Effects of the abused inhalant toluene on ethanol-sensitive potassium channels expressed in oocytes

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Effects of the abused inhalant toluene on ethanol-sensitive potassium channels expressed in oocytes Angelo M. Del Re a , Alejandro M. Dopico b , John J. Woodward a,⁎ a

Department of Neurosciences and Center for Drug and Alcohol Programs, Medical University of South Carolina, 173 Ashley Avenue, Suite 403, Charleston, SC 29425, USA b Department of Pharmacology, The University of Tennessee Health Science Center, Memphis, TN 38163, USA

A R T I C LE I N FO

AB S T R A C T

Article history:

Toluene (methylbenzene) is representative of a class of industrial solvents that are

Accepted 13 March 2006

voluntarily inhaled as drugs of abuse. Previous data from this laboratory and others have

Available online 13 April 2006

shown that these compounds alter the function of a variety of ion channels including ligandgated channels activated by ATP, acetylcholine, GABA, glutamate and serotonin, as well as

Keywords:

voltage-dependent sodium and calcium channels. It is less clear what effects toluene may

Abused solvent

have on potassium channels that act to reduce the excitability of most cells. Previous studies

Electrophysiology

have shown that ethanol potentiates the function of both the large conductance, calcium-

Oocyte

activated potassium channel (BK) and specific members of the G-protein-coupled inwardly

Recombinant ion channel

rectifying potassium channels (GirKs). Since toluene and other abused inhalants share many behavioral effects with ethanol, it was hypothesized that toluene would also enhance the function of these channels. This hypothesis was tested using two-electrode voltage-clamp electrophysiology to measure the activity of BK and GirK potassium channel currents expressed in Xenopus laevis oocytes. As reported previously, ethanol potentiated currents in oocytes expressing either BK or GirK2 channels. In contrast, toluene caused a concentrationdependent inhibition of BK channel currents with 3 mM producing approximately 50% inhibition of control currents. Currents in oocytes injected with GirK2 mRNA were also inhibited by toluene while those expressing GirK1/2 and 1/4 channels were minimally affected. In oocytes co-injected with mRNA for GirK2 and the mGluR1a metabotropic receptor, exposure to glutamate potentiated currents evoked by a high-potassium solution. Toluene inhibited these glutamate-activated currents to approximately the same degree as those induced under basal conditions. The results of these studies show that toluene has effects on BK and GirK channels that are opposite to those of ethanol, suggesting that these channels are unlikely to underlie behaviors that these two drugs of abuse share. © 2006 Elsevier B.V. All rights reserved.

1.

Introduction

The inhalation of volatile solvents for their intoxicating effects is widely recognized as a problem of drug abuse. Inhalant

abuse is prevalent worldwide, particularly among children and adolescents likely due to the low cost and relative ease of obtaining compounds that contain volatile solvents. Although several volatile chemicals are subject to abuse (for a review,

⁎ Corresponding author. Fax: +1 843 792 7353. E-mail address: [email protected] (J.J. Woodward). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.03.031

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see Dinwiddie, 1994), toluene, or methylbenzene, is considered a prototype compound for this class, and it is a common component in many commercial solvent-based products such as paints and adhesives. Despite the observation that toluene abuse resembles that of other CNS depressants including ethanol, until recently, relatively little was known about this drug's cellular and molecular sites of action. In previous studies from this laboratory and others, toluene and related solvents have been shown to modulate the activity of a wide variety of ion channels involved in regulating cellular excitability and cell-to-cell signaling. Thus, toluene inhibits channels gated by acetylcholine, ATP and glutamate (Bale et al., 2002; Woodward et al., 2004; Cruz et al., 1998), while it enhances currents in channels activated by GABA, glycine and serotonin (Beckstead et al., 2000; Lopreato et al., 2003). In addition, toluene alters the function of voltage-dependent sodium (Cruz et al., 2003) and calcium channels (Tillar et al., 2002, Shafer et al., 2005) and has recently been shown to inhibit gap junction channels (Del Re and Woodward, 2005). In many cases, the modulation of channel activity by toluene and related solvents is subunit-selective, suggesting that these compounds may target specific sites on these proteins. In the present study, we determined the effects of toluene on two potassium channels that are important regulators of cell excitability. Large-conductance, calcium-activated potassium channels (BK) are known for their role in the regulation of cell membrane potential and are intimately involved in modulating cellular excitability. These channels are found ubiquitously throughout the brain as well as in vascular and non-vascular smooth muscle (Knaus et al., 1996). BK channels regulate the duration of action potentials in neurons and are involved in regulating hormone release and vascular tone in non-neural tissue (Lang and Ritchie, 1987; Robitaille and Charlton, 1992;

Vergara et al., 1998; reviewed in Faber and Sah, 2003). G-proteinactivated inwardly rectifiying potassium channels (GirK; Kir3.X) are highly expressed in both brain and cardiac tissue where they participate in the control of membrane potential (Hille, 1992; Jan and Jan, 1997). Interestingly, ethanol has been shown to potentiate the activity of native and recombinant BK and GirK channels at concentrations that are associated with behavioral signs of intoxication (Dopico et al., 1996; Lewohl et al., 1999). Based on similarities between some of the behavioral and subjective effects of toluene and ethanol, we predicted that toluene would also potentiate BK and GirK channel activity. As discussed below, however, the results of this study show that toluene inhibits the function of both of these channels.

2.

Results

2.1.

Toluene inhibits BK channels expressed in oocytes

The Xenopus oocyte expression model is ideal for investigating the pharmacological properties of recombinant ion channels since oocytes express relatively few endogenous channels of their own (for a review, see Weber, 1999). For example, administration of a relatively large, depolarizing pulse elicited negligible net currents in non-injected oocytes held under voltage-clamp (Fig. 1A). In contrast, oocytes injected with mRNA for BK channels demonstrated large and sustained outward currents when administered the same depolarizing pulse (Fig. 1B). A small but consistent rundown of BK current was generally observed during administration of the first few depolarizing pulses. Currents stabilized within minutes to approximately 70–80% of the initial current amplitude from the first pulse and remained constant with repeated stimulations

Fig. 1 – Large-conductance, calcium-activated potassium (BK) channels expressed in oocytes are inhibited by the abused inhalant toluene. (A) Non-injected oocytes showed negligible net currents when given a +130 mV voltage step (500 ms) from a holding potential of − 80 mV. (B) The same voltage step elicited large outward currents in oocytes injected with mRNA for the BK α subunit. These currents were reversibly blocked by perfusing the oocytes with the BK channel iberiotoxin (IBTX, 100 nM). (C) BK currents were enhanced during perfusion with a solution containing ethanol (100 mM). (D) In contrast, toluene (2 mM) markedly inhibited BK currents, and this effect was reversed upon washout.

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(data not shown). All drug solutions were administered after this stable current was maintained for at least 2 min (usually within 4 consecutive pulses). To verify that these currents were due to expression of the BK channel, recordings were performed in the presence of iberiotoxin (IBTX, 100 nM), a relatively selective antagonist of BK channels. As shown in Fig. 1B, voltage-activated currents in BK-injected oocytes were markedly inhibited by IBTX. Following washout of the IBTX, currents recovered to pre-drug control levels. Previous studies have demonstrated that ethanol and longchain alcohols such as butanol, hexanol and heptanol potentiate BK channel currents (Dopico et al., 1996; Chu and Treistman, 1997). In the present study, ethanol was used as a positive control to verify the ability of alcohols to modulate BK channel activity. As expected, BK channel activity was enhanced by approximately 40% in the presence of 100 mM ethanol (Fig. 1C). This potentiation was completely reversed after washout of the ethanol-containing solution. In contrast to the results observed with ethanol, BK currents were inhibited when oocytes were exposed to solutions containing toluene (Fig. 1D). As summarized in the concentration–response graph shown in Fig. 2, toluene inhibited BK currents at concentrations of 1 mM and above,

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and at 5 mM, the highest concentration tested, toluene inhibition of BK currents was approximately 50% (Fig. 2A). Concentrations greater than 5 mM were not tested due to the limited solubility of toluene in aqueous solutions (approximately 6 mM). The inhibitory effects of toluene were completely reversed following washout of the drug, demonstrating that, at the concentrations tested, toluene does not cause a loss of membrane integrity or cell viability. To further examine the effects of toluene on BK channel function, a series of depolarizing voltage steps from a holding potential of −70 mV were given in the absence and presence of 3 mM toluene. As shown in the top panel of Fig. 2B, jumping from −70 mV to holding potentials of +20 mV and beyond elicited large outward currents that were inhibited by toluene. However, when these data were normalized as a percent of the current obtained at the +50 mV holding potential, the voltage activation curves were similar in the absence and presence of toluene (Fig. 2B, lower panel).

2.2.

Toluene inhibits GirK2 (Kir3.2) in oocytes

The effects of toluene in oocytes injected with mRNA for various GirK subunits were investigated next. In extracellular

Fig. 2 – Modulation of BK currents by toluene or ethanol. (A) Summary of effects of toluene or ethanol on BK currents. Values represent BK current in the presence of toluene (closed squares) or ethanol (open triangle) expressed as a percent of the non-drug control current (mean ± SEM; n = 5 oocytes for all values). (B) Effect of toluene on voltage activation profile for BK channels. Top panel represents a representative experiment showing activation of BK currents in the absence (open circles) or presence (closed circles) of toluene (3 mM). Values are steady-state currents (in μA) measured at the end of a 500 ms pulse from −70 mV to the indicated membrane potential value. The bottom panel shows voltage activation data normalized as a percent of the current obtained at + 50 mV in the absence (open circles) and presence (closed circles) of toluene (3 mM). Values are the mean (±SEM) from three independent experiments.

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solution containing physiological levels of Na+ and K+, activation of GirK channels results in relatively small outward currents. When the extracellular solution is switched to one containing a higher concentration of K+ (hK, with a concomitant reduction in sodium), large inward currents are observed upon activation of GirK channels (Dascal et al., 1993). Noninjected oocytes exhibited small inward currents (∼40 nA) when voltage-clamped at −80 mV and exposed to hK solution alone (data not shown). In contrast, microamp currents were observed during hK application in oocytes injected with any of the tested combinations of GirK subunits. These currents were stable during several minutes of hK perfusion and allowed oocytes to be exposed to several different solutions to test for modulation of channel activity. An example of this approach is shown in Fig. 3 and shows currents in an oocyte injected with GirK2 mRNA in the absence and presence of toluene or ethanol (Fig. 3A). Application of the hK solution induced a large and stable inward current that was reversibly inhibited by toluene at concentrations of 1 and 3 mM. In the same oocyte, 100 mM ethanol caused a significant potentiation of the current. This effect was also rapidly reversed upon washout of the ethanol

containing solution. Fig. 3B shows a similar result using a slightly different test protocol in which GirK2-injected oocytes were exposed sequentially to hK solutions in the absence and presence of 3 mM toluene. In contrast to the inhibition observed in GirK2-injected oocytes, toluene (3 mM) had little effect on currents measured in oocytes injected with GirK1 and 2 subunits (Fig. 3C). Fig. 3D summarizes the effects of toluene on currents generated by GirK2-, 1/2- and 1/4-injected oocytes. While GirK2 currents were reliably inhibited by toluene, both GirK1/2 and 1/4 channels were relatively unaffected by toluene at concentrations up to 3 mM.

2.3. Toluene effects on group I mGluR-mediated GirK currents G-protein-coupled receptors potentiate GirK channel currents when mRNA for these receptors is co-expressed with GirK channels in oocytes (Saugstad et al., 1996; Dascal, 1997). The increase in current amplitude under these conditions is thought to arise by the agonist-induced increase in the number of free βγ G-protein subunits that then interact with

Fig. 3 – Toluene inhibition of currents in oocytes expressing different GirK channel subunits. Oocytes were injected with mRNA for GirK2 or equal amounts of GirK1 and GirK2, and the membrane potential was held at −80 mV using two-electrode voltage-clamp electrophysiology. (A) Large slowly desensitizing inward currents were evoked by switching the external solution to one containing high potassium (96 mM; hK). Application of toluene (1 mM or 3 mM) in hK solution reversibly inhibited these currents. In the same oocyte, hK-evoked currents were enhanced by exposure to ethanol (100 mM). (B) Toluene caused a similar inhibition of hK currents in GirK2-injected oocytes when it was applied using a sequential application protocol. (C) Currents from a GirK1/2-injected oocyte were minimally affected by 3 mM toluene. (D) Summary figure showing subunit-dependent effects of toluene on GirK channels expressed in oocytes. Values represent the percent inhibition of control currents by 1 mM or 3 mM toluene and are expressed as the mean ± SEM (n = 8–11 for each value). Symbol (*): value significantly different (P < 0.05; ANOVA with post hoc t test) from those of GirK1/2 and GirK1/4.

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the intracellular termini of the GirK channel. In order to determine whether this interaction may alter the effects of toluene on GirK channel currents, oocytes were injected with GirK2 channel subunits together with mRNA encoding the mGluR1a metabotropic glutamate receptor. Non-injected oocytes produced small inward currents in the presence of hK or hK plus glutamate, suggesting that these cells do not express functional glutamate receptors (data not shown). Oocytes injected with mRNAs for both GirK2 channels and mGluR1a produced large inward currents when perfused with hK solution, similar to oocytes injected with GirK2 alone (Fig. 4A). When solutions were switched to hK-containing glutamate (100 μM), the inward current was immediately enhanced and showed some desensitization as previously reported (Saugstad et al., 1996). Each initial glutamate

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application to oocytes injected with GirK2/mGluR1a mRNA also evoked a rapidly inactivating current that likely results from activation of a calcium-dependent chloride current (ICaCl) that is endogenously expressed in oocytes. Surprisingly, in oocytes co-injected with GirK2 mRNA and that for mGluR1a, basal currents evoked by the hK solution in the absence of glutamate were no longer sensitive to toluene (Fig. 4B). However, glutamate-induced currents in these oocytes were inhibited by toluene with the same onset and washout profile as observed in oocytes expressing GirK2 channels alone (Fig. 4B). This finding was consistently observed for all concentrations of toluene tested and was reproducible over several multiple recording sessions. Toluene's inhibition of the glutamate-induced GirK2 current was measured as a percent of the total agonist-induced current. The magnitude of this

Fig. 4 – Effects of toluene on GirK2 currents in oocytes co-injected with the metabotropic glutamate receptor mGluR1a. (A) Representative trace from a voltage-clamped oocyte during application of hK solution and subsequent exposure to glutamate (100 μM). Note the rapidly desensitizing inward current upon initial application of glutamate that results from mGluR1 activation of the endogenous calcium-dependent chloride channel (ICaCl) and the sustained slowly desensitizing GirK channel current. (B) Representative trace showing effect of toluene (3 mM) on glutamate-evoked GirK2 currents. (C) Summary figure showing the effects of toluene on glutamate-activated currents in oocytes expressing GirK2 and mGluR1a. Values represent the percent inhibition of glutamate-induced GirK2 current and are expressed as the mean ± SEM (n = 5–8). The inhibition of basal hK-induced currents in oocytes injected with only GirK2 is shown for comparison and is taken from Fig. 3. Toluene inhibition of GirK2 currents was not significantly affected by co-expression of mGluR1a.

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inhibition was not significantly different from that observed in oocytes injected with GirK2 mRNA alone (Fig. 4C).

3.

Discussion

The results of the present study demonstrate that toluene inhibits rather than enhances the function of two potassium channels that are involved in regulating cellular excitability. The modulation of BK and GirK2 channels by toluene is a novel finding of these studies and adds to the growing list of ion channels found to be modulated by toluene and related abused inhalants.

3.1.

BK channels

Hu et al. (2001) revealed that BK channels in rat brain are targeted primarily to nerve terminals implying an important role in neural transmission. K+ flux through calcium-activated BK channels serves as a negative feedback mechanism and helps to repolarize the pre-synaptic membrane. To date, many modulators of BK channel activity have been identified, including ethanol (Dopico et al., 1996) and volatile anesthetics (Pancrazio et al., 1993). The potentiation of BK currents by ethanol would be expected to reduce neuronal excitability (Wong and Prince, 1981; MacDermott and Weight, 1982) and may contribute to the ethanol-induced depression of neuronal activity and behavior observed in C. elegans (Crowder, 2004). Alternatively, volatile anesthetics, such as halothane and isoflurane, typically inhibit BK currents, although these compounds can have opposing effects on BK-mediated hyperpolarization depending on the preparations used (MacIver and Kendig, 1991; Fujiwara et al., 1988; Pearce, 1996). Surprisingly, in the present study, toluene, often considered to have a CNS depressant profile similar to that of ethanol, inhibited BK channels expressed in oocytes. Based on this effect, toluene would be expected to enhance rather than inhibit transmitter release by reducing the feedback inhibition that BK channels normally provide. However, any effect of toluene on BK conductance and transmitter release might be countered by toluene's inhibition of voltage-sensitive sodium and calcium channels (Cruz et al., 2003; Tillar et al., 2002; Shafer et al., 2005) that are involved in action-potentialdependent neurotransmitter release.

3.2.

GirK channels

The second major finding of this study is that toluene inhibited GirK channels in a subunit-dependent manner. Inward rectifying potassium channels are found in both pre-synaptic nerve terminals and post-synaptic membranes (Ponce et al., 1996) and GirK1, 2 and 3 subunits are widely expressed, with some regional specificity, in the central nervous system (Kobayashi et al., 1995; Liao et al., 1996). These channels are particularly enriched in post-synaptic dendritic spines (Drake et al., 1997; Luscher et al., 1997; Dutar et al., 2000; Takigawa and Alzheimer, 2002) and provide inhibitory hyperpolarization following activation by various G-protein-coupled receptors (Tabata et al., 2005). Similar to BK currents, GirK2 currents are potentiated by ethanol (Lewohl et al., 1999) but are inhibited by volatile

anesthetics (Weigl and Schriebmayer, 2001; Yamakura et al., 2001). As reported in the present study, toluene also inhibited GirK channel activity, and this effect was subunit-dependent, with an inhibition of neuronal GirK2 but not cardiac GirK1/4 channel types. This subunit dependence of toluene sensitivity is similar to that reported for volatile anesthetics by Yamakura et al. (2001). The degree of toluene inhibition of GirK currents was not substantially altered during mGluR1a agonist-induced activation, suggesting that toluene inhibition is mediated primarily by sites on the GirK channel proteins. However, it should be noted that toluene did not cause an appreciable inhibition of basal GirK currents in those oocytes co-injected with mGluR1a mRNA. A similar finding was reported for the effects of 0.1 mM halothane on GirK1/4 channels co-expressed with muscarinic receptors (Weigl and Schriebmayer, 2001). While the mechanisms underlying these effects are currently unknown, they suggest that modulation of GirK channel function by toluene and volatile anesthetics is complex and is probably influenced by interactions of the channel with other cellular proteins and signaling molecules. Although toluene was shown to inhibit the function of both BK and GirK potassium channels, it is unclear whether these actions contribute to any of the behavioral actions of toluene following voluntary inhalation. In rats, blood levels of toluene approach 0.8 mM 15 min following inhalation of toluene vapor at concentrations similar to those encountered during huffing (Riegel and French, 2002). Brain toluene levels have been reported to be twice those observed in blood (Bruckner and Peterson, 1981), suggesting that brain levels of toluene may reach low millimolar concentrations within minutes of voluntary inhalation. As reported in the present study, BK and GirK channel function was inhibited by toluene at similar concentrations (3–5 mM), although this inhibition was incomplete (30–50%). As compared to other ion channels, BK and GirK channels are among the less sensitive targets for toluene and related solvents that have been identified to date. For example, IC50 values reported for toluene inhibition of NR1/2B NMDA receptors and α4β2 nAchR receptors were 0.17 mM and 0.24 mM respectively (Cruz et al., 1998; Bale et al., 2002). Values for inhibition of voltage-sensitive sodium channels, gap junction conductances and L-type calcium channels were reported to be 0.27 mM, 0.57 mM and 0.72 mM respectively (Cruz et al., 2003; Del Re and Woodward, 2005; Shafer et al., 2005). In this context, modulation of BK and GirK channel function requires relatively high concentrations of toluene that would likely cause widespread disruption of ion-channelbased signaling processes. Nonetheless, given the important role that BK and GirK channels play in regulating cellular excitability, small changes in potassium conductance through these channels could have profound effects on cell function.

4.

Experimental procedures

4.1.

Molecular biology

For studies of BK channel activity, the mouse BK channel (mslo mbr5, α subunit) cDNA was used. GirK (Kir3.1-4) subunit cDNAs were from Dr. Logothetis (Mt. Sinai School of Medicine, NY, USA) and mGluR subunit cDNAs were provided by Dr.

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Nakanishi (Kyoto Univ., Kyoto, Japan). For all mRNA synthesis, linearized cDNAs were used as the template in an in vitro transcription reaction using the appropriate sense-transcribing promoter and mMessage mMachine kit (Ambion, Inc., Austin, TX). RNA yield and purity were verified by agarose gel electrophoresis prior to use.

4.2.

Xenopus laevis oocyte preparation and injection

Adult oocyte-positive X. laevis frogs (Xenopus Express, Plant City, FL) were housed in de-chlorinated water (18–20 °C) with a 12-h/12-h light/dark cycle and fed twice weekly. Frogs were anesthetized with 0.25% ethyl m-aminobenzoate 222 (MS 222, Sigma), and a portion of the oocytes was removed. Oocytes were incubated in 1.0 mg/ml collagenase type 1A (Sigma) for 45–60 min and manually dissociated. Oocytes were injected with mRNA using a variable Nanoject injector (Drummond Scientific Co., Broomall, PA) on the same day as oocyte collection and collagenase treatment. GirK subunit combinations 1/4 and 1/2 were injected at a 1:1 ratio. Metabotropic glutamate receptor 1a mRNA was injected into some oocytes also receiving GirK2 mRNA injections. Oocytes were incubated at 17–18 °C for 3–5 days in ND96 solution containing (in mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2 and 5 HEPES, pH 7.5, supplemented with 2.5 mM Na pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin.

4.3.

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lected and analyzed on a Macintosh computer running under the IGOR-Pro graphics environment (Wavemetrics, Lake Oswego, OR) or Axograph software (Molecular Devices, Sunnyvale, CA). In each oocyte tested, drug inhibition or potentiation was calculated as a percent of the control current amplitude obtained prior to drug exposure. Solvent solutions were made by dissolving toluene or ethanol directly into the recording solution immediately prior to use. During recording, toluene- or ethanol-containing solutions were kept in sealed glass reservoirs under positive air pressure.

4.4.

Data collection and analysis

Results for the experiments are given as mean ± SEM unless indicated otherwise. Statistical analysis and non-linear regression analysis for calculating IC50 values and concentration–response curves were performed using Prism 4.0 software (GraphPad, San Diego, CA).

Acknowledgments The authors would like to thank Katherine Chike-Harris and Lalitha Kannan for technical assistance in this studies. This work was supported by grants R01 DA13951 and K02 AA00238 to JJW.

Electrophysiology and solvent preparation

Oocytes were placed in a recording chamber (total volume, ∼ 40 μl) and perfused with calcium-containing extracellular solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2 and 10 mM HEPES, pH 7.2). Microelectrodes were pulled from glass capillary tubing (OD = 1.5 mm, ID = 1.1 mm; Warner Instruments, Hampden, CT) and filled with 3 M KCl. The resistance of pipettes ranged from 0.5 to 2 MΩ. Oocytes were impaled with pipettes, and the membrane potential was clamped at −80 mV using an Axon GeneClamp amplifier (Molecular Devices, Sunnyvale, CA). BK channels were activated by administering a depolarizing voltage step (+130 mV; 500 ms) once every 30 s. Current traces were leak subtracted using the PN/4 protocol to eliminate the linear portion of the leak current during depolarization. The recording chamber was then perfused with solutions containing various concentrations of toluene (Fluka, Buchs, Switzerland) or ethanol (AAper, Shelbyville, KY) followed by a washout period. For GirK channel currents, oocytes were voltage-clamped at −80 mV and perfused with ND96 solution. Bath solutions were then switched to a high potassium (hK) solution containing (in mM): 96 KCl, 2 NaCl, 1.8 CaCl2, 1 MgCl2 and 5 HEPES, pH 7.5, to generate an inward GirK-mediated current. Once the inward basal current had reached a stable maximum, the solution was switched to one containing ethanol, toluene or glutamate. For oocytes co-expressing mGluR1α and GirK2 receptors, basal currents were elicited in hK solution followed by application of glutamate (100 μM) to evoke an agonist-evoked current. This was followed by perfusion of hK solution containing both glutamate and toluene. For most recordings, currents were filtered at 1–2 kHz, digitized at 5 kHz with a 16-bit analog-todigital interface (Instrutech, Port Washington, NY) and col-

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