γ-AMINOBUTYRIC acid type A (GABAA
) receptors are the major inhibitory neurotransmitter system in the brain and important targets for many neuromodulatory drugs, including sedatives, antiepileptics, neuro-active steroids, and general anesthetics. Each receptor-channel complex consists of five homologous subunits arranged symmetrically around a gated chloride channel. Subunits share topology with all members of the cys-loop ligand-gated ion channel superfamily: a large extracellular N-terminal domain, four transmembrane domains (TM1-4), and a large intracellular loop between TM3 and TM4.1
Genetic techniques have identified 18 different GABAA
receptor subunits: α1–6
, δ, ϵ, π, and ρ1–3
. The most common GABAA
receptor subunit composition and stoichiometry is 2α:2β:1γ.2
R(+)-Etomidate is a potent intravenous anesthetic that produces sedative/hypnotic and immobilizing actions by positively modulating GABAA
receptors in the brain and spinal cord.3
The R(+) enantiomer of etomidate displays approximately 20-fold greater anesthetic potency than S(-) both in animals and in its ability to modulate GABAA
Etomidate at clinical concentrations (2 or 3 μm)8
enhances GABA-activated GABAA
receptor function, shifting GABA dose responses leftward (i.e.
, reducing EC50
). Higher concentrations of etomidate directly activate GABAA
receptors in the absence of orthosteric agonists.5,9,10
In inhibitory postsynaptic potentials and rapid superfusion experiments, etomidate also slows GABAA
receptor-mediated current deactivation on termination of agonist application.9,11
receptors containing β2
subunits, but not receptors containing β1
subunits, are sensitive to etomidate.12,13
An amino acid at β subunit position 265 (N in β2/3
and S in β1
) strongly affects receptor sensitivity to etomidate in vitro14–16
and anesthesia in vivo
Azi-etomidate, a diaziryl photo-affinity derivative of etomidate, shows reversible pharmacological activity, including stereoselectivity, nearly identical to etomidate in animals and in molecular studies of GABAA
H]-azi-etomidate was used to photo-label affinity-purified detergent-solubilized bovine GABAA
Azi-etomidate photo-incorporated into bovine receptor at methionines in transmembrane domains on both α (α1
M236 and homologs) and β (β3
M286 and homologs), suggesting that etomidate binds at the interfaces between these subunits. This transmembrane domain interface may also form a neurosteroid binding site.22
Because azi-etomidate is predicted to selectively photo-modify specific GABAA
receptors, it could be useful in neurophysiological studies when applied together with localized photo-activation. However, the effects of photo-modifying functional GABAA
receptors are unknown.
In this article, we describe electrophysiological experiments on functional expressed GABAA receptors exposed to photo-activated azi-etomidate. We hypothesized that α1β2γ2L GABAA receptors modified with azi-etomidate would display persistent functional changes that mimic the reversible effects of etomidate or azi-etomidate binding. Experiments were performed with photo-activated azi-etomidate and with etomidate or nonactivated azi-etomidate as reversible controls. We also determined whether GABAA receptors that were irreversibly modified by azi-etomidate could be activated by etomidate and propofol, and whether high concentrations of etomidate could block azi-etomidate modification of GABAA receptor sites.
Materials and Methods
Female Xenopus laevis were housed in a veterinary-supervised environment and used in accordance with local and federal guidelines, with approval of the Massachusetts General Hospital Committee on Animal Care (Boston, MA). Frogs were anesthetized by immersion in ice-cold 0.2% tricaine (Sigma-Aldrich, St. Louis, MO) before mini-laparotomy to harvest oocytes.
R(+)-Etomidate was obtained from Bedford Laboratories (Bedford, OH). The clinical preparation in 30% propylene glycol was diluted directly into buffer. Previous studies have shown that propylene glycol at the dilutions used for these studies has no effect on GABAA
R(+)-Azi-etomidate [2-(3-methyl-3H-diaziren-3-yl)ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate] was a gift from Shaukat Husain, Ph.D. (Massachusetts General Hospital, Boston, MA) and was stored in the dark at −20°C as a 10-mm solution in methanol. The highest concentration of methanol used (0.32% v/v) did not cause measurable modulation of GABAA
receptors. Salts and buffers were purchased from Sigma-Aldrich.
Expression of GABAA Receptors
cDNAs for human GABAA
, and γ2L
subunits in pCDM8 plasmids were gifts from Dr. Paul J. Whiting, Ph.D. (Merck Sharp & Dohme Research Labs, Essex, UK). Messenger RNA was synthesized in vitro
from linearized cDNA templates and purified using commercial kits (Ambion Inc., Austin, TX). Xenopus
oocytes were microinjected with 25–50 nl (15–25 ng) of subunit mRNA mixture (1α:1β:5γ) and incubated at 16°C in ND96 (in mm: 96 NaCl, 2 KCl, 0.8 MgCl2
, 1.8 CaCl2
, 5 HEPES, pH 7.5) supplemented with penicillin/streptomycin (1% v/v) for 48–96 h before electrophysiology. Human embryonic kidney cells (HEK293) were maintained and transfected as previously described.23
A plasmid designed to drive eukaryotic expression of green fluorescent protein was mixed with the GABAA
receptor subunit plasmids to aid in identification of transfected cells. After transfection, cells were returned to culture medium and maintained for 24–72 h before electrophysiology experiments.
receptor responses to GABA were assessed in whole oocytes using two microelectrode voltage clamp electrophysiology, as previously described.24
GABA pulse durations were from 5 to 20 s, depending on the concentration of GABA used and the time to steady-state peak current.
Electrophysiology in HEK293 Cells
Whole-cell and excised outside-out patch clamp recordings were performed at –50 mV and room temperature (21–23°C) as previously described.23
Extracellular fluid (ECF) contained (in mm) 135 NaCl, 5.4 KCl, 5 HEPES, 1.6 CaCl2
, and 1 MgCl2
at pH 7.4. The intracellular (pipette) fluid contained (in mm) 140 KCl, 10 HEPES, 1 EGTA, and 1 MgCl2
at pH 7.3. Currents were stimulated using brief (0.1–1.5 s) pulses of GABA delivered via
a quad (2 × 2) superfusion pipette coupled to piezo-electric elements that switched superfusion solutions in less than 1 ms.25
Currents were digitized and recorded for later analysis.
Photo-modification in Oocytes
Photo-excitation of azi-etomidate for receptor modification experiments in oocytes was achieved using a long-wavelength (365 nm peak) ultraviolet (UV) lamp (0.16 ampere handheld; Model UVL-56, UVP, Upland, CA) that was positioned 4 cm above a Petri dish containing oocytes in ND96 solutions. Six groups of oocytes expressing GABAA receptors were pretreated for 5 min as follows: Group 1 was maintained in ND96 electrophysiology buffer; Group 2 was incubated in ND96 buffer and exposed to 365 nm UV light; Group 3 was incubated in ND96 containing 3.2 μm azi-etomidate; Group 4 was incubated in ND96 containing 3.2 μm azi-etomidate during exposure to 365 nm UV light; Group 5 was incubated in ND96 containing 3.2 μm azi-etomidate and 10 μm GABA; and Group 6 was incubated in ND96 containing 3.2 μm azi-etomidate and 10 μm GABA during exposure to 365 nm UV light. Immediately after pretreatment, oocytes were transferred with minimal buffer to 15-ml conical centrifuge tubes containing 10 ml ND96. After the closed tubes were incubated on a rocking platform for 3 min, the ND96 was aspirated, leaving minimal liquid, and replaced with fresh ND96. This wash procedure was repeated 10 times, and the washed oocytes were returned to ND96 in new Petri dishes.
Photo-modification in HEK293 Cells
Azi-etomidate stock was diluted in ECF with or without GABA (10 μm) and added to prewashed HEK293 cells, which were on glass coverslips in 35-mm Petri dishes. Cells were irradiated for 5 min with 365 nm UV light from the handheld lamp at a distance of 4 cm, then washed with a continuous stream of ECF flowing (3 ml/min) over the coverslip on a tilted Petri dish for 10 min. The coverslip and cells were then transferred to a flow-chamber mounted on the x-y slide manipulator of an inverted microscope stage, where cells were further washed with continuous ECF flow (1 ml/min) for at least another 10 min before patch-clamp recordings. An individual cell/patch photo-modification procedure was also performed on isolated voltage-clamped cells and excised outside-out patches during electrophysiological experiments. ECF containing azi-etomidate with or without GABA was continuously superfused onto the voltage-clamped cell or membrane patch via one channel of the 2 × 2 quad pipette apparatus, while irradiating with 365-nm bandpass-filtered light from the microscope’s fluorescence lamp source (a 100-watt short-arc mercury xenon lamp: model USH-102D; Ushio Inc., Tokyo, Japan). After modification, the voltage-clamped cell or patch was washed in ECF and tested at intervals for current elicited with a pulse of 1 mm GABA until a steady-state response (less than 10% change in peak current) was observed.
Data Analysis and Statistics
Electrophysiological traces were analyzed offline. Baseline leak was subtracted to correct peak currents. Deactivation time constants in HEK293 cells were derived using nonlinear least-squares fits to a multi-exponential decay function with up to 3 components: I(t) = A1 × exp(−t/τ1) + A2 × exp(−t/τ2) + A3 × exp(−t/τ3) + C. The number of components for each fit was determined by comparison of single-, double-, and triple-exponential fits, using an F test to choose the best exponential fit model with a confidence value of P = 0.99 (Clampfit8.0; Molecular Devices, Sunnyvale, CA). Most deactivation fits before modification required two components, and a handful were best fit by three components. Deactivation after photo-modification, with a few exceptions, was fitted with a single exponential decay function. To enable averaging and comparisons when different numbers of exponential components were fitted, we calculated the weighted average deactivation time constant: τw = [A1 × τ1 + A2 × τ2 + A3 × τ3]/[A1 + A2 + A3]. Results are reported as mean ± SD unless otherwise indicated. Group comparisons were performed using either two-tailed Student’s t test (with independent variances) or ANOVA with Tukey post hoc multiple comparisons test in MS Excel (Microsoft Corp., Remond, WA) with an add-on statistical toolkit (StatistiXL, Broadway Nedlands, Australia).
Reversible Azi-Etomidate Effects on α1β2γ2L GABAA Receptor-mediated Currents Elicited with Rapid Agonist Jumps
GABA-activated currents recorded from control (ECF-washed) HEK293 cells and excised membrane patches displayed features that are similar to those previously reported.23
GABA at 10 μm elicited 10-20% of the maximal current stimulated with 1 mm GABA (fig. 1A
). After terminating the GABA superfusion, GABAA
receptor-mediated current deactivation demonstrated two phases, with a weighted average time constant (τw
) ranging from 55 to 100 ms (average 70 ± 13 ms). The addition of 1–10 μm azi-etomidate enhanced peak responses to low GABA and dramatically prolonged current deactivation (fig. 1B
). These effects were reversed after washing with ECF (fig. 1C
). Combined peak current data displayed a GABA EC50
of 45 μm, and coapplication of 3.2 μm azi-etomidate produced approximately a sixfold decrease in GABA EC50
). This is less than the 10-fold reduction in GABA EC50
previously reported with 3.2 μm etomidate10
and consistent with the slightly lower potency of azi-etomidate compared with etomidate.6
To reduce the intracellular accumulation of azi-etomidate during long exposures, we tested the effects of high azi-etomidate concentrations on excised outside-out membrane patches. When superfused with 100–200 μm azi-etomidate alone, voltage-clamp recordings from excised patches demonstrated currents that rapidly increased and slowly desensitized (fig. 1E
). Peak currents directly activated with 200 μm azi-etomidate were approximately 15% of those elicited with 1 mm GABA. Azi-etomidate–activated currents desensitized to approximately 10% of peak in 60–80 s.
Irreversible Modification of GABAA Receptors in Xenopus Oocytes
Initial photo-modification studies were performed on α1
receptors expressed in Xenopus
oocytes. Groups of oocytes were subjected to different photo-modification protocols, then extensively washed to remove azi-etomidate before functional testing using two-microelectrode voltage clamp electrophysiology. Our principle measurement was the relative current elicited with low (10 μm) vs.
maximal (1 mm) GABA (I10
). Results are summarized in figure 2A
. In control oocytes incubated in ND96 buffer (Group 1), the I10
ratio averaged 0.15 (±0.037). Oocytes treated with 365 UV light and those exposed to azi-etomidate (3.2 μm) with or without UV light for 5 min (Groups 2, 3, and 4) displayed I10
ratios that were not significantly different from Group 1. Oocytes pretreated with azi-etomidate plus GABA and UV light (Group 6) gave an I10
ratio of 0.26 (±0.083), which was significantly different from control (P
= 0.02), whereas those pretreated with azi-etomidate plus GABA in the absence of UV light (Group 5) gave an I10
ratio that was not significantly different from control. These oocyte studies demonstrate a GABA-dependent, photo-dependent, irreversible modification of GABAA
receptors by azi-etomidate that mimics the reversible effects of etomidate and azi-etomidate.
Irreversible enhancement of I10/I1000 ratios was also observed in oocyte-expressed receptors after treatment with 1 μm photo-activated azi-etomidate in the presence of GABA, whereas exposure to 1 μm azi-etomidate and GABA in the absence of UV light did not increase I10/I1000. However, after 5 min oocyte incubation in 10 μm azi-etomidate (without photo-activation) or 10 μm etomidate, GABAA receptor gating remained significantly enhanced after more than 1 h of washing. This pseudo-irreversible receptor modulation, under conditions in which no covalent modification was occurring, was likely the result of very slow washout of the anesthetics from Xenopus oocytes, which are extremely large (1 mm in diameter) cells.
Irreversible Modification of GABA-activated Currents in HEK293 Cells Expressing α1β2γ2L GABAA Receptors
HEK293 cells were used to further explore the irreversible effects of azi-etomidate on GABAA receptors. Compared with oocytes, HEK293 cells are small and have high surface/volume ratios, enabling rapid drug application and washout. Initially, GABAA receptors expressed in HEK cells cultured on glass coverslips were photo-modified with azi-etomidate in Petri dishes before electrophysiology. Results were therefore from different sets of cells that had undergone different pretreatments.
Compared with GABA-activated currents from control cells (ECF or ECF + UV; fig. 2B
), currents recorded from cells exposed for 5 min to photo-activated azi-etomidate (3.2-32 μm) plus 10 μm GABA, then extensively washed, displayed both increased I10
and prolonged current deactivation (fig. 2C
). Deactivation of currents from azi-etomidate–modified cells displayed slow mono-exponential kinetics. GABA concentration response curves measured in cells first exposed to 3.2 μm photo-activated azi-etomidate (+10 μm GABA) were characterized by a GABA EC50
values approximately threefold smaller than that derived from control cells (fig. 2D
summarizes the I10
results from experiments using HEK293 cells treated in dishes and washed for at least 20 min. Deactivation rate data (not shown) follow a similar pattern, depending on cell treatment before electrophysiological testing. Cells exposed to photo-activated azi-etomidate (3.2 μm) in the presence of GABA (Azi + GABA + UV) displayed I10
that was significantly higher than control, but I10
was unchanged when GABA was absent (Azi + UV). Exposure to azi-etomidate without UV light (with or without GABA) produced no significant change in receptor function. Similarly, exposure to UV light plus etomidate (up to 100 μm), with or without 10 μm GABA (Eto + UV ± GABA), resulted in currents similar to those of control cells.
We also tested whether excess etomidate (100 μm) could prevent the irreversible enhancement of GABA-activated currents caused by photo-activated azi-etomidate by competing for binding sites. After cells or outside-out patches were pretreated with photo-activated azi-etomidate (3.2 or 10 μm), 10 μm GABA and 100 μm etomidate followed by extensive washing, GABA-activated currents unexpectedly revealed high I10
ratios (fig. 2E
; Azi + Eto + GABA + UV) and prolonged deactivation. Compared with photo-modification with Azi + GABA + UV, both of these measures of irreversible receptor modification were greater when excess etomidate was present (P
= 0.006). One explanation considered for these observations is that UV light and azi-etomidate somehow prolonged etomidate washout from HEK293 cells and patches. If so, then cells and patches subjected to different periods of wash after treatment with photo-activated azi-etomidate plus excess etomidate should display diminishing evidence of modification with time. However, by varying wash time before and during electrophysiological studies, we found no evidence of slow drug washout.
For additional photo-modification studies, we applied azi-etomidate to individual cells or patches via
one channel of the rapid-superfusion apparatus and irradiated with 365 nm bandpass-filtered light from the fluorescence lamp and optics of the electrophysiology setup microscope. GABA-activated currents from cells or excised patches were obtained both before and after exposure to photo-activated azi-etomidate and various wash intervals. After exposure to photo-activated azi-etomidate, GABA-activated currents from cells revealed evidence of irreversible effects, including increased I10
ratios and prolonged deactivation (fig. 3A vs.
3B). Photo-modified GABAA
receptors were also more sensitive to direct activation by both etomidate (not shown) and propofol (fig. 3C vs. 3D
). Photo-modification on the microscope stage appeared to be more efficient than in Petri dishes, consistent with the higher intensity of focused 365 nm light combined with continuous superfusion of azi-etomidate. Five-minute exposure to as little as 0.1 μm photo-activated azi-etomidate plus 10 μm GABA resulted in prolonged current deactivation. Based on I10
ratios, modification using 1 μm azi-etomidate on the microscope stage yielded results similar to modification with 3.2 μm azi-etomidate in Petri dishes (compare fig. 3E and 2E
). Modification of GABA-activated currents was also apparent after exposure to 32 μm photo-activated azi-etomidate in the absence of GABA (fig. 3E
The individual cell modification experiments further revealed that peak GABA-elicited current (I1000
) was greatly reduced immediately after exposure to photo-activated azi-etomidate. Recovery of I1000
during wash occurred on a timescale of minutes (fig. 4A–D
), but I1000
did not recover to full preexposure amplitude. After exposure to 365 nm light, 3.2 μm azi-etomidate, and 10 μm GABA, I1000
recovered to approximately 50% of preexposure control (fig. 3B, 4E
). Cells exposed to this protocol using 1 μm azi-etomidate recovered 75–90% of their preexposure I1000
(fig. 4A, 4E
), whereas 10 μm azi-etomidate reduced I1000
to 15–25% of control (fig. 4E
). Cells exposed to 32 μm photo-activated azi-etomidate plus 10 μm GABA had little or no current response after up to 20 min of washing. The time course for recovery of I1000
was similar after photo-modification with 1.0, 3.2, or 10 μm azi-etomidate, taking 6–10 min to reach steady state (fig. 4F
). Examination of deactivation kinetics during azi-etomidate washout showed that as amplitude recovered, deactivation slowed (fig. 4D
Using the individual excised patch modification technique, we again tested whether 32-fold excess etomidate (100 μm) prevented the irreversible effects of 5-min exposure to 3.2 μm photo-activated azi-etomidate plus 10 μm GABA. After at least 15 min of wash, maximal GABA-activated currents displayed peak currents that averaged only 8 ± 6.5% (n = 3) below pretreatment controls, whereas deactivation remained prolonged (fig. 5A and B
). This represents a significant (P
< 0.003) block of one effect produced by photo-modification with 3.2 μm azi-etomidate (the decrease in I1000
, which averaged 52% when etomidate was absent). However, 32-fold excess etomidate did not block the prolonged deactivation caused by 3.2 μm azi-etomidate modification. Further experiments using 0.32 μm azi-etomidate + 10 μm GABA + UV light demonstrated that 320-fold excess etomidate (100 μm) significantly reduced the persistent prolongation of deactivation in (fig. 5C–F
We demonstrated that exposure to photo-activated azi-etomidate irreversibly alters the electrophysiological properties of α1
receptors expressed in oocytes and HEK293 cells, indicating that receptor photo-modification by this anesthetic photolabel proceeds with apparent high efficiency. Covalent modification with photo-activatable agonists has been combined with ligand-gated ion channel electrophysiology in a small number of previous studies,26–29
and we recently used a photo-activatable anesthetic to investigate a site linked to nicotinic acetylcholine receptor desensitization.30
Recently, persistent GABAA
receptor modulation and anesthetic effects were reported for several photo-activatable neurosteroid analogs without evidence of receptor modification.31
To our knowledge, this is the first report of irreversible functional modulation of GABAA
receptors by a covalent photolabel anesthetic.
Our experiments show that photo-modification with azi-etomidate causes several persistent functional changes in α1
receptors that mimic the reversible effects of etomidate and azi-etomidate (fig. 1 and 2
). Azi-etomidate photo-modification irreversibly potentiated the activation of GABAA
receptors at low GABA concentrations (I10
), reduced GABA EC50
, and slowed deactivation. These changes all are most likely the result of stabilization of open-channel states,9,10
as also observed with anesthetic barbiturates32
and neuroactive steroids.33
Importantly, persistent gating enhancement was only evident when receptors were exposed to both azi-etomidate and 365 nm light (fig. 3E
) and was greatly increased by the addition of GABA (fig. 2
), consistent with the established positive allosteric interaction between etomidate and GABA sites. Our studies also showed no direct effects of 365 nm light on GABAA
receptors. Chang et al 34
previously reported that irradiation with short-wave UV light (265 nm) alone produced persistent gating enhancement of rat α1
receptors in Xenopus
oocytes and noted that such changes were not caused by 365 nm light. Visible light (480 nm) also has no effect on GABAA
Furthermore, sensitization of GABAA
receptors to 365 nm light cannot explain our observations because irradiation in the presence of nonphoto-reactive modulators, etomidate and GABA, produced no irreversible effects.
Azi-etomidate photo-modification also produced both reversible and irreversible loss of receptor function. This was revealed in experiments in which maximal GABA responses (I1000
) before and after azi-etomidate photo-modification were compared in the same single cell or patch (fig. 3 and 4
). During wash after azi-etomidate modification, the reversible I1000
loss recovered independently of the photolabel concentration (fig. 4F
), indicating that drug washout is not the rate-limiting step in recovery. Instead, this observation suggests slow recovery of a population of reversibly desensitized receptors that accumulate during exposure to azi-etomidate plus GABA. Furthermore, during and after washout, GABA-activated currents display slow deactivation, indicating that the recovering receptors are mostly photo-modified.
The fraction of irreversibly inactivated GABA-responsive receptors increased with the photo-activated azi-etomidate concentration (fig. 4E
). This result suggests that a high degree of azi-etomidate modification permanently drives GABAA
receptors into a desensitized state, which we demonstrated during persistent exposure to high azi-etomidate concentrations (fig. 1E
). Thus, azi-etomidate modification produces two distinct irreversible functional changes in GABAA
receptors. Some receptors reversibly desensitize and recover with wash, and these display enhanced GABA sensitivity and slow deactivation (enhanced gating). Another group of receptors permanently desensitize under conditions that favor more modification.
These results confirm that azi-etomidate covalently modifies etomidate sites on GABAA
receptors, but taken alone, these results do not rule out the possibility that other sites on GABAA
receptors are also modified. Biochemical analysis of detergent-solubilized bovine GABAA
receptors showed that most photo-incorporated [3
H]-azi-etomidate was at only two amino acids,21
suggesting that azi-etomidate is also a selective photolabel. It should be noted that the apparent efficiency of photo-labeling of bovine receptors was low, whereas our electrophysiological results suggest that a large fraction of receptors are modified by photo-activated azi-etomidate. The combination of selectivity and efficient photo-modification may make azi-etomidate potentially useful for studies of sensitive GABAA
receptor subtypes in neural circuits. Using highly localized photo-activation with azi-etomidate applied at different concentrations could provide a means for either irreversibly increasing (via
enhanced gating) or irreversibly decreasing (via
desensitization) the activity of a selected population of receptors. Photo-activatable neuroactive steroid analogs have recently been used in this kind of experiment.31
Excess etomidate during azi-etomidate photo-modification blocked the development of irreversible effects, indicating competition and confirming that these effects are mediated by etomidate sites. In single patch modification experiments using a moderate azi-etomidate concentration, 32-fold excess etomidate reduced the amount of irreversible desensitization (I1000
loss) but not the prolongation of deactivation (fig. 5
). Our initial results in cells treated in Petri dishes also indicated that 10- to 32-fold excess etomidate paradoxically increased the irreversible gating effects of azi-etomidate. A reduction in receptor gating effects was observed only when photo-modification was performed with lower azi-etomidate concentrations and etomidate was in 320-fold excess (fig. 5
). Our results suggest that functional competition studies using azi-etomidate and other potentially competitive ligands would need to be carefully designed and interpreted.
Our results also have important mechanistic interpretations regarding the interaction between etomidate and GABAA
receptors. Previous investigations have suggested that there is more than one etomidate site per GABAA
receptor channel, based largely on observations of GABA-enhancing and direct activation at different concentrations.5,10
Photo-labeling with [3
H]-azi-etomidate is consistent with the existence of two similar sites, formed at the interfaces between the two α and two β subunits.21
Some published models of the α1
receptor structure do not predict this interfacial etomidate site,35
pointing to the need for additional structural information. Nonetheless, the presence of two or more etomidate sites per receptor-channel complex is reinforced by our observation that photo-modification with azi-etomidate produces two distinct irreversible effects under different conditions. These observations indicate that at least two sequential modification steps occur: partially modified receptors, perhaps at a single etomidate site, display irreversibly enhanced gating, while modification at multiple sites produces irreversible desensitization. Our etomidate competition results also indicate the presence of multiple etomidate sites per GABAA
receptor, because competition by excess etomidate did not reduce all modification effects in parallel. Azi-etomidate photo-modification also dramatically enhanced subsequent direct activation of GABAA
receptors by etomidate or propofol (fig. 3
), another anesthetic that is thought to act via
the same sites. This result implies that there are at least two etomidate sites per GABAA
receptor, because modification of a lone site should occlude the site and prevent subsequent binding by ligands.
Previous studies have proposed cooperative interactions among etomidate sites. Our photo-modification results, particularly the characteristics of partially modified receptors, represent the first direct experimental evidence for positive cooperativity. Partially modified receptors display enhanced gating by etomidate and propofol, a direct demonstration that modification of an etomidate site promotes the reversible actions of ligands at unmodified sites. Remarkably, photo-modification increases the direct-activating efficacies of etomidate and propofol to levels comparable to that of GABA (fig. 3D
). Millimolar concentrations of etomidate and propofol are not as efficacious as GABA in gating unmodified receptors, so the efficacy increase after photo-modification is unlikely the result of enhanced binding site occupancy. Covalently tethered azi-etomidate apparently exerts a greater allosteric effect on channel gating than saturating noncovalent concentrations of the same ligand. Our etomidate competition results further suggest the presence of reciprocal positive cooperativity among etomidate binding sites. To explain the paradoxical increase in irreversible gating effects after photo-modification in the presence of excess etomidate, the competing ligand must induce an increase in receptor affinity for azi-etomidate that offsets its competition for site occupancy. Given that 32-fold excess etomidate enhanced, whereas 320-fold excess etomidate inhibited, apparent photo-modification by low concentrations of azi-etomidate, we infer that high occupancy of etomidate sites increases affinity for ligands by more than 32-fold and less than 320-fold.
For neurosteroids, which induce dual effects at GABAA
receptors similar to those of etomidate, Hosie et al22
proposed the existence of two classes of sites: high-affinity gating-enhancement sites and low-affinity channel activation sites. In this model, occupation of the enhancement sites allosterically enhances gating via
the activation sites. Another model10
based on symmetry allosteric principles of Monod, Wyman, and Changeux36
suggests the presence of two equivalent etomidate sites that exhibit low affinity in closed receptors and high affinity in open (and desensitized) receptors. Reciprocal positive cooperativity among sites is incorporated into this model via
their common linkage to the functional state. Enhanced etomidate activation after partial modification is easily explained by either of these models. Because reciprocal positive cooperativity is inherent in the two-site allosteric model,10
excess etomidate is also predicted to decrease the rate of formation of doubly modified (desensitizing) receptors, whereas accumulation of singly modified (enhanced gating) receptors could be increased if the allosteric affinity increase (estimated at approximately 150-fold) outweighs competition. Models with two classes of sites can easily be adapted to include reciprocal cooperativity. Another plausible but less likely explanation for our results is that significant asymmetry exists among different etomidate sites and that etomidate and azi-etomidate preferentially bind to different sites. If so, high concentrations of etomidate could preferentially occupy one site while allowing or allosterically enhancing azi-etomidate binding at the second site.
In summary, photo-activated azi-etomidate irreversibly alters the function of α1β2γ2L GABAA receptors, mimicking several reversible etomidate effects. GABAA receptor photo-modification by azi-etomidate requires UV photo-activation, is enhanced in the presence of GABA, and is reduced in the presence of excess etomidate. Functional GABAA receptors modified with azi-etomidate display both enhanced sensitivity to low GABA and prolonged deactivation. In addition, we show that high concentrations of azi-etomidate can deeply desensitize GABAA receptors and that modification with photo-activated azi-etomidate can lead to permanent loss of receptor function, presumably via desensitization. Our data provide evidence for at least two degrees of receptor modification, implying the presence of at least two etomidate sites per GABAA receptor, where the stoichiometry of etomidate site modification is linked to different irreversible functional effects. Furthermore, our results indicate that etomidate binding sites display reciprocal positive cooperativity.
The authors thank Dr. Shakut Husain, Ph.D. (Department of Anesthesia & Critical Care, Massachusetts General Hospital, Boston, Massachusetts), for azi-etomidate; Aiping Liu, M.S. (Senior Lab Technician, Department of Anesthesia & Critical Care, Massachusetts General Hospital), for technical assistance; and Jonathan B. Cohen, Ph.D. (Professor, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts), for helpful discussions.
1. Absalom NL, Lewis TM, Schofield PR: Mechanisms of channel gating of the ligand-gated ion channel superfamily inferred from protein structure. Exp Physiol 2004; 89:145–53
2. McKernan RM, Whiting PJ: Which GABAA-receptor subtypes really occur in the brain? Trends Neurosci 1996; 19:139–43
3. Grasshoff C, Rudolph U, Antkowiak B: Molecular and systemic mechanisms of general anaesthesia: The “multi-site and multiple mechanisms” concept. Curr Opinion Anaesthesiol 2005; 18:386–91
4. Ashton D, Wauquier A: Modulation of a GABA-ergic inhibitory circuit in the in vitro hippocampus by etomidate isomers. Anesth Analg 1985; 64:975–80
5. Tomlin SL, Jenkins A, Lieb WR, Franks NP: Stereoselective effects of etomidate optical isomers on gamma-aminobutyric acid type A receptors and animals. Anesthesiology 1998; 88:708–17
6. Husain SS, Ziebell MR, Ruesch D, Hong F, Arevalo E, Kosterlitz JA, Olsen RW, Forman SA, Cohen JB, Miller KW: 2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate: A derivative of the stereoselective general anesthetic etomidate for photolabeling ligand-gated ion channels. J Med Chem 2003; 46:1257–65
7. Belelli D, Muntoni AL, Merrywest SD, Gentet LJ, Casula A, Callachan H, Madau P, Gemmell DK, Hamilton NM, Lambert JJ, Sillar KT, Peters JA: The in vitro and in vivo enantioselectivity of etomidate implicates the GABAA receptor in general anaesthesia. Neuropharmacology 2003; 45:57–71
8. Arden JR, Holley FO, Stanski DR: Increased sensitivity to etomidate in the elderly: initial distribution versus altered brain response. Anesthesiology 1986; 65:19–27
9. Yang J, Uchida I: Mechanisms of etomidate potentiation of GABAA receptor-gated currents in cultured postnatal hippocampal neurons. Neuroscience 1996; 73:69–78
10. Rusch D, Zhong H, Forman SA: Gating allosterism at a single class of etomidate sites on alpha1beta2gamma2L GABA-A receptors accounts for both direct activation and agonist modulation. J Biol Chem 2004; 279:20982–92
11. Uchida I, Kamatchi G, Burt D, Yang J: Etomidate potentiation of GABAA receptor gated current depends on the subunit composition. Neurosci Lett 1995; 185:203–6
12. Sanna E, Murgia A, Casula A, Biggio G: Differential subunit dependence of the actions of the general anesthetics alphaxalone and etomidate at gamma-aminobutyric acid type A receptors expressed in Xenopus laevis oocytes. Mol Pharmacol 1997; 51:484–90
13. Hill-Venning C, Belelli D, Peters JA, Lambert JJ: Subunit-dependent interaction of the general anaesthetic etomidate with the gamma-aminobutyric acid type A receptor. Br J Pharmacol 1997; 120:749–56
14. Siegwart R, Jurd R, Rudolph U: Molecular determinants for the action of general anesthetics at recombinant alpha(2)beta(3)gamma(2)gamma-aminobutyric acid(A) receptors. J Neurochem 2002; 80:140–8
15. Belelli D, Lambert JJ, Peters JA, Wafford K, Whiting PJ: The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid. Proc Natl Acad Sci U S A 1997; 94:11031–6
16. Moody EJ, Knauer C, Granja R, Strakhova M, Skolnick P: Distinct loci mediate the direct and indirect actions of the anesthetic etomidate at GABA-A receptors. J Neurochem 1997; 69:1310–3
17. Jurd R, Arras M, Lambert S, Drexler B, Siegwart R, Crestani F, Zaugg M, Vogt KE, Ledermann B, Antkowiak B, Rudolph U: General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. FASEB J 2003; 17:250–2
18. Reynolds DS, Rosahl TW, Cirone J, O’Meara GF, Haythornthwaite A, Newman RJ, Myers J, Sur C, Howell O, Rutter AR, Atack J, Macaulay AJ, Hadingham KL, Hutson PH, Belelli D, Lambert JJ, Dawson GR, McKernan R, Whiting PJ, Wafford KA: Sedation and anesthesia mediated by distinct GABA(A) receptor isoforms. J Neurosci 2003; 23:8608–17
19. O’Meara GF, Newman RJ, Fradley RL, Dawson GR, Reynolds DS: The GABA-A beta3 subunit mediates anaesthesia induced by etomidate. Neuroreport 2004; 15:1653–6
20. Liao M, Sonner JM, Husain SS, Miller KW, Jurd R, Rudolph U, Eger EI II: R (+) etomidate and the photoactivable R (+) azietomidate have comparable anesthetic activity in wild-type mice and comparably decreased activity in mice with a N265M point mutation in the gamma-aminobutyric acid receptor beta3 subunit. Anesth Analg 2005; 101:131–5
21. Li GD, Chiara DC, Sawyer GW, Husain SS, Olsen RW, Cohen JB: Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog. J Neurosci 2006; 26:11599–605
22. Hosie AM, Wilkins ME, da Silva HM, Smart TG: Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. Nature 2006; 444:486–9
23. Scheller M, Forman SA: Coupled and uncoupled gating and desensitization effects by pore domain mutations in GABA(A) receptors. J Neurosci 2002; 22:8411–21
24. Rusch D, Forman SA: Classic benzodiazepines modulate the open-close equilibrium in alpha1beta2gamma2L gamma-aminobutyric acid type A receptors. Anesthesiology 2005; 102:783–92
25. Forman SA: A hydrophobic photolabel inhibits nicotinic acetylcholine receptors via open-channel block following a slow step. Biochemistry 1999; 38:14559–64
26. Brown RL, Gerber WV, Karpen JW: Specific labeling and permanent activation of the retinal rod cGMP-activated channel by the photoaffinity analog 8-p-azidophenacylthio-cGMP. Proc Natl Acad Sci U S A 1993; 90:5369–73
27. Mourot A, Rodrigo J, Kotzyba-Hibert F, Bertrand S, Bertrand D, Goeldner M: Probing the reorganization of the nicotinic acetylcholine receptor during desensitization by time-resolved covalent labeling using [3H]AC5, a photoactivatable agonist. Mol Pharmacol 2006; 69:452–61
28. He Y, Karpen JW: Probing the interactions between cAMP and cGMP in cyclic nucleotide-gated channels using covalently tethered ligands. Biochemistry 2001; 40:286–95
29. Leite JF, Blanton MP, Shahgholi M, Dougherty DA, Lester HA: Conformation-dependent hydrophobic photolabeling of the nicotinic receptor: electrophysiology-coordinated photochemistry and mass spectrometry. Proc Natl Acad Sci U S A 2003; 100:13054–9
30. Forman SA, Zhou QL, Stewart DS: Photo-activated 3-azioctanol irreversibly desensitizes muscle nicotinic ACh receptors via interactions at αE262. Biochemistry 2007; 46:11,911–8
31. Eisenman LN, Shu HJ, Akk G, Wang C, Manion BD, Kress GJ, Evers AS, Steinbach JH, Covey DF, Zorumski CF, Mennerick S: Anticonvulsant and anesthetic effects of a fluorescent neurosteroid analog activated by visible light. Nature Neurosci 2007; 10:523–30
32. Steinbach JH, Akk G: Modulation of GABA(A) receptor channel gating by pentobarbital. J Physiol 2001; 537:715–33
33. Akk G, Bracamontes JR, Covey DF, Evers A, Dao T, Steinbach JH: Neuroactive steroids have multiple actions to potentiate GABAA receptors. J Physiol 2004; 558:59–74
34. Chang Y, Xie Y, Weiss DS: Positive allosteric modulation by ultraviolet irradiation on GABA(A), but not GABA(C), receptors expressed in Xenopus oocytes. J Physiol 2001; 536:471–8
35. Campagna-Slater V, Weaver DF: Anaesthetic binding sites for etomidate and propofol on a GABAA receptor model. Neurosci Lett 2007; 418:28–33
36. Monod J, Wyman J, Changeux J: On the nature of allosteric transitions: A plausible model. J Mol Biol 1965; 12:88–118
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