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ANESTHETIC PHARMACOLOGY

Anesthetic-Like Modulation of a -Aminobutyric Acid Type A, Strychnine-Sensitive Glycine, and N-Methyl-d-Aspartate Receptors by Coreleased Neurotransmitters

Pavle S. Milutinovic, MS^*, Liya Yang, PhD, Robert S. Cantor, PhD, Edmond I. Eger, II, MD, and James M. Sonner, MD

From the *University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Department of Anesthesia and Perioperative Care, University of California, San Francisco; Department of Chemistry, Dartmouth College, Hanover, New Hampshire.

Address correspondence to James M. Sonner, MD, Department of Anesthesia and Perioperative Care, Room S-455i, University of California, San Francisco, CA 94143-0464. Address e-mail to sonnerj{at}anesthesia.ucsf.edu.

	Abstract

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INTRODUCTION: A mechanism of anesthesia has recently been proposed which predicts that coreleased neurotransmitters may modulate neurotransmitter receptors for which they are not the native agonist in a manner similar to anesthetics.

METHODS: We tested this prediction by applying acetylcholine to a NR1/NR2A N-methyl-d-aspartate receptor, glycine to a wild-type ₁ß₂ and anesthetic-resistant ₁(S270I)ß₂ -amino-butyric acid (GABA) type A receptor, and GABA to a homomeric ₁ wild type and anesthetic-resistant ₁ S267I glycine receptor. Receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping.

RESULTS: We found inhibition of N-methyl-d-aspartate receptor function by acetylcholine, enhancement of glycine receptor function by GABA, and enhancement of GABA type A receptor function by glycine. As expected of compounds with anesthetic activity, GABA showed far less potentiation (enhancement) of the function of the anesthetic-resistant S267I glycine receptor than that of the wild-type receptor. Glycine potentiated the function of wild-type GABA type A receptors but inhibited the function of the anesthetic-resistant S270I GABA type A receptor.

CONCLUSIONS: These results show that neurotransmitters that are coreleased onto anesthetic-sensitive receptors may modulate the function of receptors for which they are not the native agonist via an anesthetic-like mechanism. These findings lend support to a recent theory of anesthetic action.

	Introduction

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Considerable evidence indicates that inhaled anesthetics act by inhibiting excitatory and potentiating (enhancing) inhibitory currents through postsynaptic ligand-gated ion channels (1–3). The mechanism by which these actions are accomplished remains unknown (4). Proposed mechanisms may be categorized as direct or indirect, in that anesthetics might act either by binding directly to well-defined sites on these proteins, or indirectly by dissolving into (or adsorbing onto) the bilayers of the postsynaptic membrane, and altering various physical properties of the bilayer that are coupled to protein activity.

An indirect mechanism of anesthesia has been suggested (5) based on a hypothetical second mechanism of neurotransmitter action. It is proposed that, in addition to rapid binding of a neurotransmitter to extracellular sites on the receptor, inducing a conformational change that results in activation, the neurotransmitter also acts indirectly on its receptor by adsorbing into the postsynaptic membrane and changing bilayer physical properties that influence the transitions and equilibria among receptor conformational states. This additional membrane-mediated effect differs from agonist binding in that (1) it is less specific with respect to molecular characteristics (2), the adsorption and desorption from the bilayer might be quite slow, so that its influence on channel activity could extend over long periods and (3) it may not exhibit saturation until extremely high aqueous concentrations have been reached. Many of the characteristics of desensitization and deactivation observed electrophysiologically, both for inhibitory and excitatory channels, could be predicted by this mechanism, including the continued dependence on neurotransmitter concentration as it is increased well above binding saturation (5). In particular, it could account both for the increased peak currents and the delayed deactivation upon washout of neurotransmitter observed in inhibitory channels, as well as the decreased peak currents and very rapid deactivation commonly found in excitatory channels (6–9).

In the present work, we explored an additional neurobiological consequence of this theory. This proposed mechanism predicts that a second "noncognate" neurotransmitter, i.e., one that does not bind to that receptor's agonist sites, applied at concentrations corresponding to estimated peak concentrations at the postsynaptic membrane in the synaptic cleft, should alter the activity of a particular receptor in a manner similar to anesthetics. The release of a second neurotransmitter occurs physiologically: -amino butyric acid (GABA) and glycine are coreleased at some synapses (10,11), as are acetylcholine (ACh) and glutamate (12,13).

We hypothesized that the second, noncognate neurotransmitter to which anesthetic-sensitive receptors are exposed during neurotransmitter corelease would have anesthetic-like effects on the receptor bound by the native neurotransmitter. We tested this on recombinant receptors, using inhibitory ₁ glycine and ₁ß₂ GABA type A (GABA_A) receptors, and the excitatory NR1/NR2A N-methyl-d-aspartate (NMDA) receptor as model systems. These receptors are representative of cys-loop and glutamate receptor families, which are postulated to mediate the actions of inhaled anesthetics. We studied these particular receptor subtypes because they have been widely studied in investigations of inhaled anesthetic mechanisms (14–16). To confirm anesthetic-like modulation of glycine and GABA_A receptors, we also applied noncognate neurotransmitters to anesthetic-resistant mutant glycine and GABA_A receptors.

	MATERIALS AND METHODS

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Methods for Study of Glycine Receptors
Studies using frogs oocytes were approved by the committee on animal research at the University of California, San Francisco. The glycine receptor clones were a gift of Professor R. A. Harris (University of Texas, Austin). Stage V and VI Xenopus laevis oocytes were defolliculated by gentle rotation in 500 U/mL collagenase type 1 (Worthingtom Biochemical Corporation, Lakewood, NJ) for 1 h at room temperature. Homomeric human ₁ glycine receptors subcloned into PBK-CMV were expressed by microinjection of 0.25–1 ng cDNA into X. laevis oocytes. Oocytes were maintained at 18°C in modified Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 20 mM HEPES, 0.82 mM MgSO₄, 0.33 mM Ca(NO₃)₂, 0.41 mM CaCl₂, with 5 mM sodium pyruvate, 50 µg/mL gentamycin, 50 U/mL penicillin, and 50 µg/mL streptomycin, filtered and adjusted to pH = 7.4). All reagents were purchased from Sigma-Aldrich (St. Louis, MO). The purity of the neurotransmitters was as follows: glycine (99+%), GABA (99%), glutamate (99%), ACh (99%).

One to 4 days after injection, two-electrode voltage clamping was performed on oocytes (GeneClamp 500B; Molecular Devices, Axon Instruments, Foster City, CA). Experiments were performed using frog Ringer's solution as perfusate (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl₂, 10 mM HEPES, filtered and adjusted to pH 7.4). An automated perfusion system delivered solutions (Automate ValveBank perfusion system, San Francisco, CA). Recordings were obtained in a 250-µL recording chamber at flow rates of 2–3 mL/min. Signals were filtered using a 4-pole low-pass Bessel filter set at a 50–100 Hz cutoff before sampling at 100–1000 Hz. Water-injected and uninjected oocytes served as controls.

Oocytes were voltage clamped at –80 mV. Stable inward currents in response to glycine was verified by application of glycine for 20 s followed by a 5–6 min washout, which was repeated three times. To control for osmolarity, sucrose was added to frog Ringer's solution when needed. Osmolarity was checked using a vapor pressure osmometer (VAPRO 5520 from Wescor Inc., Logan, UT). GABA was applied to oocytes for 100 s, followed by coapplication of glycine with GABA for 20 s. Return to baseline response to agonist was confirmed. Potentiation of currents in homomeric ₁ wild type and anesthetic resistant ₁S267I mutant glycine receptors was studied using an EC₅ concentration of agonist after washin of 5 mM GABA.

Methods for Study of GABA_A and NMDA Receptors
The procedure for expressing ₁ß₂ GABA_A and NR1/NR2A NMDA receptors in X. laevis oocytes and studying them using two-electrode voltage clamping has been described elsewhere (17). We modified this protocol by washing in glycine and ACh for 100 s to evaluate their modulatory effects on GABA_A and NMDA receptors, respectively, and comparing these currents to isosmotic sucrose controls. Potentiation of currents in ₁ß₂ wild type and anesthetic-resistant ₁(S270I)ß₂ mutant GABA_A receptors was studied using an EC₂₀ concentration of agonist after washin of 5 mM glycine.

Data were analyzed by analyses of variance or by nonlinear regression to a Hill equation. P < 0.05 was considered significant.

	RESULTS

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GABA Potentiates Glycine Receptor Function
For homomeric human ₁ glycine receptors, preexposure to 0.5–10 mM GABA potentiated currents in response to 30 µM glycine (Fig. 1A). This effect did not saturate [F_4,23 = 9.62, P < 0.001 for the analysis of variance testing the null hypothesis that potentiations at all concentrations of GABA were the same]. In the absence of glycine, 5 and 10 mM GABA (but not lower concentrations) produced small but statistically significant currents through glycine receptors: 5 mM GABA produced 0.44% ± 0.13% (mean ± se) and 10 mM GABA produced 0.92% ± 0.25% of saturating currents.

View larger version (13K): [in this window] [in a new window]   Figure 1. Effect of noncognate neurotransmitter concentration on agonist activated currents in glycine, -amino butyric acid type A (GABAA) and N-methyl-d-aspartate (NMDA) receptors. Data are expressed as means ± se; all potentiations or changes in current are significantly different than zero. A, Xenopus laevis oocytes expressing homomeric human 1 glycine receptors were exposed to GABA for 100 s, or perfusates containing an isosmotic concentration of sucrose. Larger GABA concentrations enhanced (potentiated) chloride current through this receptor in response to coapplication of 30 µM glycine with GABA for 20 s. The anesthetic-like potentiation of current is concentration-dependent and does not show saturation. B, Results from a similar study performed on 1ß2 GABAA receptors exposed to glycine for 100 s, or perfusates containing sucrose. Glycine potentiated currents through GABAA receptors in response to 30 µM GABA plus glycine for 20 s, but less so than GABA potentiated glycine receptor function in panel A. C, Exposure of NR1/NR2A NMDA receptors to acetylcholine for 100 s decreased the currents evoked by coapplication of agonist (100 µM glutamate and 10 µM glycine) with acetylcholine for 20 s, compared with controls exposed to sucrose and agonist.

GABA Shifts the Glycine Concentration-Response Curve to the Left
GABA 1 mM increased currents evoked by glycine compared to currents in control oocytes perfused with 1 mM sucrose (Fig. 2A): at every concentration below saturation, glycine-evoked currents with 1 mM GABA exceeded those with sucrose controls. GABA 1 mM significantly shifted the glycine EC₅₀ from 190 ± 4 µM (EC₅₀ ± se) to 151 ± 4 µM, a value 79% of normal. The Hill number was 2.2. Figure 3A shows a current tracing demonstrating the effect of GABA on glycine receptor function.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of noncognate neurotransmitter on the agonist concentration-response relation for glycine and -amino butyric acid type A (GABAA) receptors. A, 1 mM GABA (open triangles) shifted the concentration-response relation for 1 glycine receptors to the left relative to 1 mM sucrose (closed diamonds; an isosmotic control). GABA was applied for 100 s before being coapplied with glycine for 20 s; all other perfusates contained sucrose in a concentration isosmotic to GABA. All currents were normalized to saturating currents. GABA significantly shifted the glycine EC50, from 190 ± 4 µM (open square) with sucrose to 151 ± 4 µM (closed circle). B, The concentration-response relation for 1ß2 GABAA receptors exposed to 10 mM glycine (open circles) or 10 mM sucrose (open squares) are compared. Glycine was applied for 100 s before being coapplied with GABA for 20 s. Glycine shifted the GABA EC50 from 59.4 ± 4.4 µM (closed square) with sucrose to 35.9 ± 2.2 µM (closed circle). Currents were normalized to the saturating currents.

View larger version (10K): [in this window] [in a new window]   Figure 3. Current tracings. A, Exposure of 1 glycine receptors with 0.5 mM -amino butyric acid (GABA) for 100 s enhanced peak currents evoked by coapplication of GABA with 30 µM glycine. Perfusates that did not contain GABA contained 0.5 mM sucrose to maintain osmolarity. B, Exposure of 1ß2 GABAA receptors with 10 mM glycine for 100 s enhanced peak currents evoked by coapplication of glycine with 100 µM GABA. Perfusates that did not contain glycine contained 10 mM sucrose to maintain osmolarity. C, Exposure of NR1/NR2A NMDA receptors with 10 mM acetylcholine chloride for 100 s inhibited peak currents produced by coapplication of acetylcholine with agonist (100 µM glutamate and 10 µM glycine) for 20 s. Perfusates that did not contain acetylcholine chloride contained 20 mM sucrose to maintain osmolarity, which was confirmed with a vapor pressure osmometer.

GABA Potentiates the Function of Anesthetic-Resistant Mutant Glycine Receptors Less Than Wild-Type Receptors
Current evoked by 5 mM GABA did not differ between wild-type and anesthetic-resistant mutant (₁ S267I) glycine receptors (Fig. 4A, left two bar graphs). GABA 5 mM produced less potentiation in response to EC₅ glycine in the anesthetic-resistant mutant receptor than the wild-type channel (Fig. 4A, right two bar graphs).

View larger version (11K): [in this window] [in a new window]   Figure 4. Study of anesthetic-resistant mutant -amino butyric acid type A (GABAA) and glycine receptors confirms the anesthetic effect of noncognate neurotransmitters. Data are expressed as mean ± se. A, 5 mM GABA produces no significant difference in current, expressed as a percent of the maximal current produced by a saturating concentration of glycine, between wild-type and anesthetic-resistant mutant homomeric 1 S267I glycine receptors (first two bars on graph; n = 5 oocytes). There is reduced potentiation (enhancement) of glycinergic currents in response to an EC5 concentration of glycine after washin of 5 mM GABA for the mutant (n = 6 oocytes) compared with that for the wild-type (n = 5 oocytes) receptor, consistent with an anesthetic-like mechanism (last two bars on graph). B, 5 mM glycine produces no significant difference in current (expressed as a percent of the maximal current) between wild-type and anesthetic-resistant mutant 1 S270I containing 1ß2 GABAA receptors (first two bars on graph; n = 4 oocytes). There is inhibition of GABAergic currents in response to an EC20 concentration of GABA after washin of 5 mM glycine for the mutant receptor (n = 6 oocytes) and potentiation for the wild-type (n = 6 oocytes) receptor, consistent with an anesthetic-like mechanism (last two bars on graph).

Glycine Potentiates GABA Receptor Function
Preexposure of ₁ß₂ GABA_A receptors to 5–40 mM glycine potentiated currents produced by 30 µM GABA, an effect which did not saturate (F_3,13 = 9.62, P = 0.001) (Fig. 1B). Glycine produced currents through GABA_A receptors in the absence of GABA: 5 mM glycine elicited a statistically significant current of 0.74% ± 0.12% maximal (mean ± se), similar to that reported for volatile anesthetics (18). This effect was subtracted out to calculate potentiations.

Glycine Shifts the GABA Concentration-Response Curve to the Left
Perfusion of oocytes with 10 mM glycine versus 10 mM sucrose significantly potentiated GABAergic currents at all concentrations below saturating concentrations of GABA (Fig. 2B). Glycine 10 mM significantly shifted the EC₅₀ for the GABA_A receptor by 40%, from 59.4 ± 4.4 µM (EC₅₀ ± se) to 35.9 ± 2.2 µM. The Hill number was 1.6. Figure 3B shows a current tracing of the effect of glycine on GABA_A receptors.

Glycine Potentiates Anesthetic-Resistant Mutant GABA_A Receptors Less Than Wild-Type Receptors
Current evoked by 5 mM glycine did not differ between wild-type and anesthetic-resistant mutant GABA_A receptors containing ₁S270I subunits (Fig. 4B). EC₂₀ GABA in oocytes preexposed to 5 mM glycine produced inhibition in the anesthetic-resistant mutant and potentiation in the wild-type channel (Fig. 4B).

ACh Inhibits NMDA Receptor Function
Inhibition of NR1/NR2A NMDA receptor function by ACh in concentrations ranging from 2.5 to 10 mM was observed [F_2,11 = 7.74 (P = 0.008)]. Ten millimolar ACh inhibited NMDA receptor function by 47.6% ± 10.6% (mean ± se; Figs. 1C and 3C). ACh alone did not affect NMDA receptor currents.

	DISCUSSION

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Anesthetic-like Modulation of Receptors by Coreleased Neurotransmitters
We found that preexposure of an NMDA receptor to ACh inhibited cation currents evoked by glutamate application; that preexposure of a GABA_A receptor to glycine enhanced chloride currents evoked by subsaturating GABA application; that preexposure of a glycine receptor to GABA enhanced chloride currents evoked by subsaturating glycine application. These are anesthetic-like effects. These findings suggest that noncognate neurotransmitters might affect synaptic events during neurotransmitter corelease. If so, this mechanism would especially favor enhancement of glycine receptor currents, since their function is potentiated more and at lower concentrations of the coreleased neurotransmitter than that of GABA_A receptors. This possibly accounts for why GABA and glycine corelease is common in the hindbrain (11) and spinal cord (10), which are rich in glycinergic synapses.

The extent to which neurotransmitter corelease might affect synaptic function in vivo depends on neurotransmitter concentrations in synapses. What are likely peak synaptic neurotransmitter concentrations? Neurotransmitter concentration in synaptic vesicles is high; for example, the serotonin concentration in vesicles is approximately 270 mM (19), ACh concentration in vesicles is estimated to be 250 mM (5), and vesicular concentrations of other neurotransmitters may be similar to these. As an order-of-magnitude estimate, the volume of a synaptic cleft of thickness 20 nm and diameter 200 nm is only roughly an order of magnitude more than that of neurotransmitter vesicles, so even if the neurotransmitter were uniformly distributed in the synapse, it would only result in a factor of 10 decrease in concentration over the vesicular value, assuming that all the neurotransmitter leaves the vesicle upon fusion with the presynaptic membrane. Although this is only a rough estimate, it is thus quite plausible that neurotransmitters could achieve the millimolar concentrations of noncognate neurotransmitter, which we found modulates glycine, GABA_A, and NMDA receptor function. Although the aqueous concentration of neurotransmitter in the synapse decreases rapidly, it is the rates at which neurotransmitter adsorbs and desorbs from the membrane that will determine the extent to which this mechanisms operates in vivo. These rates are unknown, even for artificial lipid bilayers.

Although they are preferentially localized to the inner leaflet, approximately 20% of the lipids of synaptic membranes are anionic, primarily containing phosphatidylserine head groups, which can interact strongly with the GABA zwitterion. The specific binding constant of GABA to phophatidylserine head groups has been measured to be 5 x 10^–3 M (20), which implies that half of all phosphatidylserine head groups have GABA bound when the aqueous GABA concentration is 200 µM: approximately, the concentration at which glycine receptor function was potentiated by GABA in our studies.

Alternative Interpretations of These Results
Our findings were predicted by an indirect, bilayer-mediated theory of anesthetic action. However, other explanations for our results are possible. For example, noncognate neurotransmitters may bind directly to sites on the receptors to produce anesthetic-like modulation of channel function. However, putative inhaled anesthetic binding sites on GABA_A and glycine receptors (21) have been modeled extensively, and have never been proposed to be low affinity GABA and glycine binding sites; nor has the putative binding site on the NMDA receptor (22) been suggested to be a low affinity binding site for ACh. Nonetheless, our data cannot exclude binding to an allosteric site on the receptor.

The noncognate neurotransmitters possibly act as agonists, binding to the canonical agonist site to produce the anesthetic-like effects we observed. Two lines of evidence, presented in Figures 2 and 4, show that this is not the case. In Figure 2, the magnitude of the effects of the noncognate neurotransmitters is shown. These effects are not consistent with an agonist action of the noncognate neurotransmitter. For example, 1 mM GABA potentiated glycine receptor function (Fig. 2), but did not evoke currents by itself. Given the sensitivity with which currents are measured, this places an upper bound of at most a 1% increase in current from 1 mM GABA when added to 250 µM glycine. This is smaller than the 13% difference in currents actually observed. Similar calculations show that 10 mM glycine should increase GABA_A receptor currents evoked by 100 µM GABA by only approximately 1% rather than the 22% actually observed.

Studies on anesthetic-resistant mutant receptors support this conclusion. Figure 4 shows that 5 mM glycine has the same agonist effect on wild-type and anesthetic-resistant mutant GABA_A receptors. However, glycine potentiated the function of wild-type GABA_A receptors while inhibiting the function of mutant GABA_A receptors. Thus, potentiation of GABA_A receptor function by glycine is mechanistically similar to that of inhaled anesthetics, not that of agonists. Likewise, 5 mM GABA had the same agonist effect on wild-type and anesthetic-resistant mutant glycine receptors, but potentiation of receptor function was much less in the mutant compared with that in the wild-type receptor, consistent with an anesthetic-like modulation of channel function, not an agonist effect.

Neurotransmitters May Exert a Selective Pressure for the Response to Anesthetics
Our findings together with the theory that motivated our experiments may help explain why animals evolved the capacity to be anesthetized. The response to the wide range of anesthetic compounds by animals in different phyla (23–25) suggests a common origin of the anesthetic site of action at least several hundred million years ago. In the absence of natural selection, the capacity to respond to anesthetics should have diminished or disappeared over this span of time as a result of spontaneous mutation (26); hence, natural selection likely acts to conserve the response to anesthetics. What is the source of the selective pressure? If neurotransmitters affect bilayer properties, and their receptors are adapted to these changes and use them to regulate their conformational equilibrium, and if anesthetics mimic this effect, then neurotransmitters will exert a selective pressure for receptors, which respond to anesthetics. That is, the "endogenous anesthetics" in animals would be neurotransmitters, the survival advantage conferred by their membrane-mediated fine-tuning of the time-dependence of ion currents explaining the well-known selection pressure for anesthetic sensitivity (26).

Continuous selection for ligand-gated ion channels, which are adapted to the indirect effects of their native neurotransmitters on bilayer properties, may also explain why synaptic transmission, which is mediated by ion channels exposed to neurotransmitters, is more sensitive to inhaled anesthetic action than is axonal conduction, which is mediated by channels that are not exposed to neurotransmitters (27). Our hypothesis may also explain why extrasynaptic receptors, which are exposed to, and perhaps adapted to, the indirect, bilayer-mediated effects of lower concentrations of neurotransmitter which spill out of the synapse, would therefore respond to lower concentrations of inhaled anesthetics than synaptic receptors (28).

	ACKNOWLEDGMENTS

 
The authors thank Howard Nash for his critical review of this manuscript.

	Footnotes

 
Accepted for publication April 2, 2007.

Supported by NIGMS R01 GM069379 (to J.S.).

The first two authors contributed equally to this work.

Dr. Eger is a paid consultant to Baxter Healthcare Corp.

Reprints will not be available from the author.

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