physiological concentrations of zinc reduce taurine-activated glyr responses to drugs of abuse

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Neuropharmacology xxx (2013) 1e9

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Neuropharmacology

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Physiological concentrations of zinc reduce taurine-activated GlyRresponses to drugs of abuse

Dean Kirson, Garrett L. Cornelison, Ashley E. Philpo, Jelena Todorovic, S. John Mihic*

Section of Neurobiology, Division of Pharmacology and Toxicology, Waggoner Center for Alcohol & Addiction Research, Institutes for Neuroscience and Cell& Molecular Biology, University of Texas at Austin, MC A4800, 2500 Speedway, Austin, TX 78712, USA

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a r t i c l e i n f o

Article history:Received 25 April 2013Received in revised form26 July 2013Accepted 29 July 2013

Keywords:EthanolIsofluraneTolueneZincAllosteric modulationGlycine receptor

Abbreviations: EC5, effective concentration produGlyR, glycine receptor; MBS, Modified Barth’s SalineSNK, StudenteNewmaneKeuls; VTA, ventral tegment* Corresponding author. Tel.: þ1 512 232 7174; fax:

E-mail address: [email protected] (S.J. Mih

0028-3908/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2013.07.025

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a b s t r a c t

Taurine is an endogenous ligand acting on glycine receptors in many brain regions, including the hip-pocampus, prefrontal cortex, and nucleus accumbens (nAcc). These areas also contain low concentrationsof zinc, which is known to potentiate glycine receptor responses. Despite an increasing awareness of therole of the glycine receptor in the rewarding properties of drugs of abuse, the possible interactions ofthese compounds with zinc has not been thoroughly addressed. Two-electrode voltage-clamp electro-physiological experiments were performed on a1, a2 a1b and a2b glycine receptors expressed in Xenopuslaevis oocytes. The effects of zinc alone, and zinc in combination with other positive modulators on theglycine receptor, were investigated when activated by the full agonist glycine versus the partial agonisttaurine. Low concentrations of zinc enhanced responses of maximally-effective concentrations of taurinebut not glycine. Likewise, chelation of zinc from buffers decreased responses of taurine- but not glycine-mediated currents. Potentiating concentrations of zinc decreased ethanol, isoflurane, and tolueneenhancement of maximal taurine currents with no effects on maximal glycine currents. Our findingssuggest that the concurrence of high concentrations of taurine and low concentrations of zinc attenuatethe effects of additional modulators on the glycine receptor, and that these conditions are more repre-sentative of in vivo functioning than effects seen when these modulators are applied in isolation.

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1. Introduction

The glycine receptor (GlyR) is a member of the cys-loop receptorsuperfamily of ligand-gated ion channels. It has a pentamericstructure composed of either a (homomeric) or a & b (heteromeric)subunits arranged around a central anion-conducting pore. GlyRare responsible for the majority of fast inhibitory neurotransmis-sion in the brainstem and spinal cord, with g-aminobutyric acidtype A (GABAA) receptors primarily fulfilling this role in the brain.However, GlyR are also found in many brain regions including thehippocampus, nAcc and prefrontal cortex (Baer et al., 2009; Jonssonet al., 2012, 2009; Lu and Ye, 2011; Lynch, 2004;Malosio et al., 1991;van den Pol and Gorcs, 1988; Waldvogel et al., 2007). Because GlyRare found in brain regions associated with hypnosis, memory andthe rewarding properties of drugs of abuse, it is reasonable to

cing 5% of maximal effect;; nAcc, nucleus accumbens;al area.þ1 512 232 2525.

ic).

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., et al., Physiological concen.doi.org/10.1016/j.neurophar

hypothesize that these receptors may play a role in the effects ofthese agents in vivo.

A wide variety of compounds act as allosteric modulators of theGlyR, including alcohols, inhaled drugs of abuse and anesthetics,neurosteroids, tropeines, divalent cations, and many others(Beckstead et al., 2000; Cheng and Kendig, 2002; Downie et al.,1996; Harvey et al., 1999; Laube et al., 1995; Molander et al.,2007, 2005; Yamashita et al., 2001; Yevenes and Zeilhofer, 2011).Thus, the GlyR has emerged as a logical target for the possibledevelopment of pharmacological agents to treat substance abuse(Tipps et al., 2010). Allosteric modulators exert their greatestenhancing effects on low concentrations of glycine that are unlikelyto be seen at GlyR in vivo except at the initiation or tail-end ofsynaptic events or perhaps at extrasynaptic receptors (Scimemi andBeato, 2009). Indeed these compounds have negligible effectswhen testedwith saturating concentrations of glycine (Kirson et al.,2012; McCracken et al., 2010).

The sulfonic acid taurine acts as a partial agonist at the GlyR,possessing approximately 50% of the efficacy of glycine (Lape et al.,2008). Taurine is the secondmost abundant amino acid in the brain,and has been implicated as an endogenous ligand of the GlyR inmultiple brain regions (Albrecht and Schousboe, 2005; Dahchour

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Fig. 1. Zn2þ affects currents elicited by maximally-effective concentrations of taurinebut not glycine. A) Sample tracings showing the effect of a maximally-effective con-centration of glycine applied in the presence or absence of Zn2þ. The tracing shows 15 sco-applications of 10 mM glycine with either 2.5 mM tricine or 100 nM Zn2þ followinga 30 s preincubation with tricine or Zn2þ. Each of these applications was preceded andfollowed by applications of 10 mM glycine alone in buffer containing backgroundlevels of Zn2þ. Horizontal bars over tracings indicate time of exposure to glycine, tri-cine, or Zn2þ. Washouts 10 min in duration separated agonist applications. B) Sampletracing showing the effect of a maximally-effective concentration of taurine applied inthe presence or absence of Zn2þ. The tracing follows the same protocol as in panel A.Horizontal bars over tracing indicate time of exposure to taurine, tricine, or Zn2þ. C)Summary of the effects of Zn2þ on brief applications of maximally-effective concen-trations of glycine or taurine. The y-axis represents the percent current potentiationobserved in the presence or absence of Zn2þ compared with that produced by glycineor taurine in background levels of Zn2þ. Data are shown as mean � S.E.M. of 9e11oocytes. *, p < 0.05.

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et al., 1996; Ericson et al., 2006; Mori et al., 2002; Rodríguez-Navarro et al., 2009). Although glycine receptors are found synap-tically in a variety of brainstem nuclei (Ferragamo et al., 1998; Limet al., 2000) and in cerebellum (Dieudonne, 1995) they are alsofound extrasynaptically, where taurine and b-alaninemay be actingas the endogenous agonists (Mori et al., 2002). Taurine may reachconcentrations as high as 20 mM in astrocytes, from which it isreleased by osmoregulatory mechanisms (Albrecht and Schousboe,2005). Extracellular taurine concentrations measured by micro-dialysis range from 1 to 100 mM but, by their nature, likely under-estimate concentrations found locally around astrocytes.

Taurine plays a role in the effects of ethanol in the nAcc(Ericson et al., 2011). Although allosteric modulators have no ef-fects when tested using saturating concentrations of full agonistsat the GlyR, this is not true when saturating concentrations ofpartial agonists are tested (Albrecht and Schousboe, 2005; Kirsonet al., 2012; Scimemi and Beato, 2009). In this paper we exam-ined the possible interactions of these allosteric modulators withthe ubiquitous GlyR modulator, zinc, on glycine- and taurine-activated GlyR.

Zinc modulation of the GlyR is biphasic in nature, with con-centrations below 10 mM enhancing responses, while higher con-centrations inhibit GlyR functioning (Harvey et al., 1999; Laubeet al., 2000, 1995; McCracken et al., 2010). Zinc is present at lowand variable levels in standard buffer solutions (Kay, 2004;McCracken et al., 2010), and is also present throughout the brainat low nM concentrations known to potentiate GlyR responses(Frederickson et al., 2006a, b; McCracken et al., 2010). Althoughzinc is packaged into some synaptic vesicles, even upon releaseconcentrations remain below 10 mM (Frederickson et al., 2006a).Zinc modulation of the glycine-activated GlyR has been extensivelystudied but the interactions between zinc and the taurine-activatedGlyR have not been characterized to the same extent, especially inconjunction with ethanol or other modulators. Previous studies ofzinc modulation of taurine-activated GlyR responses focused onadding zinc at both enhancing and inhibiting concentrations,without controlling for the background zinc likely to be found atbiologically-relevant concentrations (Laube et al., 2000). In thisstudy we further characterize zinc modulation, as well as comparezinc’s interactions with other allosteric modulators on the glycine-vs. taurine-activated GlyR.

2. Materials and methods

2.1. Reagents

All chemicals were purchased from SigmaeAldrich (St. Louis, MO) except iso-flurane which was obtained from Anaquest (New Providence, NJ).

2.2. Oocyte isolation and cDNA injection

Xenopus laevis were obtained from Nasco (Fort Atkinson, WI) and housed at19 �C on a 12-h light/dark cycle. During surgery, performed in accordance with theAssociation for Assessment and Accreditation of Laboratory Animal Care regulations,portions of ovaries were removed and placed in isolation media containing 108 mMNaCl, 1 mM EDTA, 2 mM KCl, and 10 mM HEPES. Forceps were used to manuallyremove the thecal and epithelial layers from stage V and VI oocytes. The oocytefollicular layer was removed using a 10 min incubation in 0.5 mg/ml type 1Acollagenase in buffer containing 83mMNaCl, 2 mMMgCl2, and 5mMHEPES. Animalpoles of oocytes were injected with human glycine a1, a2, a1b or a2b receptorsubunit cDNAs (1.5 ng/30 nl) in a modified pBK-cytomegalovirus vector (Mihic et al.,1997), using a micropipette (10e15 mm tip size) attached to an electronically-activated microdispenser. When heteromeric receptors were to be studied, a andb subunit cDNAs were injected in a 1:20 a:b concentration ratio to ensure incor-poration of the b subunits into receptors. Oocytes were stored in the dark at 19 �C in96-well plates containing modified Barth’s saline (MBS) [88 mM NaCl, 1 mM KCl,2.4 mMNaHCO3,10mMHEPES, 0.82 mMMgSO4$7H2O, 0.33mM Ca(NO3)2, 0.91 mMCaCl2 at pH 7.5] supplemented with 2 mM sodium pyruvate, 0.5 mM theophylline,10 U/ml penicillin, 10 mg/l streptomycin and 50 mg/l gentamicin, and sterilized bypassage through a 0.22 mm filter.

Please cite this article in press as: Kirson, D., et al., Physiological concenabuse, Neuropharmacology (2013), http://dx.doi.org/10.1016/j.neurophar

2.3. Two-electrode voltage-clamp electrophysiology

Oocytes expressed GlyR within 24 h, and all electrophysiological measurementswere made within 5 days of cDNA injection. Before electrophysiological recording,oocytes were placed in a 100 ml bath with the animal poles facing upwards andimpaled with two high-resistance (0.5e10 MU) glass electrodes filled with 3 M KCl.Cells were voltage-clamped at �70 mV using an OC-725C oocyte clamp (WarnerInstruments, Hamden, CT) and perfused with MBS at a rate of 2 ml/min using aMasterflex USA peristaltic pump (Cole Parmer Instrument Co., Vernon Hills, IL)through 18-gauge polyethylene tubing. All drug solutions were prepared in MBS,MBS þ 2.5 mM tricine, or MBS þ 100 nM ZnCl. When saturating concentrations ofagonists were applied, applications lasted for 15 s for the short-application exper-iments and for 26 min for the continuous application experiments. For experimentsusing submaximal concentrations of taurine, concentrations that yielded 5 percentof the maximally-effective taurine response (EC5) were applied for 30 s. For short-application experiments, modulators were co-applied with agonists following a30 s preincubation of modulator alone. For continuous application experiments,modulators were co-applied with agonist for 2 min following agonist applicationalone. All drug applications during short-application experiments were followed bysix to tenminwashout periods to allow for complete receptor resensitization. Loss ofvolatile compounds through tubing and evaporation from bath was previouslymeasured (Beckstead et al., 2002, 2000; Mihic et al., 1994; Yamakura et al., 1999). Allconcentrations reported are the bath concentrations to which the oocytes were


Fig. 2. Zn2þ affects currents elicited by long exposures to maximally-effective con-centrations of taurine but not glycine. A) Sample tracing showing the effects of 2.5 mMtricine or 100 nM Zn2þ co-applied with saturating concentrations of glycine after10 min of continuous glycine application. Two minutes applications of 10 mM glycinein either 2.5 mM tricine or 100 nM Zn2þ were preceded and followed by 10 mM glycinein buffer containing background levels of Zn2þ. Horizontal bars over the tracing indi-cate time of exposure to glycine, tricine, or Zn2þ. B) Sample tracing showing the effectsof 2.5 mM tricine or 100 nm Zn2þ co-applied with saturating concentrations of taurine,after 10 min of continuous taurine application. The tracing follows the same protocolas in panel A. Horizontal bars over the tracing indicate time of exposure to taurine,tricine, or Zn2þ. C) Summary of the effects of maximally-effective concentrations ofglycine or taurine in the presence or absence of Zn2þ during continuous agonist ex-posures. The y-axis represents the percent current enhancement observed in thepresence or absence of Zn2þ compared with the glycine or taurine current levelimmediately preceding tricine or zinc co-application. Data are shown as mean � S.E.M.of 4e6 oocytes. *, p < 0.05.

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exposed. Data were acquired at a rate of 1 kHz using a Powerlab 4/30 digitizer withLabChart version 7 software (ADInstruments, Bella Vista, NSW, Australia).

2.4. Data analysis

Peak currents were measured and used in data analysis. Currents observed inthe presence of agonist plus modulators were compared with currents generated byagonist without the modulator of interest present. Experimental values are listed asthe mean � S.E.M. Significant differences between experimental conditions weredetermined using ANOVA or repeated measures ANOVA and post-hoc tests, asindicated. SigmaPlot version 11.0 (Systat Software, San Jose, CA) was used for sta-tistical testing.

3. Results

3.1. Chelation of endogenous zinc decreases responses to taurine

We previously showed that various allosteric modulators ofGlyR, such as ethanol and some anesthetics, have different effects


on currents generated by saturating concentrations of glycineversus taurine (Kirson et al., 2012). In order to compare zincmodulation of GlyR currents generated by maximally-effectiveconcentrations of glycine and taurine, we compared currentsgenerated by co-applications of agonist and either 100 nM zinc or2.5 mM of the zinc chelator tricine with currents generated byagonist alone. Fig. 1A shows a sample tracing of successive 15 sapplications of 10 mM glycine in the presence of a backgroundconcentration of zinc, 2.5 mM tricine, or 100 nM zinc. Fig. 1B showsthe same experimental protocol as in 1A but with 100 mM taurineinstead employed as the agonist. Addition of 100 nM zinc enhancedsaturating taurine currents almost 60% (Fig. 1C) while leavingsaturating glycine-mediated currents unchanged. Elimination ofbackground levels of zinc in the perfusion buffer by co-applicationof tricine decreased saturating taurine currents while having noeffects on saturating glycine currents. A two-way ANOVA showed asignificant interaction effect between the concentration of zinc inthe buffer and the agonist tested [F(1,37) ¼ 34.4, p < 0.001]. AStudenteNewmaneKeuls (SNK) multiple comparison post-hoc testshowed significant differences between glycine and taurine both inthe presence of 2.5 mM tricine [q ¼ 4.6, p < 0.01] and 100 nM zinc[q ¼ 7.1, p < 0.001], as well as a significant effect of zinc concen-tration when taurine was the agonist [q ¼ 12.3, p < 0.001].

We next looked at the effects of zinc modulation using satu-rating agonist concentrations that were applied continuously for10 min, followed by 2 min co-applications of agonist and either2.5 mM tricine or 100 nM zinc. This continuous agonist applicationapproach allows for channels to equilibrate between the open anddesensitized states (Fig. 2A, B). Under these conditions all receptorshave bound agonist and are either activated (opening) or desensi-tized. In this experimental paradigm the effects of modulators canthus only be due to their effects on channel opening/closing ki-netics (Popen) or desensitization/resensitization rates and not onpossible effects on agonist affinity. As seen in Fig. 2C, the effectsexhibited by zinc trended in the same directions as those seen inFig. 1C, but to a smaller degree. Co-application of 100 nM zincproduced greater potentiation of saturating taurine currentscompared to saturating glycine currents. Co-application of tricineshowed a decrease in saturating taurine responses compared to anegligible increase in saturating glycine. Similar to the shortapplication experiments, a two-way ANOVA showed a significantinteraction effect between the concentration of zinc in the bufferand the agonist tested [F(1,16) ¼ 34.3, p < 0.001]. A SNK multiplecomparison post-hoc test revealed significant differences betweenglycine and taurine both in the presence of 2.5 mM tricine [q ¼ 7.1,p < 0.001] and 100 nM zinc [q ¼ 4.6, p < 0.01], as well as a sig-nificant effect of zinc concentration when taurine was the agonist[q ¼ 11.6, p < 0.001].

3.2. Zinc and ethanol interactions

Physiologically-relevant low nM concentrations of zinc enhanceethanol modulation of GlyR currents generated by submaximal butnot maximally-effective concentrations of glycine (McCrackenet al., 2010). As these two modulators are likely to be presentconcurrently at GlyR in vivo, we compared the effects of enhancingconcentrations of zinc on ethanol modulation of GlyR activated bymaximally-effective glycine or taurine concentrations. We firstlooked at the effects of 15 s co-applications of maximally-effectiveconcentrations of agonist and 200 mM ethanol in the presence ofbackground levels of zinc and also in the presence of 2.5 mM tricineor 100 nM zinc. Fig. 3A shows that ethanol modulation of themaximally-effective taurine response is present in all threedifferent zinc concentrations. However, 100 nM zinc significantlydecreased [Two-way Repeated Measures (RM) ANOVA with SNK


Fig. 3. Zn2þ affects ethanol potentiation of currents elicited by maximally-effectiveconcentrations of taurine but not glycine. A) Sample tracings showing the effect ofZn2þ on brief applications of maximally-effective concentrations of taurine. Taurine(100 mM) was co-applied with 200 mM ethanol for 15 s following a 30 s preincubationwith 200 mM ethanol. Ethanol applications were flanked by 15 s applications ofmaximally-effective taurine applied alone. Series of applications were carried out inbuffer containing background levels of Zn2þ, 2.5 mM tricine or 100 nM Zn2þ. Hori-zontal bars over tracings indicate time of exposure to taurine, tricine, Zn2þ or ethanol.B) Summary of the effects of 200 mM ethanol on brief applications of maximally-effective concentrations of glycine or taurine in the presence of a background levelof Zn2þ, the absence of Zn2þ produced by tricine, or the addition of 100 nM Zn2þ. They-axis represents the percent current potentiation observed with ethanol co-application, in background Zn2þ, 2.5 mM tricine or 100 nM Zn2þ. Data are shown asmean � S.E.M. of 6 oocytes. C) Summary of the effects of 50 mM ethanol on briefapplications of maximally-effective concentrations of taurine in the presence of abackground level of Zn2þ, the absence of Zn2þ produced by tricine, or the addition of100 nM Zn2þ. The y-axis represents the percent current potentiation observed withethanol co-application, in background Zn2þ, 2.5 mM tricine or 100 nM Zn2þ. Data areshown as mean � S.E.M. of 8 oocytes. *, p < 0.05.

Fig. 4. Zinc/ethanol interactions on GlyR composed of a variety of different subunits.Summaries of the effects of ethanol on brief applications of maximally-effective con-centrations of glycine or taurine in the presence of a background level of Zn2þ, theabsence of Zn2þ produced by tricine, or the addition of 100 nM Zn2þ on the a1b GlyR(A) a2 GlyR (B) or a2b GlyR (C). The y-axis represents the percent current potentiationobserved with ethanol co-application, in background Zn2þ, 2.5 mM tricine or 100 nMZn2þ. Data are shown as mean � S.E.M. of 5e8 oocytes. *, p < 0.05.


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multiple comparison procedure; q ¼ 5.0, p < 0.01; q ¼ 4.6, p < 0.01,respectively] the degree of ethanol percent potentiation of a satu-rating taurine concentration compared to the potentiation seen ineither the background zinc level or after zinc chelation. In back-ground levels of zinc, or in the presence of 2.5 mM tricine, or100 nM zinc, 200 mM ethanol produced significantly greaterenhancement of responses in saturating taurine than glycine[q ¼ 12.9, p < 0.001; q ¼ 11.6, p < 0.001; q ¼ 8.3, p < 0.001,respectively], as there was negligible inhibition of glycine currentsobserved instead (Fig. 3B). The same trends were observed using50 mM ethanol, just with a lower degree of ethanol potentiation


(Fig. 3C) [q ¼ 3.85, p < 0.05, comparing taurine alone with taurineplus zinc].

We next looked at the combined effects of zinc and ethanol onreceptors comprised of different subunits. The heteromeric a1bGlyR is the predominant adult form found in brainstem and spinalcord of mammals and is likely to be found synaptically, as the bsubunit is involved in anchoring the GlyR via its interactions withgephyrin (Kirsch and Betz, 1995). As shown in Fig. 4A, the effects ofcombined ethanol and zinc on the heteromeric channel exhibit thesame trends as those seen on the homomeric channel but withslightly increased enhancement of taurine currents for the het-eromeric channel. A Two-way RM ANOVA with SNK multiplecomparison procedure showed ethanol potentiation of currents in100 nM zinc significantly decreased from both background zinc[q ¼ 3.8, p < 0.05] and 2.5 mM tricine [q ¼ 3.1, p < 0.05], as well assignificantly greater ethanol percent potentiation of taurine vsglycine currents [q ¼ 7.5, p ¼ 0.001]. We also performed equivalentexperiments on a2 homomeric (Fig. 4B) and a2b (Fig. 4C) hetero-meric receptors. Two-way RM ANOVAs with SNK multiple com-parison procedures showed significantly greater ethanol percent


Fig. 5. Zn2þ decreases isoflurane and toluene potentiation of currents elicited bymaximally-effective concentrations of taurine but not glycine. These experiments werecarried out in the same manner as the ethanol experiments described in Fig. 3. A)Effect of isoflurane on currents elicited by brief applications of maximally-effectiveglycine (10 mM) or taurine (100 mM) concentrations in the presence or absence ofZn2þ. Agonist was co-applied with 0.55 mM isoflurane for 15 s following a 30 s pre-incubation with 0.55 mM isoflurane. Data are shown as mean þ S.E.M. of 6 oocytes. B)Effect of toluene on currents elicited by brief applications of maximally-effectiveconcentrations of glycine (10 mM) or taurine (100 mM) in the presence or absenceof Zn2þ. Agonist was co-applied with 0.42 mM toluene for 15 s following a 30 s pre-incubation with 0.42 mM toluene. Data are shown as mean � S.E.M. of 4 oocytes. *,p < 0.05.

Fig. 6. Zn2þ affects ethanol potentiation of low concentrations of full and partial ag-onists. A) Ethanol potentiation of the effects of 5% maximal (EC5) glycine- or taurine-mediated currents in the presence of 2.5 mM tricine (i.e., in the absence of Zn2þ). EC5

glycine and taurine were co-applied with either 50 mM or 200 mM ethanol following a30 s preincubation with ethanol. Data are shown as mean þ S.E.M. of 5 oocytes. B)Ethanol potentiation of EC5 glycine and taurine is enhanced by Zn2þ. EC5 glycine ortaurine were co-applied with 200 mM ethanol following preincubation with ethanol,in the presence of either background Zn2þ (left two bars) or 2.5 mM tricine (right twobars). Data are shown as mean þ S.E.M. of 9 oocytes. *, p < 0.05.


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potentiation of taurine vs glycine currents [a2, q ¼ 7.14, p < 0.01;a2b, q ¼ 4.6, p < 0.05]. We found that taurine had very low efficacyon GlyR containing a2 subunits: 13.2 � 5.4% in a2 homomers and6.1 � 1.4% in a2b heteromeric GlyR, compared to the effects pro-duced by a saturating concentration of glycine.

3.3. Zinc and isoflurane interactions

Because biologically-relevant concentrations of zinc affectethanol potentiation of saturating taurine currents, we investigatedother allosteric modulators of the GlyR for zinc-modulator in-teractions. The inhaled volatile anesthetic isoflurane was testedusing the same experimental protocols as those used for ethanol.Fig. 5A shows the results of 15s co-applications of 0.55 mM iso-flurane with either 10 mM glycine or 100 mM taurine in thepresence of background levels of zinc, 2.5 mM tricine or 100 nMzinc. As for ethanol, isoflurane potentiation of saturating taurinecurrents in either background zinc or 2.5 mM tricine were verysimilar, while the addition of 100 nM zinc significantly decreasedthe isoflurane percent potentiation from those levels [Two-way RM


ANOVAwith SNKmultiple comparison procedure; q¼ 4.1, p< 0.02;q ¼ 4.4, p < 0.05, respectively]. Saturating glycine-mediated cur-rents wereminimally affected by isofluranewhether in backgroundlevels of zinc, 2.5 mM tricine, or 100 nM zinc. Isoflurane enhance-ment was significantly different between glycine and taurine in thepresence of a background level of zinc [q ¼ 7.4, p < 0.001] and in2.5 mM tricine [q ¼ 7.8, p < 0.001].

3.4. Zinc and toluene interactions

The next allosteric modulator tested was the inhaled drug ofabuse toluene. Fig. 5B shows the results of the brief co-applicationsof 0.42 mM toluene with either 10 mM glycine or 100 mM taurinein the presence of background levels of zinc, 2.5 mM tricine, or100 nM zinc. Toluene had negligible effects on 10 mM glycinecurrents and its effects on 100 mM taurine currents were similar toeffects seen with ethanol and isoflurane. Again, similar to ethanoland isoflurane, toluene percent potentiation of saturating taurine-mediated currents was significantly reduced in the presence of100 nM zinc, compared to the other two zinc conditions [Two-wayRM ANOVA with SNK multiple comparison procedure; q ¼ 6.3,p< 0.002; q¼ 5.7, p< 0.002, respectively]. Toluene enhancement ofsaturating taurine was significantly greater than effects on satu-rating glycine in background levels of zinc [q ¼ 8.8, p < 0.001],


Fig. 7. Zn2þ levels do not significantly affect desensitization of glycine- or taurine-activated glycine receptors. A) Zn2þ does not affect the rate of desensitization produced bymaximally-effective concentrations of glycine or taurine. Percent decrease in peak current observed 5 s post-peak is graphed for each agonist/Zn2þ level combination. Data areshown as mean þ S.E.M. of 26e35 oocytes. B) Zn2þ and ethanol do not affect the rate of desensitization produced by maximally-effective concentrations of glycine or taurine.Percent decrease in peak current seen 5 s post-peak in the presence of 200 mM ethanol is graphed for each agonist/Zn2þ level combination. Data are shown as mean þ S.E.M. of 6oocytes. C) Zn2þ levels have no effect on isoflurane changes in desensitization rates of taurine-activated glycine receptor currents. Percent decrease in peak current observed 5 spost-peak in the presence of 0.55 mM isoflurane is graphed for each agonist/Zn2þ level combination. Data are shown as mean þ S.E.M. of 6 oocytes. D) Zn2þ and toluene do notinteract to affect desensitization rates of maximally-effective concentrations of glycine and taurine. Percent decrease in peak current seen 5 s post-peak in the presence of 0.42 mMtoluene is graphed for each agonist/Zn2þ level combination. Data are shown as mean þ S.E.M. of 5 oocytes. *, p < 0.05.


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2.5 mM tricine [q ¼ 7.2, p < 0.001], and 100 nM zinc [q ¼ 3.3,p < 0.05].

3.5. Zinc & ethanol interactions at a low taurine concentration

We previously found that zinc enhances ethanol potentiation oflow concentrations of glycine, and that the degree of ethanolenhancement is reduced when zinc is chelated by tricine(McCracken et al., 2010). Although we saw minor ethanol effectswith changing zinc levels at saturating concentrations of glycine,we did see changes in ethanol potentiation of saturating concen-trations of taurine based on the level of zinc present (Fig. 2B). Thuswe extended the observations made in the McCracken et al. (2010)paper, this time using EC5 concentrations of taurine. We first testedwhether ethanol potentiation of EC5 taurine currents in a1 GlyRdepends on the concentration of zinc. Fig. 6A shows that when zincis chelated with tricine, the degree of ethanol potentiation of EC5taurine-mediated currents is the same as EC5 glycine-mediatedcurrents for both 50 mM and 200 mM ethanol. We next tested ifchelation of zinc significantly reduces ethanol potentiation of EC5taurine-mediated currents as it does for glycine-mediated currents.Fig. 6B shows the results of 200 mM ethanol effects on EC5 glycineand taurine currents, with and without the addition of 2.5 mMtricine. With no differences between agonists, ethanol potentiationwas significantly lower in 2.5 mM tricine compared to background


levels of zinc [Two-way ANOVA with SNK multiple comparisonprocedure; q ¼ 3.7, p < 0.05].

3.6. Zinc/modulator interaction effects on desensitization rates

We next looked at the effects of changing zinc concentration ondesensitization rates. Desensitization rates were determined bycomparing the peak current to that measured five seconds post-peak for saturating glycine and taurine currents, in the presenceof background levels of zinc or in the presence of 2.5 mM tricine or100 nM zinc. Changing the zinc concentration did not affect thedesensitization rates of glycine-activated currents in a1 GlyR(Fig. 7A). Taurine-mediated desensitizationwas significantly slowerthan for glycine-mediated currents in background zinc [Two-wayANOVA with SNK multiple comparison procedure; q ¼ 8.5,p < 0.001], as well as in 2.5 mM tricine [q ¼ 10.4, p < 0.001], and in100 nM zinc [q ¼ 4.2, p < 0.01]. Additionally, desensitization oftaurine-mediated currents in 2.5 mM tricine and 100 nM zinc weresignificantly different [q ¼ 3.7, p < 0.05].

Because drugs of abuse and zinc are likely to be foundconcomitantly at receptors in vivo, we examined the effects ofdifferent allosteric modulators on desensitization rates in thepresence of background zinc, 2.5 mM tricine, or 100 nM zinc. Fig. 7Bshows the desensitization rates produced by 10 mM glycine and100mM taurine appliedwith 200mMethanol in the three different



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zinc concentrations. Taurine þ ethanol current desensitization wassignificantly slower than that seen in glycine þ ethanol currents inbackground zinc [Two-way RM ANOVA with SNK multiple com-parison procedure; q ¼ 7.7, p < 0.001], 2.5 mM tricine [q ¼ 11.0,p < 0.001], and 100 nM zinc [q ¼ 5.1, p < 0.01]. Additionally,desensitization of glycine þ ethanol currents in 2.5 mM tricine and100 nM zinc were significantly different [q ¼ 4.3, p < 0.05].

Fig. 7C shows the results for desensitization experimentsinvolving isoflurane. Desensitization was similar for 10 mM glycinewith 0.55 mM isoflurane currents and 100 mM taurine with0.55 mM isoflurane currents, with the only significant differencefound between glycine þ isoflurane and taurine þ isoflurane in100 nM zinc buffer [Two-way RM ANOVA with SNK multiplecomparison procedure; q ¼ 3.5, p < 0.05]. Fig. 7D shows the effectsof toluene on desensitization mediated by either glycine or taurine.There was a significant effect of agonist [Two-way RM ANOVAwithSNK multiple comparison procedure; q ¼ 4.2, p < 0.05].

4. Discussion

The dopaminergic projection from the ventral tegmental area(VTA) to the nAcc is critical to the perception of the rewardingproperties of drugs of abuse, and ethanol’s actions on the GlyRmay be particularly important in these brain regions. For example,extracellular dopamine levels increase in the nAcc followingethanol (Imperato and Di Chiara, 1986) or glycine (Molander et al.,2005) administration and glycine perfusion into the nAcc of ratsdecreases alcohol consumption (Molander et al., 2005). Manyvolatile agents such as isoflurane and toluene also have abuse li-ability (Johnston et al., 2012; Wilson et al., 2008). Toluene alsoincreases extracellular dopamine levels in the VTA and nAcc whenperfused into the former (Riegel et al., 2007). Most studiesexamining the effects of these agents on GlyR functioning do so bydetermining the effects of single modulators in isolation. However,since zinc is ubiquitous in cerebrospinal and interstitial fluids atGlyR-potentiating levels, the effects of drugs of abuse on GlyRin vivo will always be seen in combination with zinc effects. In factone could go so far as to say that studying these compounds in theabsence of zinc would represent an artificial situation unlikely tobe found in vivo. Since taurine and glycine may act as GlyR agonistsin brain regions such as the nAcc, we examined the effects ofcombinations of allosteric modulators on GlyR activated by theseagonists. Maximally-effective concentrations of taurine but notglycine were affected by zinc chelation (Figs. 1 and 2). This is likelythe result of glycine producing a very high probability of channelopening (intra-cluster Po w 1), with maximally-effective concen-trations keeping the channel open almost 100% of the time beforeit desensitizes, regardless of the presence of any allosteric modu-lator. As a partial agonist, taurine has a much lower Po (w0.5) atmaximally-effective concentrations (Lape et al., 2008), allowing forzinc to exhibit its enhancing effects. The zinc potentiation seenafter 10 min of continuous taurine perfusion could be attributableto zinc: (1) increasing Po; (2) enhancing conductance; (3)decreasing the desensitization rate or; (4) increasing the rate ofGlyR resensitization. Previous studies performed using low agonistconcentrations show that zinc does increase Po but it does notaffect conductance (Laube et al., 2000). Our data (Fig. 7A) showsthat zinc can also increase the desensitization rate in taurine-activated GlyR, which would tend to counteract its Po enhancingeffects. In this way, zinc acts in a very similar fashion to other GlyRallosteric modulators (Kirson et al., 2012). The decrease in re-sponses seen when zinc is chelated by tricine suggests that sub-maximal glycine responses and all taurine responses areoverestimated when not controlling for background zinc concen-trations in buffer solutions.


Because zinc is ubiquitous in the CNS, the effects of any drugs ofabuse or other allosteric modulators of interest on GlyR functioningrequire comparison in the presence and absence of potentiatingconcentrations of zinc. In the presence of a maximally-effectiveconcentration of glycine, changing the zinc concentration had noeffect on responses for any of the modulators tested, since 10 mMglycine has already produced a maximal response. However, effectson maximally-effective concentrations of taurine were expectedand seen. If zinc and other positive modulators were acting in anadditive or synergistic manner, we would expect the 100 nM zinccondition to give a higher degree of potentiation than our standardbuffers, with chelation of zinc by 2.5 mM tricine giving a lowerdegree of potentiation than both other conditions. However we didnot find this to be the case for maximally-effective concentrationsof taurine with alcohol, isoflurane, or toluene.

Chelation of zinc with tricine did not change the alcohol, iso-flurane, or toluene potentiation of a maximally-effective taurineconcentrationwhen compared to the effects seen in the presence ofbackground levels of zinc (Figs. 3B, 5A and 5B). However, at sub-maximal concentrations of glycine or taurine, chelation of back-ground zinc does decrease ethanol potentiation of GlyR responses(Fig. 6B). The most parsimonious explanation for the lack of changeseen at higher levels of taurine would be that background levels ofzinc during those particular experiments were low. However, theaddition of 100 nM zinc to buffers, ensuring that the concentrationof zinc is in the potentiating range, led to a significant reduction inthe ethanol, isoflurane, and toluene potentiation of taurine-activated GlyR responses (Figs. 3B, 5A and B). This finding cannotbe explained by simple competition between modulators as zinc isnot believed to bind the receptor at the same locations as alcohols,anesthetics, and inhalants (Beckstead et al., 2000; Laube et al.,2000; Lynch et al., 1998; Mihic et al., 1997; Yamakura et al., 1999).However, zinc does increase the Po of the taurine-activated GlyR(Laube et al., 2000), in effect mimicking the glycine-bound GlyR.When this occurs and the GlyR Po approaches 1 any other positivemodulator present will as a result produce decreased percentenhancement compared to when less zinc is present.

Since the a2 subunit appears to be expressed at higher levelsthan a1 in higher brain regions (Jonsson et al., 2012) we studiedethanol/zinc interactions on a variety of different GlyR subtypesactivated by taurine vs glycine (Figs. 3B, 4AeC). The a2-containingreceptors were previously shown to be less sensitive to ethanol(Mascia et al., 1996), and zinc (Miller et al., 2005), than those thatcontain a1 subunits and this was also reflected in our experimentsusing amaximally-effective concentration of taurine.Weperformedthese experiments in the absence and presence of the b subunitwhich anchors the GlyR to gephyrin in the synapse (Kirsch and Betz,1995). Badanich et al. (2013) found that in the orbitofrontal cortex,where a2-containing GlyR are thought to predominate (Jonssonet al., 2012), there was no effect of the glycine receptor antagoniststrychnine on neuronal holding current, but strychnine did antag-onize ethanol-induced changes in holding current and spike firing.The most parsimonious explanation for these findings is that thereare insufficient extracellular glycine or taurine concentrations tosignificantly affect holding current, unless ethanol is present. It isreasonable to hypothesize that ethanol promotes the release ofglycine or taurine and it may also then enhance the effects of thesecompounds on the GlyR, although the Badanich et al. (2013) studydoes not address the relative importance of each phenomenon. Iftaurine is the responsible agonist then our findings raise twopossible complicating issues. First, like Shmieden et al. (1992), wefound that, relative to glycine, taurine has very low efficacy on a2-containing GlyR, much lower than its efficacy on a1-containingGlyR. Even at saturating concentrations, taurine could at best haveonly modest effects on a2 GlyR function. The next issue is that



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ethanol weakly enhances taurine-mediated currents in a2 and a2bGlyR. These findings are difficult to reconcile with the possibilitythat the effects seen in the Badanich et al. (2013) study aremediatedby taurine on a2-containing receptors in the orbitofrontal cortex.Further studies addressing these issues are warranted.

5. Conclusions

In summary, low concentrations of zinc potentiated GlyR re-sponses frommaximally-effective concentrations of taurine but notglycine. Chelation of zinc reduced GlyR responses produced bymaximally-effective concentration of taurine but not glycine, andthus any experiments conducted in the absence of zinc chelationwill tend to overestimate taurine efficacy. This would also be truewhen submaximal glycine concentrations are used. At the lowconcentrations tested, zinc decreased the enhancing effect oftaurine-mediated responses for all drugs of abuse tested whenapplied in combination with them. As taurine is likely to be anendogenous GlyR ligand in brain regions affected by these com-pounds, the presence of low concentrations of zinc in these regionsresults in GlyR responses that are not the result of a simple sum-mation of responses to single modulators, and this needs to betaken into consideration when investigating the role of the GlyRin vivo. It is highly likely that previously published studies of thisreceptor have in reality been studying the zinc-modulated GlyR. Totruly understand how allosteric modulators act at the GlyR onemust examine their actions in the context of the physiologicalmilieu at the receptor, reflecting in vivo conditions, as well asstudying each modulator’s contribution in isolation.

Acknowledgments

This research was supported by National Institute on AlcoholAbuse & Alcoholism grant 5P01AA020683.

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physiological concentrations of zinc reduce taurine-activated glyr responses to drugs of abuse

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