Neuroscience 297 (2015) 68–77

MANIPULATIONS OF EXTRACELLULAR LOOP 2 IN a1 GLYR ULTRA-SENSITIVE RECEPTORS (USERS) ENHANCE SENSITIVITY TO , ETHANOL, AND , BUT NOT

A. NAITO, a K. H. MUCHHALA, a J. TRANG, a (volatile), propofol (intravenous), and lidocaine (local), L. ASATRYAN, b J. R. TRUDELL, c G. E. HOMANICS, d,e known to enhance -induced chloride currents using a b R. L. ALKANA AND D. L. DAVIES * two-electrode voltage clamp electrophysiology. Loop 2 a Department of Pharmacology and Pharmaceutical Sciences, mutations produced a significant 10-fold increase in isoflu- University of Southern California, School of Pharmacy, 1985 Zonal rane and lidocaine sensitivity, but no increase in propofol Avenue, Los Angeles, CA 90089, USA sensitivity compared to WT a1 GlyRs. Interestingly, we also b Titus Family Department of Clinical Pharmacy and found that structural manipulations in the C-terminal end of Pharmaceutical Economics and Policy, University of Loop 2 were sufficient and selective for a1 GlyR modulation Southern California, School of Pharmacy, 1985 Zonal Avenue, Los by ethanol, isoflurane, and lidocaine. These studies are the Angeles, CA 90089, USA first to report the extracellular region of a1 GlyRs as a site of lidocaine action. Overall, the findings suggest that c Department of , Beckman Program for Molecular Loop 2 of 1 GlyRs is a key region that mediates isoflurane and Genetic Medicine, Stanford University, Stanford a University Medical Center, Stanford, CA 94305, USA and lidocaine modulation. Moreover, the results identify d important amino acids in Loop 2 that regulate isoflurane, Department of , University of Pittsburgh, lidocaine, and ethanol action. Collectively, these data indi- 6060 Biomedical Science Tower 3, Pittsburgh, PA 15261, USA cate the commonality of the sites for isoflurane, lidocaine, e Department of Pharmacology and Chemical Biology, University and ethanol action, and the structural requirements for of Pittsburgh, 6060 Biomedical Science Tower 3, Pittsburgh, allosteric modulation on a1 GlyRs within the extracellular PA 15261, USA Loop 2 region. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. Abstract—We recently developed ultra-sensitive ethanol receptors (USERs) as a novel tool for investigation of single receptor subunit populations sensitized to extremely low ethanol concentrations that do not affect other receptors Key words: a1 GlyRs, Loop 2, isoflurane, lidocaine, ethanol, in the nervous system. To this end, we found that mutations -gated . within the extracellular Loop 2 region of glycine receptors (GlyRs) and c-aminobutyric acid type A receptors (GABAARs) can significantly increase receptor sensitivity INTRODUCTION to micro-molar concentrations of ethanol resulting in up to a 100-fold increase in ethanol sensitivity relative to Glycine receptors (GlyRs), one of the major inhibitory wild-type (WT) receptors. The current study investigated: receptor systems in the adult (1) Whether structural manipulations of Loop 2 in a1 mammalian (CNS) (Dutertre GlyRs could similarly increase receptor sensitivity to other et al., 2012), are fast-acting ligand-gated ion channels ; and (2) If mutations exclusive to the C-terminal (LGICs) that belong to the Cys-loop superfamily, whose end of Loop 2 are sufficient to impart these changes. We expressed a1 GlyR USERs in Xenopus oocytes and tested members also include the closely related c-aminobutyric the effects of three classes of anesthetics, isoflurane acid-type A (GABAA), nicotinic acetylcholine (nACh), and 5-hydroxytryptamine3 (5-HT3) receptors (Ortells and Lunt, 1995; Xiu et al., 2005). GlyRs mediate inhibitory *Corresponding author. Address: Titus Family Department of Clinical neurotransmission in the , brain stem, cerebral Pharmacy and Pharmaceutical Economics and Policy, School of cortex, ventral tegmental area, , dor- Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA. Tel: +1-323-442-1427; fax: +1-323-442- sal raphe, and amygdala, and are considered a site of 1704. action for allosteric modulators including, , gen- E-mail address: [email protected] (D. L. Davies). eral anesthetics (volatile and intravenous), neuroactive Abbreviations: EC, extracellular; EC2,effective concentration at 2% of steroids, endocannabinoids, divalent cations, and aver- maximal response; EC50, half-maximal effective concentration; GABAAR, c-aminobutyric acid type A receptor; GlyR, glycine mectins (Downie et al., 1996; Rajendra et al., 1997; receptor; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Lynch, 2004; Dutertre et al., 2012; Maguire et al., 2014). IC, intracellular; Imax, maximum current; MAC, minimum alveolar 1 subunit-containing GlyRs are widely expressed in the concentration; OFC, orbitofrontal cortex; SEM, standard error of the a mean; TM, transmembrane; USER, ultra-sensitive ethanol receptor; spinal cord and brain stem and have been implicated in WT, wild type. playing a role in the immobilizing effects of ethanol and http://dx.doi.org/10.1016/j.neuroscience.2015.03.034 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

68 A. Naito et al. / Neuroscience 297 (2015) 68–77 69 action by inhibiting motor responses to noxious potentiation by intoxicating (10–20 mM) concentrations stimuli and mediating ethanol-induced loss of righting of ethanol (Wallner et al., 2003). With this in mind, we reflex (Antognini and Schwarz, 1993; Williams et al., studied the differences in the sequences 1995; Legendre, 2001; Ye et al., 2009; Aguayo et al., between GABAARs containing the d subunit and GlyR 2014). a1 subunits and found major differences within the Loop A large body of evidence implicates sites within the 2 regions of the EC domain of these two receptors. We transmembrane (TM), extracellular (EC), and therefore conducted a series of investigations that intracellular (IC) domain of GlyRs and GABAARs as focused on the EC domain of a1 GlyRs. Notably, we iden- important targets of ethanol and anesthetic action. Site- tified EC Loop 2 (positions 50–59) as a critical site of etha- directed mutagenesis and radioligand binding studies, nol action that mediates ethanol sensitivity of GlyRs and for example, have identified both inter- and intra-subunit GABAARs (Crawford et al., 2007, 2008; Olsen et al., regions within the TM domains of a1 GlyRs, and a2 and 2007; Perkins et al., 2008, 2009, 2012; Naito et al., 2014). b1 subunits of GABAARs as critical sites of action for Over the course of these aforementioned ethanol, as well as volatile and general anesthetics investigations, we found that manipulation of the (Mihic et al., 1997; Krasowski et al., 1998; Mascia et al., structural features of EC Loop 2 conferred a significant 2000). Current evidence suggests that some of these increase in ethanol sensitivity of up to 100-fold as anesthetics act, in part, via binding at different regions compared to homomeric recombinant WT a1 GlyRs and within the TM domain or the central pore region of the resulted in the discovery of ultra-sensitive ethanol receptors (Cummins, 2007). For example, ethanol and receptors (USERs) (Perkins et al., 2009; Naito et al., volatile anesthetics (isoflurane, and its structural isomer, 2014). When expressed in oocytes and mammalian cells enflurane) are reported to share overlapping sites of in vitro, USERs respond to extremely low micro-molar action in the TM2 and TM3 domain that are distinctly dif- ethanol concentrations – concentrations too low to affect ferent from the site of action of the intravenous anesthetic, native receptors. Remarkably, despite this significant propofol (Mascia et al., 1996; Mihic et al., 1997; increase in ethanol sensitivity, these USERs featured Krasowski et al., 1998, 2001; Nury et al., 2011; Sauguet minimal changes relative to WT a1 GlyRs in general et al., 2013). Mutations in the TM domain of GABAARs receptor characteristics including the maximum current at positions 270 or 291 in a2, or at positions 265 or 286 amplitude (Imax), Hill slope, and agonist sensitivity mea- in b2 rendered these receptors insensitive to isoflurane, sured by the half maximal effective concentration while preserving receptor sensitivity to propofol (EC50). In addition, similar Loop 2 manipulations also pro- (Krasowski et al., 1998). Thus, these findings suggest that duced USERs in homomeric a2 GlyRs and heteromeric the sites of ethanol and isoflurane action on GABAARs dif- a1 and c2 subunits of a1b2c2 GABAARs (Naito et al., fers from those of propofol action. 2014). Interestingly, reversion of the residues in the C- The structural homology among Cys-loop receptors terminal region of Loop 2 in both a1 and a2 GlyR implies that the sites of ethanol and anesthetic action USERs (positions 55–59) back to WT, significantly identified in GABAARs may also correlate to GlyRs. increased ethanol sensitivity by reducing the threshold Isoflurane and propofol enhance glycine-induced and increasing the magnitude of response, without alter- chloride currents of GlyRs, suggesting their involvement ing agonist EC50 sensitivity relative to WT a1 GlyRs in the actions of these anesthetics (Downie et al., 1996; (Naito et al., 2014). Structural manipulation of the C-term- Krasowski and Harrison, 1999). Prior studies found that inal region of Loop 2 in c2 GABAARs eliminated ethanol a point mutation at position 52 in the EC Loop 2 region sensitivity; thus implicating this region as an important of a1 GlyRs altered ethanol sensitivity (Davies et al., regulator of ethanol sensitivity (Naito et al., 2014). 2004; Crawford et al., 2007, 2008; Perkins et al., 2008), Overall, these findings demonstrate the importance of but did not affect propofol sensitivity (Mascia et al., the EC Loop 2 region of ethanol action across multiple 1996). These differences have also been reported in subunits of GlyRs and GABAARs and could be useful in GABAARs (Bali and Akabas, 2004). In addition, recent elucidating the role that these receptor subunits play in studies have characterized position 380 in the large IC mediating the behavioral manifestations of ethanol. loop between TM 3 and TM 4 of a1 GlyRs as an important While USERs are ultra-sensitive to the effects of the site for propofol action, but not for other anesthetics general anesthetic, ethanol, their selectivity to other (alcohols, , trichloroethanol, and isoflurane) anesthetics has not been investigated. The present study (Moraga-Cid et al., 2011). Furthermore, lidocaine, a local tested the hypothesis that structural manipulations of anesthetic, potentiates GlyRs, implicating their involve- Loop 2, particularly the C-terminal end, of human a1 ment in lidocaine action (Hara and Sata, 2007). Recent GlyRs are important for transducing the potentiating studies report that lidocaine-induced potentiation of GlyR effects of three classes of anesthetics – volatile, currents was abolished when the S267 in the TM intravenous, and local. Based on prior studies that domain of a1 GlyRs, a previously reported target site for identify common sites among ethanol and isoflurane, we ethanol (Mihic et al., 1997), was mutated (Hara and expect that the Loop 2 manipulations that increased Sata, 2007). Taken together, these studies indicate that ethanol sensitivity of a1 GlyR USERs would similarly ethanol and certain anesthetics have overlapping sites of increase receptor sensitivity to the volatile anesthetic, action on a1 GlyRs that are distinct from those of propofol. isoflurane, but would not significantly alter the effects of Interestingly, previous studies indicate that GABAARs the intravenous anesthetic, propofol. Similarly, since containing the d subunit are especially sensitive to earlier studies have demonstrated that an ethanol site in 70 A. Naito et al. / Neuroscience 297 (2015) 68–77 the TM domain of a1 GlyRs modulated the effects of the Table 1. Loop 2 sequence alignment and mutations for the human WT local anesthetic, lidocaine, we tested the effects of this and a1 GlyR USERs. Loop 2 of a1 GlyR spans exon 3 (indicated by drug in a1 GlyR USERs to elucidate its sites of action on solid line) and exon 4 (indicated by dotted line). Mutations present in each USER are indicated in bold. Loop 2 of USER 1 represents the a1 GlyRs. We investigated the potentiating effects of Loop 2 sequence of d GABAARs. [A: , D: Aspartate, E: isoflurane, lidocaine, and propofol on a1 GlyR USERs Glutamate, H: Histidine, I: Isoleucine, M: Methionine, N: Asparagine, R: using two-electrode voltage clamp electrophysiology. Arginine, S: Serine, T: , Y: Tyrosine] A long-term goal of our ongoing USER investigations is the creation of knock-in mice that would allow us to a1 GlyR Loop Mutations 2Sequence evaluate the effectiveness of the Loop 2 mutations in vivo. Expression of Loop 2 in GlyRs is controlled by WT two exons: exon 3 for the N-terminal end and exon 4 for USER 1 S50H, A52S, T54A, T55N, the C-terminal end. Since mutations exclusively D57E, R59T contained within a single exon would greatly simplify the USER 2 S50H, T54A, T55N, D57E, R59T development of knock-in mice, we developed a new 1 a USER 3 S50H, A52S, T54A, T55N GlyR USER (i.e., USER 4) to investigate whether the USER 4 D57E, R59T C-terminal region of Loop 2 is sufficient to impart changes in a1 GlyR sensitivity to ethanol and other anesthetics. Overall, the findings supported the hypothesis and provide additional insights into the was performed by subcloning human a1 GlyR into mam- structure–activity relationship of anesthetics on a1 GlyRs. malian vector pCIS2 or pBK-CMV using Quick Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, EXPERIMENTAL PROCEDURES USA) and verified by partial sequencing (DNA Core Facility, University of Southern California) as described Materials previously (Davies et al., 2003). Stage V or VI Xenopus oocytes were injected with 1 GlyR WT or USER cDNA Stage V or VI Xenopus oocytes were purchased a (1 ng/32 nl). Oocytes were stored in incubation medium commercially from EcoCyte Bioscience (Austin, TX, USA). [ND96 supplemented with 2 mM sodium pyruvate, Gentamycin, 3-aminobenzoic acid ethyl ester, glycine, and 50 mg/ml of gentamycin and 10-ml heat-inactivated propofol were purchased from Sigma (St. Louis, MO, HyCloneÒ horse serum (VWR, San Dimas, CA, USA), USA). Isoflurane (USP grade) was purchased from Abbott adjusted to pH 7.5] in Petri dishes (VWR, San Dimas, Laboratories (North Chicago, IL, USA), lidocaine was CA, USA). All solutions were sterilized by passage through purchased from Tocris Bio-Techne (Minneapolis, MN, 0.22- M filters. Injected oocytes were stored at 18 C and USA), and ethanol was purchased from Decon Labs, Inc. l ° used in electrophysiological experiments 24–48 h after (King of Prussia, PA, USA). Glycine stock solutions were injection for a period of 1 week. prepared from powder and diluted with Modified Barth’s Solution (MBS) containing (in mM) 88 NaCl, 1 KCl, 10 HEPES, 0.82 MgSO4, 2.4 NaHCO3, 0.91 CaCl2, and 0.33 Whole cell two-electrode voltage clamp recordings Ca(NO3)2, adjusted to a final pH of 7.5 (Davies et al., Two-electrode voltage clamp recording was performed 2003). 1 mM stock solutions of isoflurane, lidocaine, and using techniques according to those previously reported propofol were prepared in DMSO and serially diluted with (Davies et al., 2003; Perkins et al., 2009). Briefly, oocytes buffer (DMSO 60.1%) immediately prior to testing. Pilot were voltage clamped at a membrane potential of 70 mV studies found that DMSO at this concentration, with or with- using oocyte clamp OC-725C (Warner Instruments;À out glycine, had no appreciable effect on a1 GlyR currents in Hamden, CT, USA) and the oocyte recording chamber WT and USERs. was continuously perfused with MBS ± anesthetic (isoflu- rane, lidocaine, or propofol), ethanol and/or glycine using a Mutagenesis and expression of a1 GlyR in oocytes Dynamax peristaltic pump (Rainin Inst Co., Emeryville, Amino acid sequences of the Loop 2 regions of human a1 CA, USA) at 3 ml/min using an 18-gauge polyethylene tube GlyR wild type (WT) and USERs are shown in Table 1. (Becton Dickinson, Sparks, MD, USA) and resultant cur- Loop 2 is defined as positions 50–59 in the a1 GlyR rents were recorded. subunit (Mihic et al., 1997; Perkins et al., 2009), and spans exon 3 (positions 50–56) and exon 4 (positions 57–59) Anesthetic concentration responses. Concentration (Table 1). a1 GlyR USERs were developed according to response curves for isoflurane, lidocaine, and propofol methods described previously (Perkins et al., 2009). were generated with oocytes expressing a1 GlyR WT or Briefly, we replaced the Loop 2 region of human a1 USERs by measuring the ClÀ currents elicited by agonist GlyRs with that of the ethanol-sensitive Loop 2 of d concentrations producing 2% of the maximal effect (EC2) GABAARs using site-directed mutagenesis to produce a1 ±5%. EC2 glycine concentrations were applied until ClÀ GlyR USER 1 (Table 1). Using Loop 2 of a1 GlyR USER currents were within 10% of each other. We studied EC2 1 as a template, we reverted specific non-conserved amino concentrations because GlyR responses are quite acid residues back to WT a1 GlyR residues to generate sensitive to potentiation at this concentration, whereas, USERs 2, 3 (Naito et al., 2014) and 4. a1 GlyR USER 4 they are much less sensitive at EC50 glycine represents Loop 2 mutations of USER 1 at positions 57 concentrations and potentiation is not observed at Imax and 59 in exon 4 only (Table 1). Site-directed mutagenesis concentrations. EC2 was used as a control pre- and A. Naito et al. / Neuroscience 297 (2015) 68–77 71 post-anesthetic application. Once stable, oocytes were Data analysis tested for potentiation by anesthetics. Oocytes were pre- Data for each experiment were obtained from a sample size incubated with either isoflurane, lidocaine, or propofol for (n) of 6–10 oocytes. Results are expressed as 60 s followed by co-application of the tested anesthetic mean ± SEM. Graphs and statistical analyses were and glycine for 30 s at a perfusion rate of 3 ml/min generated by Prism (GraphPad Software Inc., San Diego, (Davies et al., 2004). Washout periods (5–15 min depend- CA, USA). The percent potentiation of 1 GlyR currents ing on anesthetic concentration tested) were allowed a were determined by calculating the percent change in between glycine and anesthetic applications to ensure glycine EC -induced ClÀ currents in the presence of complete receptor re-sensitization. WT and USER 2 anesthetic at each concentration tested. The threshold responses were measured across anesthetic concentra- for anesthetic sensitivity was determined by comparing tions ranging 1–400 M for isoflurane and 0.1–50 M for l l the 1 GlyR USER percent potentiation at the lowest propofol. Holding currents were not significantly affected a concentration against WT percent potentiation. EC during pre-incubation with isoflurane, lidocaine, or propofol 50 values were approximated using a non-linear, exponential, alone, i.e. in the absence of glycine. As indicated in pre- least squares (ordinary) fit analysis with a 95% confidence vious studies, the final bath concentration of isoflurane is interval. Statistical significance was determined using approximately 50% of the prepared concentration due to a one-way repeated measures analysis of variance losses during delivery (Krasowski and Harrison, 2000). (ANOVA), Bonferroni post hoc analysis, and student’s t To maintain the intended concentration of isoflurane, solu- test. Statistical significance was defined as ⁄p < 0.05. tions were prepared immediately prior to application. Furthermore, to minimize loss of isoflurane due to volatilization, containers were sealed with parafilm during RESULTS perfusion. The anesthetic concentrations tested roughly a1 GlyR USERs 1 and 2 have increased sensitivity to correspond to physiologically relevant concentrations that isoflurane cause anesthesia (immobility) in mammals (the human minimum alveolar concentration (MAC) for isoflurane is 260–320 lM; and EC50 concentrations for lidocaine, and a1 GlyR WT. We tested the effects of isoflurane on propofol are 1–1.9 lM, and 0.4 lM, respectively) (Franks WT a1 GlyRs at concentrations ranging 1–400 lM. and Lieb, 1994; Downie et al., 1996; Krasowski and These concentrations encompass clinical relevance Harrison, 1999; Miller, 2010). Although studies have based on published MAC values of inhaled anesthetics reported direct activation of GlyRs by volatile and intra- that cause immobility in 50% of mammals (Krasowski venous anesthetics, these effects typically occur at con- and Harrison, 2000). We found that isoflurane produced centrations beyond clinical relevance and have been concentration-dependent potentiation of EC2 glycine- observed to a greater extent in GABAARs (Krasowski induced ClÀ currents in WT a1 GlyRs with significant and Harrison, 1999).Therefore, this effect was not studied effects starting at 100 lM isoflurane (Fig. 1). There were in detail. no significant effects of isoflurane on these receptors at concentrations below 100 lM. Representative glycine- Ethanol concentration response. Electrophysiological activated ClÀ current tracings are shown in Fig. 2. There recordings were conducted according to the methods was minimal direct activation by isoflurane alone at con- described above for anesthetic application. centrations P100 lM(Fig. 2), however these effects

Fig. 1. a1 GlyR USERs 1 and 2 have increased sensitivity to isoflurane compared to WT. Isoflurane-induced potentiation of glycine EC2-activated ClÀ currents in Xenopus oocytes expressing a1 GlyR USERs are shown. Values for isoflurane potentiation are presented as percentage of glycine EC2 control. The glycine EC2 concentrations utilized ranged from 5 to 10 lM in USERs and 18 to 25 lM for WT. The threshold for isoflurane sensitivity in a1 GlyR USERs 1 and 2 was 10 lM, and 100 lM for a1 GlyR WT and USER 3. Significant increases in the magnitude of isoflurane response for a1 GlyR USERs compared to WT are denoted by ⁄p < 0.05. There were no significant differences in the threshold for isoflurane sensitivity in a1 GlyR USER 3 relative to WT. Each data point represents the mean ± SEM from 6 to 9 oocytes. 72 A. Naito et al. / Neuroscience 297 (2015) 68–77

Fig. 2. Representative tracings of a1 GlyR USERs in response to isoflurane. Two-electrode voltage clamp electrophysiology tracings of homomeric a1 GlyR WT and a1 GlyR USERs expressed in Xenopus oocytes in response to isoflurane. (A) a1 GlyR WT in response to 100 lM isoflurane; (B) a1 GlyR USER 1 in response to 10 lM isoflurane; (C) a1 GlyR USER 2 in response to 10 lM isoflurane; and (D) a1 GlyR USER 3 in response to 10 lM isoflurane. Effects of isoflurane were tested with EC2 glycine (5–25 lM) and the membrane potential was 70 mV. À

reducing the threshold for isoflurane sensitivity 10-fold Table 2. Approximate EC50 values for isoflurane and propofol in WT and a1 GlyR USERs. EC50 values ± standard error of the mean (SEM) from 100 lM in WT a1 GlyRs. There was a significant were approximated using standard curve-fitting analysis for isoflurane decrease in the EC50 of a1 GlyR USER 2 compared to and propofol WT (Table 2). At concentrations P10 lM, the magnitude of isoflurane potentiation significantly a1 GlyR Isoflurane EC50 (lM) Propofol EC50 (lM) increased in a1 GlyR USER 2 relative to WT (Fig. 1). WT 228.3 ± 21.5 19.3 ± 6.3 However, the magnitude of isoflurane potentiation was USER 1 78.3 ± 13.9* 17.1 ± 14.2 * lower than those produced by a1 GlyR USER 1 across USER 2 174.2 ± 15.7 10.6 ± 15.5 all tested concentrations (Fig. 1). USER 3 242.4 ± 11.6 15.2 ± 17.3 USER 4 89.4 ± 9.7* 15.1 ± 8.2

* Represent statistically significant differences in EC50 values compared to WT a1 GlyR USER 3. Isoflurane produced concentration- a1 GlyRs. dependent potentiation of EC2 glycine-induced currents starting at 100 M in 1 GlyR USER 3, indicating no were not statistically significant (<1% of maximal GlyR l a change in the threshold for isoflurane sensitivity currents, p>0.05). EC values are presented in 50 compared to WT 1 GlyRs (Fig. 1). While there was a Table 2. a degree of isoflurane-induced potentiation at 10 lM, these responses were not significant (p = 0.1). In addition, a1 GlyR USER 1. We tested the effects of isoflurane there was no significant difference in the EC50 of a1 on a1 GlyR USERs to establish whether the sites GlyR USER 3 compared to WT (Table 2). The magnitude responsible for increasing receptor sensitivity to ethanol of glycine response to isoflurane did not differ would similarly increase sensitivity to isoflurane. In significantly compared to WT a1 GlyR (Fig. 1) except at agreement with our recent ethanol findings (Naito et al., 400 lM, and was lower than that of other a1 GlyR USERs. 2014), isoflurane produced concentration-dependent potentiation of EC2 glycine-induced currents in a1 GlyR USER 1 starting at 10 lM(Figs. 1 and 2). The left shift a1 GlyR USERs 1 and 2 have increased sensitivity to in the isoflurane concentration response relative to WT lidocaine a1 GlyRs (Fig. 1) is corroborated by a significant reduc- tion in EC50 compared to WT (Table 2). There was a sig- nificant reduction in the threshold for isoflurane sensitivity a1 GlyR WT. We tested the effects of lidocaine on WT from 100 M in WT 1 GlyRs to 10 M in 1 GlyR USER l a l a a1 GlyRs at concentrations ranging 0.1–100 lM. The 1. The magnitude of glycine response to isoflurane signifi- tested concentrations were based on clinical data, in cantly increased in a dose-dependent manner in 1 GlyR a which plasma toxicity in humans is found at USER 1 relative to WT 1 GlyRs (Fig. 1). a concentrations as low as 4.5 mg/kg (Miller, 2010). Lidocaine potentiated EC2 glycine-induced ClÀ currents a1 GlyR USER 2. Isoflurane produced concentration- in WT a1 GlyRs with significant effects starting at 1 lM dependent potentiation of EC2 glycine-induced currents in (Fig. 3). There were no significant effects of lidocaine on a1 GlyR USER 2 starting at 10 lM(Fig. 1), significantly these receptors at concentrations below 1 lM. A. Naito et al. / Neuroscience 297 (2015) 68–77 73

a1 GlyRs at 0.1 and 1 lM. However, the magnitude of lidocaine potentiation did not significantly differ from that of WT at lidocaine concentrations P10 lM.

a1 GlyR USER 3. Lidocaine produced concentration- dependent potentiation of EC2 glycine-induced currents starting at 1 lM in a1 GlyR USER 3 (Fig. 3), indicating no change in the threshold for lidocaine sensitivity compared to WT a1 GlyRs. The magnitude of glycine response to lidocaine across all tested concentrations also did not differ significantly compared to WT a1 GlyRs (Fig. 3), and was lower than that of other a1 GlyR USERs. Fig. 3. a1 GlyR USERs 1 and 2 have increased sensitivity to lidocaine compared to WT. Lidocaine-induced potentiation of glycine a1 GlyR USERs exhibit no significant changes in EC2-activated ClÀ currents in Xenopus oocytes expressing a1 GlyR propofol sensitivity compared to WT USERs are shown. Values for lidocaine potentiation are presented as percentage of glycine EC2 control. The glycine EC2 concentrations We tested a range of clinically relevant propofol utilized ranged from 5 to 10 lM in USERs and 18 to 25 lM for WT. concentrations ranging from 0.1 to 50 lM. While propofol The threshold for lidocaine sensitivity for a1 GlyR USERs 1 and 2 was 0.1 lM, and 1 lM for a1 GlyR WT and USER 3. Significant appeared to inhibit EC2 glycine-induced ClÀ currents in increases in the magnitude of lidocaine response for a1 GlyR USERs a1 GlyR WT and USERs 1, 2 and 3 at 0.1 lM(Fig. 4), compared to WT are denoted by ⁄p < 0.05. Each data point these effects were not statistically significant. Propofol represents the mean ± SEM from at least 6 to 9 oocytes. potentiated WT a1 GlyR currents starting at 0.5 lM in a concentration-dependent manner (Fig. 4). Repre- sentative tracings are shown in Fig. 5. In the current a1 GlyR USER 1. Lidocaine potentiated EC2 glycine- induced currents in a1 GlyR USER 1 starting at 0.1 lM study, no significant effects of direct receptor activation (Fig. 3), significantly reducing the threshold for lidocaine by propofol were observed across tested concentrations. sensitivity from 1 lM in WT a1 GlyRs to 0.1 lM in a1 There were no significant changes in the threshold for GlyR USER 1. The pattern of glycine response to propofol sensitivity relative to WT for all a1 GlyR USERs. lidocaine followed an inverted-U profile with a peak The magnitude of glycine response to propofol in a1 response at 1 lM(Fig. 3). Similar to our previous ethanol GlyR USER 1 did not significantly differ from that of findings (Naito et al., 2014) and our current isoflurane find- WT across all concentrations tested (Fig. 4). However, in ings, a1 GlyR USER 1 demonstrated increased sensitivity a1 GlyR USERs 2 and 3, the magnitude of glycine to lidocaine with respect to threshold and magnitude of response to propofol increased significantly from that of response (up to 100 lM) relative to WT a1 GlyRs. WT a1 GlyRs and USER 1 at concentrations P50 lM (Fig. 4). EC50 values indicate no significant differences among a1 GlyR WT and all USERs (Table 2). a1 GlyR USER 2. Lidocaine potentiated EC2 glycine- induced currents in 1 GlyR USER 2 starting at 0.1 M a l Loop 2 mutations exclusive to exon 4 of 1 GlyRs are (Fig. 3), significantly reducing the threshold for lidocaine a sufficient to increase ethanol, isoflurane and sensitivity 10-fold from 1 M in WT 1 GlyRs. At l a lidocaine sensitivity concentrations below 0.1 lM, there were no significant effects of lidocaine on these USERs. The magnitude of The Loop 2 region of a1 GlyRs spans both exons 3 and 4 lidocaine potentiation was significantly greater than WT (Table 1). Although Loop 2 mutations spanning exons 3

Fig. 4. a1 GlyR USERs show no changes to propofol sensitivity compared to WT. Propofol-induced potentiation of glycine EC2-activated ClÀ currents in Xenopus oocytes expressing a1 GlyR USERs are shown. Values for propofol potentiation are presented as percentage of glycine EC2 control. There were no significant changes in the threshold for propofol sensitivity between a1 GlyR USERs and WT (0.5 lM). However, there was a significant increase in the magnitude of propofol response in a1 GlyR USERs 2 and 3 compared to WT at 50 lM (denoted by ⁄p < 0.05). Each data point represents the mean ± SEM from at least 6 to 9 oocytes. 74 A. Naito et al. / Neuroscience 297 (2015) 68–77

Fig. 5. Representative tracings of a1 GlyR USERs in response to propofol. Two-electrode voltage clamp electrophysiology tracings of homomeric a1 GlyR WT and a1 GlyR USERs expressed in Xenopus oocytes in response to propofol. (A) a1 GlyR WT in response to 0.5 lM propofol; (B) a1 GlyR USER 1 in response to 0.5 lM propofol; (C) a1 GlyR USER 2 in response to 0.5 lM propofol; and (D) a1 GlyR USER 3 in response to 0.5 lM propofol. Effects of propofol were tested with EC2 glycine (5–25 lM) and the membrane potential was 70 mV. À and 4 of a1 GlyR USERs 1 and 2 significantly increased in the C-terminal end of Loop 2 (Table 1) are sufficient isoflurane and lidocaine sensitivity, Loop 2 mutations in to alter ethanol and anesthetic sensitivity in a1 GlyR the N-terminal region, exclusive to exon 3, in a1 GlyR USER 4. USER 3 did not significantly alter sensitivity to these As illustrated in Fig. 6A, ethanol significantly anesthetic agents with regards to the threshold and potentiated EC2 glycine-induced currents starting at magnitude of response relative to WT a1 GlyRs (Fig. 1). 0.25 mM. The magnitude of response of a1 GlyR USER Thus, we tested whether mutations exclusive to exon 4 4 in response to 0.25 mM ethanol did not significantly

Fig. 6. a1 GlyR USER 4 demonstrates increased sensitivity to ethanol, isoflurane, and lidocaine, but not propofol. a1 GlyR WT and USER 4 glycine EC2-activated ClÀ currents in response to (A) 0.25 mM and 50 mM ethanol; (B) isoflurane; (C) lidocaine; (D) propofol. The threshold for ethanol sensitivity was reduced from 50 mM in a1 GlyR WT to 0.25 mM in a1 GlyR USER 4. The threshold for isoflurane sensitivity was reduced from 100 lM in WT to 10 lM in a1 GlyR USER 4. The threshold for lidocaine sensitivity was reduced from 1 lM in WT to 0.1 lM in a1 GlyR USER 4. There were no significant changes to the threshold for propofol sensitivity and the magnitude of propofol response in a1 GlyR USER 4 relative to WT. Each data point represents the mean ± SEM from at least 6 to 9 oocytes. A. Naito et al. / Neuroscience 297 (2015) 68–77 75 differ from the response of WT a1 GlyRs exposed to 2 as a key site responsible for the enhancement of GlyR 50 mM ethanol (Fig. 6A). In addition, isoflurane function by ethanol, isoflurane, and lidocaine. produced concentration-dependent potentiation of EC2 The Loop 2 mutations spanning both exons 3 and 4 glycine-induced currents starting at 10 lM(Fig. 6B), (positions 50–59) in a1 GlyR USERs 1 and 2 that indicating a decrease in the threshold for isoflurane significantly increased ethanol sensitivity in prior studies sensitivity relative to WT a1 GlyRs. Furthermore, EC50 (Naito et al., 2014), also increased isoflurane and lido- significantly decreased in a1 GlyR USER 4, compared caine sensitivity relative to WT a1 GlyRs by demonstrat- to WT (Table 2). Thus, a1 GlyR USER 4 exhibited a ing a 10-fold decrease in threshold (Figs. 1 and 3). In similar increase in isoflurane sensitivity as that seen in contrast, Loop 2 mutations that are exclusive to the a1 GlyR USERs 1 and 2 with regard to the threshold, N-terminal region (exon 3) in a1 GlyR USER 3 shared but not the extent of the magnitude of response. similar characteristics in the threshold and magnitude of Although there was no incremental increase in the potentiation to WT a1 GlyRs in response to isoflurane magnitude of isoflurane potentiation relative to a1 GlyR and lidocaine (Figs. 1 and 3). Interestingly, the same USERs 1 and 2, the responses remained significantly mutations in exon 3 of a1 GlyR USER 3 in earlier studies greater than that of WT a1 GlyRs at 10 lM and 100 lM imparted ultra-sensitivity to ethanol and normalized the isoflurane (Figs. 1 and 6B). agonist sensitivity back to WT levels (Naito et al., 2014). Lidocaine produced concentration-dependent These findings suggest that certain regions within Loop potentiation of EC2 glycine-induced currents in a1 GlyR 2 may differentially regulate allosteric modulation of a1 USER 4 starting at 0.1 lM(Fig. 6C). Similar to a1 GlyR GlyRs. In fact, merely two point mutations, D57E and USERs 1 and 2, the threshold for lidocaine sensitivity R59T, at the C-terminal end of Loop 2 in exon 4 of a1 was reduced 10-fold relative to WT a1 GlyRs. At GlyR USER 4 were sufficient to significantly increase concentrations below 0.1 lM, there were no significant receptor sensitivity to ethanol, isoflurane, and lidocaine effects of lidocaine on these receptors. While the (Fig. 6A–C). Furthermore, the difference between main- magnitude of lidocaine potentiation increased in a1 taining the WT residues at positions 57 and 59 of the GlyR USER 4 relative to WT, these results were not C-terminal end of Loop 2 (USER 3), and mutating this significantly greater than those produced by USER 1 region (USER 4), seems to control isoflurane and lido- (Figs. 3 and 6C). In agreement with our initial findings caine enhancement of a1 GlyRs. Altogether, these with propofol, the threshold for propofol sensitivity, the results suggest that structural changes at the C-terminal EC50 (Table 2), as well as the magnitude of propofol end of Loop 2 appear to be a key determinant that is response in a1 GlyR USER 4 did not significantly differ both sufficient, and importantly, selective to increase from that of WT a1 GlyRs and other a1 GlyR USERs ethanol, isoflurane, and lidocaine sensitivity of a1 (Fig. 6D). GlyRs. Furthermore, this study is the first to report an extracellular site of action for lidocaine on a1 GlyRs. DISCUSSION Interestingly, there were no significant differences in the threshold for propofol sensitivity between WT and The present study tested the hypothesis that structural USER a1 GlyRs (Fig. 3). While propofol enhanced the manipulations of EC Loop 2 that affect ethanol sensitivity magnitude of potentiation of a1 GlyR USERs at of human a1 GlyR USERs would also affect sensitivity to concentrations P0.5 lM relative to WT, there were no the volatile anesthetic, isoflurane, but would not significant differences in the magnitude of propofol- significantly alter the effects of the intravenous induced potentiation of a1 GlyR USERs compared to WT anesthetic, propofol. We also tested the effects of at clinically relevant concentrations between 0.5 and lidocaine on these USERs to understand the possible 10 lM(Fig. 3). These findings indicate that the mutations sites and mechanisms of this local anesthetic on a1 in Loop 2 that increase ethanol, isoflurane, and/or GlyRs. This was accomplished by characterizing the lidocaine sensitivity in a1 GlyRs do not influence propofol selectivity of a1 GlyR USERs to volatile (isoflurane), sensitivity, and further reinforce findings by Krasowski local (lidocaine), and intravenous (propofol) anesthetics. and colleagues that the sites in the a2 or b1 subunits of In agreement with earlier findings, ethanol, isoflurane, GABAARs that abolish isoflurane potentiation do not and lidocaine sites of action do not appear to overlap affect propofol potentiation (Krasowski et al., 1998). More with those of propofol action on a1 GlyRs, as indicated importantly, these results highlight the selective nature of by the lack of change in the threshold for propofol Loop 2 mutations to increase a1 GlyR sensitivity to certain sensitivity of a1 GlyR USERs with respect to WT a1 anesthetic agents, but not all. Recent studies have GlyRs (Figs. 1, 3, 4 and 6). These findings suggest that reported propofol sites at the interface between the EC the sites of propofol action in a1 GlyRs are distinct from and TM 2 domains, specifically between adjacent TM 2 those of ethanol and isoflurane, reinforcing similar helices in the b3 subunit of GABAARs (Franks, 2015). findings reported in the TM domain of GABAARs Our current findings suggest that EC Loop 2 is not a critical (Krasowski et al., 1998; Bali and Akabas, 2004). Overall, region for propofol action and modulation of a1 GlyRs. our findings confirm that Loop 2 plays an important role The distinguishing features that account for the in mediating ethanol, isoflurane, and lidocaine action by increase in sensitivity to isoflurane and lidocaine in a1 decreasing the threshold for sensitivity and increasing GlyR USERs 1, 2, and 4 (relative to WT and USER 3) the magnitude of a1 GlyR response to these agents. appear to be attributed to the differences between the Furthermore, we identified the C-terminal region of Loop C-terminal regions of Loop 2. As reported earlier, the 76 A. Naito et al. / Neuroscience 297 (2015) 68–77 charged residue arginine R59 at the C-terminal end of excitability and neurochemical cascades in certain brain Loop 2 increases the propensity to form salt-bridge regions in the presence of low anesthetic concentrations. interactions with N-terminal residues across Loop 2, for Thus, testing these USERs in vivo would provide unam- instance, with the histidine H50 in a1 GlyR USER 3 biguous results about the role of individual receptor sub- (Naito et al., 2014). Thus, the introduction of charged resi- units in transducing the effects of anesthetics without dues at the C-terminal end may contribute to con- concerns about collateral effects on other nervous sys- formational changes to the structure of Loop 2, tems that often confound these studies. preventing accessibility of allosteric modulators from act- ing at this site. Further studies should explore the gating CONCLUSIONS properties as a result of these Loop 2 mutations on USERs to determine the functional changes induced in Collectively, our results demonstrate that the Loop 2 region a1 GlyR USERs. of a1 GlyRs is a site of ethanol, isoflurane, and lidocaine Through a1 GlyR USER 4, we demonstrate that action. Moreover, we demonstrate that subtle structural mutations within a single exon create receptors with manipulations (D57E, R59T) at the C-terminal end of high sensitivity to several anesthetic molecules. These Loop 2 are sufficient to significantly increase ethanol, findings could potentially provide a discrete DNA isoflurane, and lidocaine sensitivity in a1 GlyR USER 4. sequence for searching databases for other receptors Our results also add to the body of literature that sites of that might share high sensitivity to and ethanol, isoflurane, and lidocaine action do not overlap anesthetics. This goal is important because, so far, only with those of propofol. Importantly, these findings demonstrate the selectivity of 1 GlyR USERs in that GABAARs containing delta subunits have exhibited a sensitivity to alcohol in the range of mild intoxication increases in receptor sensitivity are not solely limited to (18 mM) (Wallner et al., 2003; Olsen et al., 2007). It is ethanol, but also apply to isoflurane and lidocaine. The possible that there are undiscovered receptors that share common structural homology among the Cys-loop the high alcohol sensitivity of USERs; these receptors superfamily of receptors implies that similar increases in might help explain the constellation of effects on coordina- anesthetic sensitivity via Loop 2 may also apply across tion, balance, fear conditioning, and memory that corre- multiple receptors and receptor subunits. Moreover, the sponds to mild intoxication. advantage of harboring mutations to a single exon in In agreement with earlier studies, the in vivo Loop 2 may promote genetic incorporation to develop expression of USERs in genetically engineered animals genetically engineered USER knock-in animals. may provide a novel tool for investigation of single Ultimately, USERs allow activation of specific receptor receptor subunit populations sensitized to extremely low subunits at concentrations that do not affect WT anesthetic concentrations that do not affect other receptors, and could potentially provide a valuable tool to receptors in the nervous system. While the current understand the functional role of specific receptors in study tested a1 GlyR USER sensitivity to anesthetics in mediating the behavioral effects of anesthetic action. the presence of EC2 agonist concentrations, a recent study reported that the effects of ethanol potentiation AUTHORSHIP CONTRIBUTIONS (30 mM) diminish in the presence of higher agonist Participated in research design: Alkana, Naito, Muchhala, concentrations (EC ) in 4 1 GABA Rs (Bowen et al., 50 a b d A Asatryan, Davies. Conducted experiments: Naito, 2015), suggesting the possibility that the effects of Muchhala, Trang. Performed data analysis: Naito, USER potentiation may be diminished in vivo. However, Muchhala, Trang. Wrote or contributed to the writing of a recent investigation reported a glycine-dependent the manuscript: Alkana, Naito, Muchhala, Asatryan, mechanism of regulating neuronal excitability in the orbi- Trudell, Homanics, Davies. tofrontal cortex (OFC) by ethanol exposure, providing the first line of evidence that ethanol-induced potentiation of GlyRs are the primary mediators of inhibitory projec- CONFLICT OF INTEREST STATEMENT tions in the OFC (Badanich et al., 2013). Importantly, The authors declare no conflict of interest. while both GABAARs and GlyRs are expressed in OFC , GABAARs are not sensitive to behaviorally rele- Acknowledgments—This work was supported in whole or in part, vant concentrations of ethanol. Thus, the inhibitory pro- by the National Institutes of Health, National Institute on jections induced by ethanol in the OFC are glycine- Alcohol Abuse and Alcoholism [Grants AA10422, AA020980, dependent and not regulated through GABAAR activity AA17243, AA022448]; American Foundation for Pharmaceutical (Badanich et al., 2013). Furthermore, application of the Education; and the USC School of Pharmacy. We thank Miriam selective GlyR antagonist, , did not significantly Fine for technical assistance in molecular biology; Dr. Curtis alter tonic glycine-mediated currents; thus implicating the T. Okamoto for guidance and review; Yihui Wang, Sahil Patel and Layla Demirchyan for laboratory assistance. existence of extrasynaptic GlyRs, and GlyR transporters that maintain low levels of extracellular glycine (Badanich et al., 2013). Additional work reported similar REFERENCES findings of extrasynaptic GlyRs in the dorsal raphe nucleus that largely govern ethanol-induced tonic inhibi- Aguayo LG, Castro P, Mariqueo T, Munoz B, Xiong W, Zhang L, tion (Maguire et al., 2014). In light of these findings, Lovinger DM, Homanics GE (2014) Altered effects of GlyR USERs have the potential to significantly affect ethanol in mice with alpha1 subunits that are A. Naito et al. / Neuroscience 297 (2015) 68–77 77

insensitive to Gbetagamma modulation. Neuropsycho- Maguire EP, Mitchell EA, Greig SJ, Corteen N, Balfour DJ, Swinny pharmacology 39:2538–2548. JD, Lambert JJ, Belelli D (2014) Extrasynaptic glycine receptors Antognini JF, Schwarz K (1993) Exaggerated anesthetic of rodent dorsal raphe serotonergic neurons: a sensitive target for requirements in the preferentially anesthetized brain. ethanol. Neuropsychopharmacology 39:1232–1244. Anesthesiology 79:1244–1249. Mascia MP, Machu TL, Harris RA (1996) Enhancement of homomeric Badanich KA, Mulholland PJ, Beckley JT, Trantham-Davidson H, glycine receptor function by long-chain alcohols and anaesthetics. Woodward JJ (2013) Ethanol reduces neuronal excitability of Br J Pharmacol 119:1331–1336. lateral orbitofrontal cortex neurons via a glycine receptor Mascia MP, Trudell JR, Harris RA (2000) Specific binding sites for dependent mechanism. Neuropsychopharmacology alcohols and anesthetics on ligand-gated ion channels. Proc Natl 38:1176–1188. Acad Sci USA 97:9305–9310. Bali M, Akabas MH (2004) Defining the propofol binding site location Mihic SJ, Ye Q, Wick MJ, Koltchine VV, Krasowski MD, Finn SE, on the GABA A receptor. Mol Pharm 65:68–76. Mascia MP, Valenzuela CF, Hanson KK, Greenblatt EP, Harris Bowen MT, Peters ST, Absalom N, Chebib M, Neumann ID, RA, Harrison NL (1997) Sites of alcohol and volatile anaesthetic McGregor IS (2015) Oxytocin prevents ethanol actions at delta action on GABA(A) and glycine receptors. Nature 389:385–389. subunit-containing GABAA receptors and attenuates ethanol- Miller RD (2010) Miller’s anesthesia. In: Miller RD, editor. Miller’s induced motor impairment in rats. Proc Natl Acad Sci U S A. anesthesia, vol. 7. Philadelphia, PA: Churchill Livingstone/ Crawford DK, Perkins DI, Trudell JR, Bertaccini EJ, Davies DL, Elsevier. p. 491–522. Alkana RL (2008) Roles for loop 2 residues of alpha1 glycine Moraga-Cid G, Yevenes GE, Schmalzing G, Peoples RW, Aguayo receptors in agonist activation. J Biol Chem 283:27698–27706. LG (2011) A Single phenylalanine residue in the main intracellular Crawford DK, Trudell JR, Bertaccini E, Li KX, Davies DL, Alkana RL loop of alpha1 gamma-aminobutyric acid type A and glycine (2007) Evidence that ethanol acts on a target in Loop 2 of the receptors influences their sensitivity to propofol. Anesthesiology extracellular domain of alpha1 glycine receptors. J Neurochem 115:464–473. 102:2097–2109. Naito A, Muchhala KH, Asatryan L, Trudell JR, Homanics GE, Cummins TR (2007) Setting up for the block: the mechanism Perkins DI, Davies DL, Alkana RL (2014) Glycine and GABA(A) underlying lidocaine’s use-dependent inhibition of sodium ultra-sensitive ethanol receptors as novel tools for alcohol and channels. J Physiol 582:11. brain research. Mol Pharm 86:635–646. Davies DL, Crawford DK, Trudell JR, Mihic SJ, Alkana RL (2004) Nury H, Van Renterghem C, Weng Y, Tran A, Baaden M, Dufresne V, Multiple sites of ethanol action in alpha1 and alpha2 glycine Changeux JP, Sonner JM, Delarue M, Corringer PJ (2011) X-ray receptors suggested by sensitivity to pressure antagonism. J structures of general anaesthetics bound to a pentameric ligand- Neurochem 89:1175–1185. gated ion channel. Nature 469:428–431. Davies DL, Trudell JR, Mihic SJ, Crawford DK, Alkana RL (2003) Olsen RW, Hanchar HJ, Pratap M, Wallner M (2007) GABA A Ethanol potentiation of glycine receptors expressed in Xenopus receptor subtypes: the ‘‘one glass of wine’’ receptors. Alcohol oocytes antagonized by increased atmospheric pressure. Alcohol 41:201–209. Clin Exp Res 27:1–13. Ortells MO, Lunt GG (1995) Evolutionary history of the ligand-gated Downie DL, Hall AC, Lieb WR, Franks NP (1996) Effects of ion-channel superfamily of receptors. Trends Neurosci inhalational general anaesthtetics on native glycine receptors in 18:121–127. rat medullary neurones and recombinant glycine receptors in Perkins DI, Trudell JR, Asatryan L, Davies DL, Alkana RL (2012) Xenopus ooctyes. BrJPharmacol 118:493–502. Charge and geometry of residues in the loop 2 beta hairpin Dutertre S, Becker CM, Betz H (2012) Inhibitory glycine receptors: an differentially affect agonist and ethanol sensitivity in glycine update. J Biol Chem 287:40216–40223. receptors. J Pharmacol Exp Ther 341:543–551. Franks NP (2015) Structural comparisons of ligand-gated ion Perkins DI, Trudell JR, Crawford DK, Alkana RL, Davies DL (2008) channels in open, closed, and desensitized states identify a Targets for ethanol action and antagonism in loop 2 of the novel propofol-binding site on mammalian gamma-aminobutyric extracellular domain of glycine receptors. J Neurochem acid type A receptors. Anesthesiology. 106:1337–1349. Franks NP, Lieb WR (1994) Molecular and cellular mechanisms of Perkins DI, Trudell JR, Crawford DK, Asatryan L, Alkana RL, Davies . Nature 367:607–614. DL (2009) Loop 2 structure in glycine and GABAA receptors plays Hara K, Sata T (2007) The effects of the local anesthetics lidocaine a key role in determining ethanol sensitivity. J Biol Chem and procaine on glycine and gamma-aminobutyric acid receptors 284:27304–27314. expressed in Xenopus oocytes. Anesth Analg 104:1434–1439. Rajendra S, Lynch JW, Schofield PR (1997) The glycine receptor. Krasowski MD, Harrison NL (1999) actions on Pharmacol Therapeut 73:121–146. ligand-gated ion channels. Cell Mol Life Sci 55:1278–1303. Sauguet L, Howard RJ, Malherbe L, Lee US, Corringer PJ, Harris RA, Krasowski MD, Harrison NL (2000) The actions of ether, alcohol and Delarue M (2013) Structural basis for potentiation by alcohols alkane general anaesthetics on GABA A and glycine receptors and anaesthetics in a ligand-gated ion channel. Nat Commun and the effects of TM2 and TM3 mutations. Br J Pharmacol 4:1697. 129:731–743. Wallner M, Hanchar HJ, Olsen RW (2003) Ethanol enhances alpha 4 Krasowski MD, Koltchine VV, Rick CE, Ye Q, Finn SE, Harrison NL beta 3 delta and alpha 6 beta 3 delta gamma-aminobutyric acid (1998) Propofol and other intravenous anesthetics have sites of type A receptors at low concentrations known to affect humans. action on the g-aminobutyric acid type A receptor distinct from Proc Natl Acad Sci USA 100:15218–15223. that for isoflurane. Mol Pharm 53:530–538. Williams KL, Ferko AP, Barbieri EJ, Di Gregorio GJ (1995) Glycine Krasowski MD, Nishikawa K, Nikolaeva N, Lin A, Harrison NL (2001) enhances the central properties of ethanol in mice. Methionine 286 in transmembrane domain 3 of the GABAA Pharmacol Biochem Behav 50:199–205. receptor beta subunit controls a binding cavity for propofol and Xiu X, Hanek AP, Wang J, Lester HA, Dougherty DA (2005) A unified other alkylphenol general anesthetics. Neuropharmacology view of the role of electrostatic interactions in modulating the 41:952–964. gating of cys loop receptors. J Biol Chem 280:41655–41666. Legendre P (2001) The inhibitory synapse. Cell Mol Life Ye JH, Sokol KA, Bhavsar U (2009) Glycine receptors contribute to Sci 58:760–793. hypnosis induced by ethanol. Alcohol Clin Exp Res 33: Lynch JW (2004) Molecular structure and function of the glycine 1069–1074. receptor . Physiol Rev 84:1051–1095.

(Accepted 17 March 2015) (Available online 28 March 2015)