an 1 or 6 Subunit. We first examined the GABA sensitivity of the four isoforms to be studied using standard concentration-response experiments. Whole-cell currents were recorded from recombinant 132L, 632L, 13, and 63 GABA_A receptors (Fig. 1, A-D). Cells were voltage-clamped at -20 mV for cells transfected with 2L subunit-containing receptors, and because of the smaller amplitudes of currents, cells were voltage-clamped at -50 mV for cells transfected with subunit-containing receptors (Wohlfarth et al., 2002; Feng et al., 2004). GABA_A receptor channel activation is not voltage-dependent at negative membrane potentials (-10 to -75 mV), although some reports show different degrees of nonlinearity (rectification) at positive potentials (Bianchi et al., 2002). In a recent report on pentobarbital modulation of 13 and 132L receptors (Feng et al., 2004), membrane potential was held at both -20 and -50 mV for each isoform, and pentobarbital-evoked effects were consistent for both receptor isoforms at both holding potentials. In addition, membrane potential was clamped from -10 to -75 mV to study neurosteroid modulation of these receptor isoforms, and no voltage-dependent effects were observed (Wohlfarth et al., 2002). As GABA concentrations were increased, the GABA-evoked whole-cell peak conductance change (G) increased (Fig. 1E). The maximal G for the 132L isoform was 270.3 ± 47.6 nS (n = 6), which was not significantly different from that for 632L receptors (206.9 ± 65.6 nS, n = 7) but was significantly greater than that for 63 (81.5 ± 25.3 nS, n = 6) (p < 0.01) and 13 (22.1 ± 11.1 nS, n = 5) (p < 0.01) receptors. The maximal G evoked by GABA from 632L receptors was not significantly different from that evoked from 63 receptors but was significantly greater than that from 13 receptors (p < 0.05). The maximal G evoked by GABA from 63 receptors was not significantly different than that evoked from 13 receptors.

View larger version (16K): [in this window] [in a new window]   Fig. 1. GABA concentration-response patterns of 2L and GABAA receptors containing either an 1 or 6 subunit subtype. A-D, representative whole-cell current traces evoked by different concentrations of GABA from recombinant 132L, 632L, 13, and 63 GABAA receptors. E, GABA concentration-response curve expressed as mean peak conductance changes (G) versus a series of GABA concentrations for 132L (n = 6, ), 632L (n = 7, ), 13 (n = 5, ), and 63 (n = 6, ) receptors. The solid line above each current trace represents the duration (4 s) of GABA application. The error bars represent means ± S.E.M.

As reported previously, 6 subunit-containing GABA_A receptors had a lower GABA EC₅₀ than 1 subunit-containing receptors (Saxena and Macdonald, 1996; Fisher et al., 1997). The EC₅₀ for 632L receptors (0.49 ± 0.14 µM) was smaller than that for 132L receptors (6.17 ± 2.33 µM) (p < 0.05). The EC₅₀ for 63 receptors (0.28 ± 0.05 µM) was also smaller than that for 13 receptors (5.24 ± 0.43 µM) (p < 0.001). No significant differences in EC₅₀ values between 632L and 63 receptors or 132L and 13 receptors were observed (Fig. 1E). The mean Hill coefficients for 132L and 632L receptors were 1.6 ± 0.2 and 1.5 ± 0.1, respectively. The mean Hill coefficients for 13 and 63 receptors were 1.0 ± 0.1 and 1.3 ± 0.1, respectively.

Differences in Direct Activation of 2L or Subunit-Containing GABA_A Receptors by Propofol. Propofol has been reported to directly activate GABA_A receptors (Orser et al., 1994; Lam and Reynolds, 1998; Pistis et al., 1999; Davies et al., 2001; Krasowski et al., 2001; Brown et al., 2002; Dong and Xu, 2002). Propofol directly activated both 2L and subunit-containing GABA_A receptors (Fig. 2, A-D). However, similar to the results obtained with GABA, propofol evoked a greater maximal G from 2L than from subunit-containing receptors regardless of whether an 1 or 6 subtype was present (Fig. 2E).

View larger version (14K): [in this window] [in a new window]   Fig. 2. Propofol evoked greater direct response from 2L than subunit-containing GABAA receptors. A-D, representative whole-cell current traces evoked by different concentrations of propofol from recombinant 132L, 632L, 13, and 63 GABAA receptors. E, propofol concentration-response curve expressed as mean peak conductance changes (G) versus a series of propofol concentrations for 132L (n = 6, ), 632L (n = 6, ), 13 (n = 7, ), and 63 (n = 5, ) receptors. The solid line above each current trace represents the duration (4 s) of propofol application. The error bars represent means ± S.E.M.

For both 2L and subunit-containing GABA_A receptors, propofol-evoked direct current showed no or minimal desensitization at propofol concentrations up to 300 µM. However, at very high concentrations (>1 mM), propofol currents were rapidly activating and showed extensive desensitization, and a "rebound" current appeared upon washout of propofol (Fig. 2, A-D). The increased desensitization and appearance of a rebound current might have resulted from propofol blocking open GABA_A receptor channels at a low-affinity site (Adodra and Hales, 1995; Davies et al., 2001). It seemed that the rate of desensitization was faster and the extent of desensitization was larger for 6 than for 1 subunit-containing receptors. The mechanisms underlying this phenomenon remain unknown. One possibility may be that the affinity of propofol to the channel-binding site is greater for 6 than for 1 subunit-containing receptors, so that more complete block is observed in 6 subunit-containing receptors. The multiphasic nature of the propofol concentration-response curve may also be partly explained by open channel block. However, the basis for the rapid change in current activation rate at high concentrations was unclear and was not further investigated.

Propofol Enhanced Peak Currents Evoked by a High Concentration of GABA from 13 More Than from 132L, 632L, and 63 GABA_A Receptors. Performing concentration-response curves in the presence of a modulator such as propofol can provide an initial assessment of possible mechanisms of action by evaluating changes in EC₅₀ and maximal current amplitudes. Propofol (3 µM) was coapplied with increasing GABA concentrations (from 0.01 to 1000 µM). Propofol slightly enhanced currents at high GABA concentrations and thus shifted the GABA concentration-response curve upward similarly (115% of maximal current) for 132L and 632L receptors (Fig. 3, A-C). The GABA EC₅₀ was not significantly altered by propofol for these receptor isoforms.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Propofol produced greater enhancement of 13 receptor than 132L, 632L, and 63 receptor currents at high GABA concentrations. A and B, examples of whole-cell current traces evoked by GABA alone as well as coapplication of GABA and propofol (3 µM) from 132L and 632L receptors. C, concentration-response curves for GABA alone (open symbols) and coapplication of GABA with 3 µM propofol (solid symbols) plotted for 132L (squares) and 632L (triangles) receptors. D and E, examples of whole-cell current traces evoked by GABA alone as well as coapplication of GABA and propofol (3 µM) from 13 and 63 receptors. F, concentration-response curves for GABA alone (open symbols) and coapplication of GABA with 3 µM propofol (solid symbols) plotted for 13 (circles) and 63 (diamonds) receptors. The solid line above each current trace represents the duration (4 s) of GABA application or GABA and propofol coapplication. n = 5 to 7 cells for each GABA or GABA + propofol concentration-response curve. The error bars represent means ± S.E.M.

Coapplication of propofol with high concentrations of GABA evoked a greater current enhancement for 13 receptors than for 63 receptors (139.1 ± 4.9% versus 106.4 ± 2.6%) (p < 0.001) (Fig. 3, D-F). The maximal enhancement for 13 receptors was also significantly greater than that for 132L (p < 0.01) and 632L (p < 0.05) receptors. The EC₅₀ for 13 receptors for GABA coapplied with propofol (7.05 ± 0.65 µM) was greater than that for GABA alone (5.24 ± 0.43 µM) (p = 0.05). The EC₅₀ for 63 receptors was unchanged by propofol (Fig. 3F). The actual amount of enhancement of GABA_A receptor current by propofol is somewhat difficult to interpret because the enhancement of 132L and 632L receptor currents is probably increased by the direct activation of propofol on these receptors. Nonetheless, these concentration-response curves give the GABA concentration dependence in the presence or absence of propofol and therefore are functionally useful.

Modulation by Propofol of Peak Currents, Desensitization, and Deactivation Evoked by a Saturating Concentration of GABA Was Similar for 132L and 632L GABA_A Receptors. We were interested in exploring the modulation by propofol of peak GABA_A receptor currents and kinetic properties evoked by a saturating GABA concentration. Long pulses of 1 mM GABA provide information about multiple phases of desensitization as well as the deactivation after washout of GABA. To better resolve the fast phase of desensitization, the cells were lifted from the recording dish to improve solution exchange rate. Propofol (10 µM) was preapplied and followed by coapplication of propofol (10 µM) and a saturating GABA concentration (1 mM), allowing controlled duration of pre-equilibration as well as resolution of the direct effect of propofol. Propofol did not potentiate the peak GABA-evoked current of either 132L (98.3 ± 1.4%, n = 6) or 632L (97.4 ± 4.7%, n = 6) receptors (Fig. 4, A-C).

View larger version (25K): [in this window] [in a new window]   Fig. 4. Propofol modulated the peak current, desensitization, and deactivation of 132L and 632L receptors similarly. A and B, representative whole-cell current traces evoked by GABA (1 mM) alone as well as coapplication of GABA (1 mM) and propofol (10 µM) with propofol preapplied from 132L and 632L receptors. The GABA control current (gray trace) was normalized to the current evoked by coapplication of GABA and propofol to demonstrate the changes in desensitization and deactivation. C, propofol did not potentiate the mean GABA peak currents of 132L (n = 6) and 632L (n = 6) receptors. The gray dashed line indicates 100%. D, propofol treatment significantly decreased the mean desensitization of 132L and 632L receptors. E, propofol treatment significantly increased the mean time constant of deactivation of 132L and 632L receptors. The solid line above each representative current trace denotes the duration of GABA application, and the black dashed line denotes that of propofol application. The error bars represent means ± S.E.M. *, significantly different from corresponding GABA control at p < 0.05; **, p < 0.01; ##, significantly different from GABA + propofol of 132L isoform at p < 0.01; and +++, significantly different from GABA control of 132L isoform at p < 0.001.

Mean desensitization of 132L receptor current induced by GABA alone was 54.5 ± 5.0%, which was significantly smaller than that of 632L receptor current (81.9 ± 3.1%) (p < 0.001). Propofol reduced the extent of desensitization for both 132L and 632L receptor currents. Mean desensitization was significantly reduced to 44.0 ± 5.6% (p < 0.01) for 132L receptors and to 72.1 ± 3.9% (p < 0.01) for 632L receptors (Fig. 4, A, B, and D). These results were consistent with prior studies of propofol on neuronal GABA_A receptors (Bai et al., 1999; Dong and Xu, 2002).

Propofol also prolonged deactivation of both 132L and 632L receptor currents. The mean weighted deactivation rate for 132L receptor currents was significantly increased from 420.6 ± 63.2 to 534.6 ± 76.1 ms by propofol (p < 0.01), and that of 632L receptor currents was significantly increased from 346.3 ± 32.8 to 533.2 ± 68.6 ms (p < 0.05) (Fig. 4, A, B, and E).

Modulation by Propofol of Peak Currents and Deactivation with a Saturating Concentration of GABA Was Different for 13 and 63 GABA_A Receptors. The preapplication and lifted cell techniques were also used to explore propofol modulation of peak currents and kinetic properties of 13 and 63 receptor currents. The mean peak current enhancement of 13 receptors by propofol (205.8 ± 33.1%, n = 7) was significantly greater than that of 63 receptors (107.6 ± 5.0%, n = 7) (p < 0.05) (Fig. 5, A-C).

View larger version (22K): [in this window] [in a new window]   Fig. 5. Propofol differentially modulated the peak current and deactivation of 13 and 63 receptors. A and B, representative whole-cell current traces evoked by GABA (1 mM) alone as well as coapplication of GABA (1 mM) and propofol (10 µM) with propofol preapplied from 13 and 63 receptors. C, propofol produced significantly greater mean enhancement from 13 (n = 7) than from 63 (n = 7) receptors. The gray dashed line indicates 100%. D, propofol treatment did not significantly affect the mean desensitization of 13 and 63 receptors. E, propofol treatment significantly increased the mean time constant of deactivation of 63 receptors but did not alter that of 13 receptors. The solid line above each representative current trace denotes the duration of GABA application, and the black dashed line denotes that of propofol application. The error bars represent means ± S.E.M. *, significantly different from corresponding 63 GABA control or 63 isoform at p < 0.05; ###, significantly different from GABA + propofol of 13 isoform at p < 0.001; and +++, significantly different from GABA control of 13 isoform at p < 0.001.

Mean desensitization of 13 receptor currents for GABA alone was 8.9 ± 3.5%, which was significantly smaller than that for 63 receptor currents (49.3 ± 5.2%) (p < 0.001) (Fig. 5, A, B, and D), consistent with previous reports (Bianchi et al., 2002). Propofol did not alter the extent of desensitization for either isoform.

The deactivation rate of 13 receptor currents after application of GABA alone was 103.9 ± 16.0 ms, which was significantly faster than that for 63 receptor currents (315.0 ± 23.8 ms) (p < 0.001) (Fig. 5, A, B, and E). Although propofol did not alter deactivation of 13 receptor currents, it increased the deactivation rate to 411.5 ± 44.3 ms for 63 receptor currents (p < 0.05).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The Enhancement of Maximal Peak Currents by Propofol Was Greater for 13 Receptors Than for 132L, 632L, and 63 Receptors. For all recombinant GABA_A receptor isoforms examined in the present study, propofol directly activated the receptors with multiphasic concentration-response curves. The mechanisms underlying these properties remain unknown. One possibility is that there are multiple receptor binding sites with different affinities for propofol. Propofol in the presence of high GABA concentrations produced a more substantial upward shift of the GABA concentration-response curve for 13 than for 132L, 632L, and 63 receptors. Thus, propofol was similar to pentobarbital and neurosteroids in exerting greater potentiation of this receptor isoform (Wohlfarth et al., 2002; Feng et al., 2004). Coapplication of 3 µM propofol with high concentrations of GABA produced a small enhancement of 132L and 632L receptor currents. A part of the enhancement might be contributed by direct activation by propofol of GABA_A receptors because propofol at this concentration evoked direct currents from 132L and 632L receptors. Consistent with this interpretation, when propofol was preapplied, and thus the direct activation current could be taken into account, 132L and 632L receptor peak currents were not increased by propofol. Thus, propofol modulation was similar to those of pentobarbital and neurosteroids (Wohlfarth et al., 2002; Feng et al., 2004), which did not potentiate maximal GABA-evoked currents. In contrast, propofol enhanced both 13 and 63 receptor peak currents. subunit-containing GABA_A receptors have been reported to be modulated by a variety of structurally different compounds (Lees and Edwards, 1998; Thompson et al., 2002; Wohlfarth et al., 2002; Wallner et al., 2003; Feng et al., 2004). The consistent observation with subunit-containing receptors is that, unlike most isoforms, the maximal currents evoked by GABA can be increased by allosteric modulators. These observations are reminiscent of the increased efficacy of positive modulators on receptor currents activated by partial agonists. There is also direct evidence that GABA is not a "full" agonist at isoforms, because the synthetic GABA analog 4,5,6,7-tetra-hydroisoxazolo[4,5-c]pyridin-3-ol activated larger currents than GABA (Adkins et al., 2001; Brown et al., 2002). These data support the idea that GABA may be a partial agonist for subunit-containing GABA_A receptors (Bianchi and Macdonald, 2003). It is noteworthy that propofol was reported to slightly enhance the saturating GABA-evoked peak currents of GABA_A receptors on native hippocampal neurons (Bai et al., 1999), but the contribution of subunit-containing receptors on these neurons was not known (Wisden et al., 1992).

Subunit-Dependent Modulation of Recombinant GABA_A Receptor Kinetic Properties by Propofol. Propofol significantly decreased the extent of desensitization and prolonged the deactivation of 132L and 632L receptors in a similar manner, suggesting that the kinetic modifications induced by propofol are predominantly dependent on the 2L rather than the subunit in these receptors. This finding is consistent with a report on the modulation of GABA_A receptor kinetic properties by propofol in native hippocampal neurons (Bai et al., 1999), because the predominant GABA_A receptor isoform is 2L in hippocampal pyramidal cells (Wisden et al., 1992). Similar alterations in kinetic properties produced by propofol were also reported for GABA_A receptors in native spinal cord neurons (Dong and Xu, 2002), implying that 2L receptors may also be predominantly present on these neurons. Although propofol decreased desensitization of 2L subunit-containing receptors, we observed prolonged current deactivation. If the decreased macroscopic desensitization reflected reduced stability of desensitized states, faster deactivation would be predicted based on the proposal that prolongation of deactivation is "coupled" with increased desensitization (Jones and Westbrook, 1995; Haas and Macdonald, 1999). A similar pattern of modulation was observed for pentobarbital modulation of 132L receptor currents (Feng et al., 2004). We and others have recently suggested that increasing gating efficacy can secondarily decrease macroscopic desensitization as well as prolong deactivation (Bianchi and Macdonald, 2001; Scheller and Forman, 2002). Consistent with this mechanism, increased gating efficacy (frequency) was reported for propofol modulation of single GABA_A receptor channels from neurons (Orser et al., 1994). Simulation studies also suggested that propofol-evoked prolongation of GABA current deactivation and reduced desensitization might be achieved by propofol stabilization of the ligand-bound pre-open state (Bai et al., 1999).

Propofol did not significantly affect desensitization but differentially modified the deactivation of 13 and 63 receptor currents, providing an additional example of independent modulation of desensitization and deactivation. Pentobarbital and neurosteroids have been reported to increase desensitization and prolong deactivation of 13 receptor currents (Wohlfarth et al., 2002; Feng et al., 2004). However, propofol did not significantly alter the desensitization and deactivation of 13 receptors in the present study, which is unexpected because any kinetic parameter that could increase maximal open probability of a nondesensitizing receptor should also prolong deactivation. We do not have an explanation for this observation. It is possible that we could not resolve changes in deactivation at the whole-cell level. The possible mechanism underlying the different effect of propofol and pentobarbital or neurosteroids on 13 receptor desensitization might be that these general anesthetics differentially modulated the rate constants of the 13 receptor desensitized state. Consistent with this possibility, these drugs had different effects on channel open states. Pentobarbital was reported to increase mean open duration of recombinant receptor single channel currents, including 13 receptors (Feng et al., 2004), whereas in contrast, propofol has been reported to increase channel open frequency (Orser et al., 1994). Propofol did not significantly modify desensitization of 63 receptor currents but significantly prolonged deactivation. The mechanisms for this propofol effect remain unclear. One parsimonious explanation may be that propofol slowed the agonist unbinding, which has been reported for another anesthetic drug, halothane (Li and Pearce, 2000). These data suggest that modulation by propofol of GABA_A receptor kinetic properties is subunit-dependent.

Implications for Propofol Actions on 2L or Subunit-Containing Receptor Currents: Multiple Anesthetic Mechanisms. General anesthetics exert their effect in the brain largely by modulating GABA_A receptor synaptic currents (Olsen and Macdonald, 2002). However, substantial recent evidence suggests that nonsynaptic or tonic forms of inhibition can have profound effects on neuronal excitability. Pentobarbital and several other general anesthetics have been reported to potentiate the currents of subunit-containing GABA_A receptors (Lees and Edwards, 1998; Brown et al., 2002; Wohlfarth et al., 2002; Feng et al., 2004). These and the present studies suggest that subunit-containing receptors are an important target for general anesthetics. subunit-containing GABA_A receptors are involved in tonic inhibition (Stell et al., 2003; Wei et al., 2003), suggesting that general anesthetics may partly exert their effect by enhancing tonic inhibition. This may be one explanation for the findings that the tonic inhibition was enhanced by propofol in hippocampal neurons (Bai et al., 2001; Bieda and MacIver, 2004). In addition, propofol as well as pentobarbital (Feng et al., 2004) decreased the desensitization and prolonged the deactivation of 2L subunit-containing receptors, which may mediate the phasic inhibition. This modification of kinetic properties by propofol has been demonstrated to prolong the synaptic currents (Bai et al., 1999). Therefore, some general anesthetics such as propofol may also exert their effect by enhancing phasic inhibition. In addition, it was reported that a clinically relevant concentration of propofol is 0.4 µM (Dong and Xu, 2002). It is noteworthy that propofol at this concentration evoked direct currents from 2L subunit-containing receptors. Thus, in addition to modulating GABA_A receptor currents, propofol may directly activate GABA_A receptors to contribute to its anesthetic effects in the brain.

The 12L receptor isoform is ubiquitously distributed in the brain and may be one of the major targets for propofol anesthetic effects. In contrast, 62L and 6 isoforms are restricted to the cerebellum (Wisden et al., 1992), a structure that may be involved in propofol anesthetic side effects such as ataxia. In the present study, we report that propofol substantially enhanced 13 receptor peak currents; however, a previous study suggested 1 receptors may be a minor isoform of subunit-containing GABA_A receptors in the brain (Poltl et al., 2003). Therefore, it is likely that 13 receptors have limited contribution to propofol effects in the brain. More importantly, the present findings that propofol similarly modulated the deactivation and/or desensitization of 132L, 632L, and 63 receptors suggest that propofol may exert similar effect on GABAergic inhibition in different regions of the brain. These data also suggest that the enhancement of tonic inhibition and phasic inhibition may be equally important for propofol anesthetic effects as well as side effects. Further experiments are needed to confirm these speculations in vivo.

	Acknowledgements

 
We thank Luyan Song for preparing the GABA_A receptor subunit cDNAs and Matt T. Bianchi and Martin J. Gallagher for critical reading of the manuscript.

	Footnotes

 
This work was supported by National Institutes of Health grant R01-NS33300 (to R.L.M.).

ABBREVIATION: HEK, human embryonic kidney.

Address correspondence to: Dr. Robert L. Macdonald, Department of Neurology, Vanderbilt University Medical Center, 6140 Medical Research Building III, 465 21st Ave., South, Nashville, Tennessee 37232-8552. E-mail: robert.macdonald{at}vanderbilt.edu

	References

Top Abstract Materials and Methods Results Discussion References

 
Adkins CE, Pillai GV, Kerby J, Bonnert TP, Haldon C, McKernan RM, Gonzalez JE, Oades K, Whiting PJ, and Simpson PB (2001) 43 GABA_A receptors characterized by fluorescence resonance energy transfer-derived measurements of membrane potential. J Biol Chem 276: 38934-38939.[Abstract/Free Full Text]

Adodra S and Hales TG (1995) Potentiation, activation and blockade of GABA_A receptors of clonal murine hypothalamic GT1-7 neurones by propofol. Br J Pharmacol 115: 953-960.[Medline]

Bai D, Pennefather PS, MacDonald JF, and Orser BA (1999) The general anesthetic propofol slows deactivation and desensitization of GABA_A receptors. J Neurosci 19: 10635-10646.[Abstract/Free Full Text]

Bai D, Zhu G, Pennefather P, Jackson MF, MacDonald JF, and Orser BA (2001) Distinct functional and pharmacological properties of tonic and quantal inhibitory postsynaptic currents mediated by -aminobutyric acid_A receptors in hippocampal neurons. Mol Pharmacol 59: 814-824.[Abstract/Free Full Text]

Bianchi MT, Haas KF, and Macdonald RL (2002) 1 and 6 subunits specify distinct desensitization, deactivation and neurosteroid modulation of GABA_A receptors containing the subunit. Neuropharmacology 43: 492-502.[CrossRef][Medline]

Bianchi MT and Macdonald RL (2001) Mutation of the 9' leucine in the GABA_A receptor 2L subunit produces an apparent decrease in desensitization by stabilizing open states without altering desensitized states. Neuropharmacology 41: 737-744.[CrossRef][Medline]

Bianchi MT and Macdonald RL (2002) Slow phases of GABA_A receptor desensitization: structural determinants and possible relevance for synaptic function. J Physiol 544: 3-18.[Abstract/Free Full Text]

Bianchi MT and Macdonald RL (2003) Neurosteroids shift partial agonist activation of GABA_A receptor channels from low- to high-efficacy gating patterns. J Neurosci 23: 10934-10943.[Abstract/Free Full Text]

Bieda MC and MacIver MB (2004) A major role for tonic GABA_A conductances in anesthetic suppression of intrinsic neuronal excitability. J Neurophysiol 92: 1658-1667.[Abstract/Free Full Text]

Brown N, Kerby J, Bonnert TP, Whiting PJ, and Wafford KA (2002) Pharmacological characterization of a novel cell line expressing human 43 GABA_A receptors. Br J Pharmacol 136: 965-974.[CrossRef][Medline]

Carlson BX, Engblom AC, Kristiansen U, Schousboe A, and Olsen RW (2000) A single glycine residue at the entrance to the first membrane-spanning domain of the -aminobutyric acid type A receptor 2 subunit affects allosteric sensitivity to GABA and anesthetics. Mol Pharmacol 57: 474-484.[Abstract/Free Full Text]

Davies PA, Kirkness EF, and Hales TG (2001) Evidence for the formation of functionally distinct GABA_A receptors. J Physiol 537: 101-113.[Abstract/Free Full Text]

Dong XP and Xu TL (2002) The actions of propofol on -aminobutyric acid_A and glycine receptors in acutely dissociated spinal dorsal horn neurons of the rat. Anesth Analg 95: 907-914.[Abstract/Free Full Text]

Feng HJ, Bianchi MT, and Macdonald RL (2004) Pentobarbital differentially modulates 13 and 132L GABA_A receptor currents. Mol Pharmacol 66: 988-1003.[Abstract/Free Full Text]

Fisher JL and Macdonald RL (1997) Functional properties of recombinant GABA_A receptors composed of single or multiple subunit subtypes. Neuropharmacology 36: 1601-1610.[CrossRef][Medline]

Fisher JL, Zhang J, and Macdonald RL (1997) The role of 1 and 6 subtype amino-terminal domains in allosteric regulation of -aminobutyric acid_A receptors. Mol Pharmacol 52: 714-724.[Abstract/Free Full Text]

Greenfield LJ Jr, Sun F, Neelands TR, Burgard EC, Donnelly JL, and Macdonald RL (1997) Expression of functional GABA_A receptors in transfected L929 cells isolated by immunomagnetic bead separation. Neuropharmacology 36: 63-73.[CrossRef][Medline]

Haas KF and Macdonald RL (1999) GABA_A receptor subunit 2 and subtypes confer unique kinetic properties on recombinant GABA_A receptor currents in mouse fibroblasts. J Physiol 514: 27-45.[Abstract/Free Full Text]

Hill-Venning C, Belelli D, Peters JA, and Lambert JJ (1997) Subunit-dependent interaction of the general anaesthetic etomidate with the -aminobutyric acid type A receptor. Br J Pharmacol 120: 749-756.[CrossRef][Medline]

Jones MV and Westbrook GL (1995) Desensitized states prolong GABA_A channel responses to brief agonist pulses. Neuron 15: 181-191.[CrossRef][Medline]

Krasowski MD, Nishikawa K, Nikolaeva N, Lin A, and Harrison NL (2001) Methionine 286 in transmembrane domain 3 of the GABA_A receptor subunit controls a binding cavity for propofol and other alkylphenol general anesthetics. Neuropharmacology 41: 952-964.[CrossRef][Medline]

Lam DW and Reynolds JN (1998) Modulatory and direct effects of propofol on recombinant GABA_A receptors expressed in xenopus oocytes: influence of - and 2-subunits. Brain Res 784: 179-187.[CrossRef][Medline]

Lees G and Edwards MD (1998) Modulation of recombination human -aminobutyric acid_A receptors by isoflurane: influence of the subunit. Anesthesiology 88: 206-217.[CrossRef][Medline]

Li X and Pearce RA (2000) Effects of halothane on GABA_A receptor kinetics: evidence for slowed agonist unbinding. J Neurosci 20: 899-907.[Abstract/Free Full Text]

McKernan RM and Whiting PJ (1996) Which GABA_A-receptor subtypes really occur in the brain? Trends Neurosci 19: 139-143.[CrossRef][Medline]

Nusser Z, Sieghart W, and Somogyi P (1998) Segregation of different GABA_A receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. J Neurosci 18: 1693-1703.[Abstract/Free Full Text]

Olsen RW and Macdonald RL (2002) GABA_A receptor complex: structure and function, in Glutamate and GABA Receptors and Transporters: Structure, Function and Pharmacology (Egebjerg J, Schousboe A, and Krogsgaard-Larsen P eds) pp 202-235, Taylor & Francis, London.

Orser BA, Wang LY, Pennefather PS, and MacDonald JF (1994) Propofol modulates activation and desensitization of GABA_A receptors in cultured murine hippocampal neurons. J Neurosci 14: 7747-7760.[Abstract]

Pistis M, Belelli D, McGurk K, Peters JA, and Lambert JJ (1999) Complementary regulation of anaesthetic activation of human (632L) and Drosophila (RDL) GABA receptors by a single amino acid residue. J Physiol 515: 3-18.[Abstract/Free Full Text]

Poltl A, Hauer B, Fuchs K, Tretter V, and Sieghart W (2003) Subunit composition and quantitative importance of GABA_A receptor subtypes in the cerebellum of mouse and rat. J Neurochem 87: 1444-1455.[Medline]

Saxena NC and Macdonald RL (1996) Properties of putative cerebellar -aminobutyric acid_A receptor isoforms. Mol Pharmacol 49: 567-579.[Abstract]

Scheller M and Forman SA (2002) Coupled and uncoupled gating and desensitization effects by pore domain mutations in GABA_A receptors. J Neurosci 22: 8411-8421.[Abstract/Free Full Text]

Stell BM, Brickley SG, Tang CY, Farrant M, and Mody I (2003) Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by subunit-containing GABA_A receptors. Proc Natl Acad Sci USA 100: 14439-14444.[Abstract/Free Full Text]

Thompson SA, Wingrove PB, Connelly L, Whiting PJ, and Wafford KA (2002) Tracazolate reveals a novel type of allosteric interaction with recombinant -aminobutyric acid_A receptors. Mol Pharmacol 61: 861-869.[Abstract/Free Full Text]

Uchida I, Li L, and Yang J (1997) The role of the GABA_A receptor 1 subunit N-terminal extracellular domain in propofol potentiation of chloride current. Neuropharmacology 36: 1611-1621.[CrossRef][Medline]

Wallner M, Hanchar HJ, and Olsen RW (2003) Ethanol enhances 43 and 63 -aminobutyric acid type A receptors at low concentrations known to affect humans. Proc Natl Acad Sci USA 100: 15218-15223.[Abstract/Free Full Text]

Wei W, Zhang N, Peng Z, Houser CR, and Mody I (2003) Perisynaptic localization of subunit-containing GABA_A receptors and their activation by GABA spillover in the mouse dentate gyrus. J Neurosci 23: 10650-10661.[Abstract/Free Full Text]

Williams DB and Akabas MH (2002) Structural evidence that propofol stabilizes different GABA_A receptor states at potentiating and activating concentrations. J Neurosci 22: 7417-7424.[Abstract/Free Full Text]

Wisden W, Laurie DJ, Monyer H, and Seeburg PH (1992) The distribution of 13 GABA_A receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. J Neurosci 12: 1040-1062.[Abstract]

Wohlfarth KM, Bianchi MT, and Macdonald RL (2002) Enhanced neurosteroid potentiation of ternary GABA_A receptors containing the subunit. J Neurosci 22: 1541-1549.[Abstract/Free Full Text]

This article has been cited by other articles:

				H. Shiratsuchi, Y. Kouatli, G. X. Yu, H. M. Marsh, and M. D. Basson Propofol inhibits pressure-stimulated macrophage phagocytosis via the GABAA receptor and dysregulation of p130cas phosphorylation Am J Physiol Cell Physiol, June 1, 2009; 296(6): C1400 - C1410. [Abstract] [Full Text] [PDF]

				S. Ellefsen, K.-O. Stenslokken, C. E. Fagernes, T. A. Kristensen, and G. E. Nilsson Expression of genes involved in GABAergic neurotransmission in anoxic crucian carp brain (Carassius carassius) Physiol Genomics, January 8, 2009; 36(2): 61 - 68. [Abstract] [Full Text] [PDF]

				X. Wang Propofol and Isoflurane Enhancement of Tonic Gamma-Aminobutyric Acid Type A Current in Cardiac Vagal Neurons in the Nucleus Ambiguus Anesth. Analg., January 1, 2009; 108(1): 142 - 148. [Abstract] [Full Text] [PDF]

				H. P. Goodkin, S. Joshi, Z. Mtchedlishvili, J. Brar, and J. Kapur Subunit-Specific Trafficking of GABAA Receptors during Status Epilepticus J. Neurosci., March 5, 2008; 28(10): 2527 - 2538. [Abstract] [Full Text] [PDF]

				C. J. Herden, N. E. Pardo, R. K. Hajela, Y. Yuan, and W. D. Atchison Differential Effects of Methylmercury on {gamma}-Aminobutyric Acid Type A Receptor Currents in Rat Cerebellar Granule and Cerebral Cortical Neurons in Culture J. Pharmacol. Exp. Ther., February 1, 2008; 324(2): 517 - 528. [Abstract] [Full Text] [PDF]

				M. T. Bianchi, E. J. Botzolakis, K. F. Haas, J. L. Fisher, and R. L. Macdonald Microscopic kinetic determinants of macroscopic currents: insights from coupling and uncoupling of GABAA receptor desensitization and deactivation J. Physiol., November 1, 2007; 584(3): 769 - 787. [Abstract] [Full Text] [PDF]

				M. Mortensen and T. G. Smart Extrasynaptic {alpha}{beta} subunit GABAA receptors on rat hippocampal pyramidal neurons J. Physiol., December 15, 2006; 577(3): 841 - 856. [Abstract] [Full Text] [PDF]

				M. Yamashita, W. Marszalec, J. Z. Yeh, and T. Narahashi Effects of Ethanol on Tonic GABA Currents in Cerebellar Granule Cells and Mammalian Cells Recombinantly Expressing GABAA Receptors J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 431 - 438. [Abstract] [Full Text] [PDF]

				K. R. Drasbek and K. Jensen THIP, a Hypnotic and Antinociceptive Drug, Enhances an Extrasynaptic GABAA Receptor-mediated Conductance in Mouse Neocortex Cereb Cortex, August 1, 2006; 16(8): 1134 - 1141. [Abstract] [Full Text] [PDF]

				I. Yamaoka, M. Doi, M. Nakayama, A. Ozeki, S. Mochizuki, K. Sugahara, and F. Yoshizawa Intravenous administration of amino acids during anesthesia stimulates muscle protein synthesis and heat accumulation in the body Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E882 - E888. [Abstract] [Full Text] [PDF]

				D. A. Wagner, M. P. Goldschen-Ohm, T. G. Hales, and M. V. Jones Kinetics and Spontaneous Open Probability Conferred by the {epsilon} Subunit of the GABAA Receptor J. Neurosci., November 9, 2005; 25(45): 10462 - 10468. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: mol.104.003426v1 66/6/1517    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Feng, H.-J. Articles by Macdonald, R. L. Search for Related Content PubMed PubMed Citation Articles by Feng, H.-J. Articles by Macdonald, R. L.