Glycine Receptors in Spinal Nociceptive Control-An Update.

Diminished inhibitory control of spinal nociception is one of the major culprits of chronic pain states. Restoring proper synaptic inhibition is a well-established rational therapeutic approach explored by several pharmaceutical companies. A particular challenge arises from the need for site-specific intervention to avoid deleterious side effects such as sedation, addiction, or impaired motor control, which would arise from wide-range facilitation of inhibition. Specific targeting of glycinergic inhibition, which dominates in the spinal cord and parts of the hindbrain, may help reduce these side effects. Selective targeting of the α3 subtype of glycine receptors (GlyRs), which is highly enriched in the superficial layers of the spinal dorsal horn, a key site of nociceptive processing, may help to further narrow down pharmacological intervention on the nociceptive system and increase tolerability. This review provides an update on the physiological properties and functions of α3 subtype GlyRs and on the present state of related drug discovery programs.

Modulation of Glycinergic Neurotransmission may Contribute to the Analgesic Effects of Propacetamol.

Treating neuropathic pain remains challenging, and therefore new pharmacological strategies are urgently required. Here, the enhancement of glycinergic neurotransmission by either facilitating glycine receptors (GlyR) or inhibiting glycine transporter (GlyT) function to increase extracellular glycine concentration appears promising. Propacetamol is a N,N-diethylester of acetaminophen, a non-opioid analgesic used to treat mild pain conditions. In vivo, it is hydrolysed into N,N-diethylglycine (DEG) and acetaminophen. DEG has structural similarities to known alternative GlyT1 substrates. In this study, we analyzed possible effects of propacetamol, or its metabolite N,N-diethylglycine (DEG), on GlyRs or GlyTs function by using a two-electrode voltage clamp approach in Xenopus laevis oocytes. Our data demonstrate that, although propacetamol or acetaminophen had no effect on the function of the analysed glycine-responsive proteins, the propacetamol metabolite DEG acted as a low-affine substrate for both GlyT1 (EC50 > 7.6 mM) and GlyT2 (EC50 > 5.2 mM). It also acted as a mild positive allosteric modulator of GlyRα1 function at intermediate concentrations. Taken together, our data show that DEG influences both glycine transporter and receptor function, and therefore could facilitate glycinergic neurotransmission in a multimodal manner.

The intracellular domain of homomeric glycine receptors modulates agonist efficacy.

Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyREM, the agonists glycine, ß-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyREM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyREM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyREM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for the ICD in pentameric ligand-gated channels.

Mechanism of gating and partial agonist action in the glycine receptor.

Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter binding. Neurotransmitters that produce maximum open channel probabilities (Po) are full agonists, whereas those that yield lower than maximum Po are partial agonists. Cys-loop receptors are an important class of neurotransmitter receptors, yet a structure-based understanding of the mechanism of partial agonist action has proven elusive. Here, we study the glycine receptor with the full agonist glycine and the partial agonists taurine and γ-amino butyric acid (GABA). We use electrophysiology to show how partial agonists populate agonist-bound, closed channel states and cryo-EM reconstructions to illuminate the structures of intermediate, pre-open states, providing insights into previously unseen conformational states along the receptor reaction pathway. We further correlate agonist-induced conformational changes to Po across members of the receptor family, providing a hypothetical mechanism for partial and full agonist action at Cys-loop receptors.

The anticonvulsant zonisamide positively modulates recombinant and native glycine receptors at clinically relevant concentrations.

GABAA and glycine receptors mediate fast synaptic inhibitory neurotransmission. Despite studies showing that activation of cerebral glycine receptors could be a potential strategy in the treatment of epilepsy, few studies have assessed the effects of existing anticonvulsant therapies on recombinant or native glycine receptors. We, therefore, evaluated the actions of a series of anticonvulsants at recombinant human homo-oligomeric glycine receptor α1, α2 and α3 subtypes expressed in Xenopus oocytes using two-electrode voltage-clamp methods, and then assessed the most effective drug at native glycine receptors from entorhinal cortex neurons using whole-cell voltage-clamp recordings. Ganaxolone, tiagabine and zonisamide positively modulated glycine induced currents at recombinant homomeric glycine receptors. Of these, zonisamide was the most efficacious and exhibited an EC50 value ranging between 450 and 560 µM at α1, α2 and α3 subtypes. These values were not significantly different indicating a non-selective modulation of glycine receptors. Using a therapeutic concentration of zonisamide (100 µM), the potency of glycine was significantly shifted from 106 to 56 µM at α1, 185 to 112 µM at α2, and 245 to 91 µM at α3 receptors. Furthermore, zonisamide (100 µM) potentiated exogenous homomeric and heteromeric glycine mediated currents from layer II pyramidal cells of the lateral or medial entorhinal cortex. As therapeutic concentrations of zonisamide positively modulate recombinant and native glycine receptors, we propose that the anticonvulsant effects of zonisamide may, at least in part, be mediated via this action.

Identification of the hypertension drug niflumic acid as a glycine receptor inhibitor.

Glycine is one of the major neurotransmitters in the brainstem and the spinal cord. Glycine binds to and activates glycine receptors (GlyRs), increasing Cl- conductance at postsynaptic sites. This glycinergic synaptic transmission contributes to the generation of respiratory rhythm and motor patterns. Strychnine inhibits GlyR by binding to glycine-binding site, while picrotoxin blocks GlyR by binding to the channel pore. We have previously reported that bath application of strychnine to zebrafish embryos causes bilateral muscle contractions in response to tactile stimulation. To explore the drug-mediated inhibition of GlyRs, we screened a chemical library of ~ 1,000 approved drugs and pharmacologically active molecules by observing touch-evoked response of zebrafish embryos in the presence of drugs. We found that exposure of zebrafish embryos to nifedipine (an inhibitor of voltage-gated calcium channel) or niflumic acid (an inhibitor of cyclooxygenase 2) caused bilateral muscle contractions just like strychnine-treated embryos showed. We then assayed strychnine, picrotoxin, nifedipine, and niflumic acid for concentration-dependent inhibition of glycine-mediated currents of GlyRs in oocytes and calculated IC50s. The results indicate that all of them concentration-dependently inhibit GlyR in the order of strychnine > picrotoxin > nifedipine > niflumic acid.

Cholesterol regulates cannabinoid analgesia through glycine receptors.

Cholesterol plays vital roles in many central physiological and pathological processes. As a key component in the cell membrane, cholesterol can regulate a variety of ion channels, including ligand-gated ion channels (LGICs). However, relatively little is known about the molecular detail and in vivo consequence of cholesterol-LGIC interaction. Here, we reveal that membrane cholesterol depletion significantly inhibits the potentiating effects of dehydroxylcannabidiol (DH-CBD) on glycine-activated currents (IGly) in HEK 293T cells expressing α1/α3 glycine receptors (GlyRs). Simvastatin considerably decreases cholesterol levels and DH-CBD-induced potentiation of IGly in the spinal cord of mice. Simvastatin also significantly decreases DH-CBD analgesia in acute and chronic pain of mice. The cholesterol levels in the dorsal horn of spinal cord, measured by mass spectrometry imaging, are specifically correlated with cannabinoid potentiation of spinal GlyRs and cannabinoid-induced analgesia. These findings suggest that spinal cholesterol is critical for the efficacy of glycinergic cannabinoid-induced analgesia.

Taurine Suppression of Central Amygdala GABAergic Inhibitory Signaling via Glycine Receptors Is Disrupted in Alcohol Dependence.

BACKGROUND: Alcohol use disorder (AUD) increases brain stress systems while suppressing reward system functioning. One expression of stress system recruitment is elevated GABAergic activity in the central amygdala (CeA), which is involved in the excessive drinking seen with AUD. The sulfonic amino acid taurine, a glycine receptor partial agonist, modulates GABAergic activity in the rewarding effects of alcohol. Despite taurine abundance in the amygdala, its role in the dysregulation of GABAergic activity associated with AUD has not been studied. Thus, here, we evaluated the effects of taurine on locally stimulated GABAergic neurotransmission in the CeA of naïve- and alcohol-dependent rats. METHODS: We recorded intracellularly from CeA neurons of naïve- and alcohol-dependent rats, quantifying locally evoked GABAA receptor-mediated inhibitory postsynaptic potentials (eIPSP). We examined the effects of taurine and alcohol on CeA eIPSP to characterize potential alcohol dependence-induced changes in the effects of taurine. RESULTS: We found that taurine decreased amplitudes of eIPSP in CeA neurons of naïve rats, without affecting the acute alcohol-induced facilitation of GABAergic responses. In CeA neurons from dependent rats, taurine no longer had an effect on eIPSP, but now blocked the ethanol (EtOH)-induced increase in eIPSP amplitude normally seen. Additionally, preapplication of the glycine receptor-specific antagonist strychnine blocked the EtOH-induced increase in eIPSP amplitude in neurons from naïve rats. CONCLUSIONS: These data suggest taurine may act to oppose the effects of acute alcohol via the glycine receptor in the CeA of naïve rats, and this modulatory system is altered in the CeA of dependent rats.

Expression of glycine receptor α3 subunit in neuropathic and inflammatory central pain sensitization

INTRODUCTION: Increasing evidence suggests that changes in the balance of excitatory/inhibitory neurotransmission are involved in the development of the majority of chronic pain forms. In this context, impairment in glycine mediated inhibitory neurotransmission is thought to play a critical role in the disinhibition that accounts for the development and maintenance of central pain hypersensitivity. AIMS: The goal of this study was to evaluate the Glycine Receptor α3 subunit (α3GlyR) expression in neuropathic (Chronic Constriction Injury, CCI) and inflammatory (Zymosan A injected) animal models of chronic pain. RESULTS AND CONCLUSION: RT-qPCR analysis of spinal cord samples showed that glra3 gene expression does not change after 3 days of CCI and 4 hours of Zymosan A injection. However, we found that protein levels evaluated by Western blot increased after inflammatory pain. These data suggest that central sensitization is differentially regulated depending on the type of pain. α3GlyR protein expression plays an important role in the first step of inflammatory pain establishment.

Identification of pathways that regulate circadian rhythms using a larval zebrafish small molecule screen.

The circadian clock ensures that behavioral and physiological processes occur at appropriate times during the 24-hour day/night cycle, and is regulated at both the cellular and organismal levels. To identify pathways acting on intact animals, we performed a small molecule screen using a luminescent reporter of molecular circadian rhythms in zebrafish larvae. We identified both known and novel pathways that affect circadian period, amplitude and phase. Several drugs identified in the screen did not affect circadian rhythms in cultured cells derived from luminescent reporter embryos or in established zebrafish and mammalian cell lines, suggesting they act via mechanisms absent in cell culture. Strikingly, using drugs that promote or inhibit inflammation, as well as a mutant that lacks microglia, we found that inflammatory state affects circadian amplitude. These results demonstrate a benefit of performing drug screens using intact animals and provide novel targets for treating circadian rhythm disorders.

Activation of glycine receptors in the lateral habenula rescues anxiety- and depression-like behaviors associated with alcohol withdrawal and reduces alcohol intake in rats.

The lateral habenula (LHb) is activated by a range of aversive states including those related to alcohol withdrawal and has glycine receptors (GlyRs), a sensitive target of alcohol. However, whether GlyRs in the LHb contribute to alcohol-related behaviors is unknown. Here, we report that rats experiencing withdrawal from chronic alcohol consumption showed higher anxiety and sensitivity to stress compared to their alcohol-naïve counterparts. Intra-LHb injection of glycine attenuated these aberrant behaviors and reduced alcohol intake upon alcohol re-access. Glycine's effect was blocked by strychnine, a GlyR antagonist, indicating that it was mediated by strychnine-sensitive GlyRs. Conversely, intra-LHb strychnine elicited anxiety- and depression-like behaviors in Naïve rats but not in withdrawal rats. Additionally, both the frequency and the amplitude of the spontaneous IPSCs were lower in LHb neurons in slices of withdrawal rats compared to naïve rats. Also, there were sporadic strychnine-sensitive synaptic events in some LHb neurons. Bath perfusion of strychnine induced a depolarizing inward current and increased action potential firings in LHb neurons. By contrast, bath perfusion of glycine or sarcosine, a glycine transporter subtype 1 inhibitor, inhibited LHb activity. Collectively, these data reveal that LHb neurons are under the tonic glycine inhibition both in physiological and pathological conditions. Activation of GlyRs reverses LHb hyperactivity, alleviates aberrant behaviors, and reduces alcohol intake, thus highlighting the GlyRs in the LHb as a potential therapeutic target for alcohol-use disorders.

Diverse small molecules prevent macrophage lysis during pyroptosis.

Pyroptosis is a programmed process of proinflammatory cell death mediated by caspase-1-related proteases that cleave the pore-forming protein, gasdermin D, causing cell lysis and release of inflammatory intracellular contents. The amino acid glycine prevents pyroptotic lysis via unknown mechanisms, without affecting caspase-1 activation or pore formation. Pyroptosis plays a critical role in diverse inflammatory diseases, including sepsis. Septic lethality is prevented by glycine treatment, suggesting that glycine-mediated cytoprotection may provide therapeutic benefit. In this study, we systematically examined a panel of small molecules, structurally related to glycine, for their ability to prevent pyroptotic lysis. We found a requirement for the carboxyl group, and limited tolerance for larger amino groups and substitution of the hydrogen R group. Glycine is an agonist for the neuronal glycine receptor, which acts as a ligand-gated chloride channel. The array of cytoprotective small molecules we identified resembles that of known glycine receptor modulators. However, using genetically deficient Glrb mutant macrophages, we found that the glycine receptor is not required for pyroptotic cytoprotection. Furthermore, protection against pyroptotic lysis is independent of extracellular chloride conductance, arguing against an effect mediated by ligand-gated chloride channels. Finally, we conducted a small-scale, hypothesis-driven small-molecule screen and identified unexpected ion channel modulators that prevent pyroptotic lysis with increased potency compared to glycine. Together, these findings demonstrate that pyroptotic lysis can be pharmacologically modulated and pave the way toward identification of therapeutic strategies for pathologic conditions associated with pyroptosis.

Ultrapotent chemogenetics for research and potential clinical applications.

Chemogenetics enables noninvasive chemical control over cell populations in behaving animals. However, existing small-molecule agonists show insufficient potency or selectivity. There is also a need for chemogenetic systems compatible with both research and human therapeutic applications. We developed a new ion channel-based platform for cell activation and silencing that is controlled by low doses of the smoking cessation drug varenicline. We then synthesized subnanomolar-potency agonists, called uPSEMs, with high selectivity for the chemogenetic receptors. uPSEMs and their receptors were characterized in brains of mice and a rhesus monkey by in vivo electrophysiology, calcium imaging, positron emission tomography, behavioral efficacy testing, and receptor counterscreening. This platform of receptors and selective ultrapotent agonists enables potential research and clinical applications of chemogenetics.

Cochlear ablation in neonatal rats disrupts inhibitory transmission in the medial nucleus of the trapezoid body.

Inhibitory circuits in the auditory brainstem undergo multiple postnatal changes that are both activity-dependent and activity-independent. We tested to see if the shift from GABA- to glycinergic transmission, which occurs in the rat medial nucleus of the trapezoid body (MNTB) around the onset of hearing, depends on sound-evoked neuronal activity. We prevented the activity by bilateral cochlear ablations in early postnatal rats and studied ionotropic GABA and glycine receptors in MNTB neurons after hearing onset. The removal of the cochlea decreased responses of GABAA and glycine receptors to exogenous agonists as well as the amplitudes of inhibitory postsynaptic currents. The reduction was accompanied by a decrease in the number of glycine receptor- or vesicular GABA transporter-immunopositive puncta. Furthermore, the ablations markedly affected the switch in presynaptic GABAA to glycine receptors. The increase in the expression of postsynaptic glycine receptors and the shift in inhibitory transmitters were not prevented. The results suggest that inhibitory transmission in the MNTB is subject to multiple developmental signals and support the idea that auditory experience plays a role in the maturation of the brainstem glycinergic circuits.

Ketone body modulation of ligand-gated ion channels.

Ketogenesis is a metabolic process wherein ketone bodies are produced from the breakdown of fatty acids. In humans, fatty acid catabolism results in the production of acetyl-CoA which can then be used to synthesize three ketone bodies: acetoacetate, acetone, and ß-hydroxybutyrate. Ketogenesis occurs at a higher rate in situations of low blood glucose, such as during fasting, heavy alcohol consumption, and in situations of low insulin, as well as in individuals who follow a 'ketogenic diet' consisting of low carbohydrate and high fat intake. This diet has various therapeutic indications, including reduction of seizure likelihood in epileptic patients and alcohol withdrawal syndrome. However, the mechanisms underlying these therapeutic benefits are still unclear, with studies suggesting various mechanisms such as a shift in energy production in the brain, effects on neurotransmitter production, or effects on various protein targets. Two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes was used to investigate the actions of ketone bodies on three ionotropic receptors: GABAA, glycine, and NMDA receptors. While physiologically-relevant concentrations of acetone have little effect on inhibitory GABA or glycine receptors, ß-hydroxybutyrate inhibits the effects of agonists of these receptors at concentrations achieved in vivo. Additionally, both acetone and ß-hydroxybutyrate act as inhibitors of glutamate at the excitatory NMDA receptor. Due to the role of hyperexcitability in the pathogenesis of epilepsy and alcohol withdrawal, the inhibitory actions of acetone and ß-hydroxybutyrate at NMDA receptors may underlie the therapeutic benefit of a ketogenic diet for these disorders.

Plasminogen binding inhibitors demonstrate unwanted activities on GABAA and glycine receptors in human iPSC derived neurons.

Plasminogen binding inhibitors (PBIs) reduce the risk of bleeding in hemorrhagic conditions. However, generic PBIs are also associated with an increased risk of seizures, an adverse effect linked to unwanted activities towards inhibitory neuronal receptors. Development of novel PBIs serve to remove compounds with such properties, but progress is limited by a lack of higher throughput methods with human translatability. Herein we apply human induced pluripotent stem cell (hiPSC) derived neurons in combination with dynamic mass redistribution (DMR) technology to demonstrate robust and reproducible modulation of both GABAA and glycine receptors. These cells respond to GABA (EC50 0.33 ±â€¯0.18 µM), glycine (EC50 11.0 ±â€¯3.7 µM) and additional ligands in line with previous reports from patch clamp technologies. Additionally, we identify and characterize a competitive antagonistic behavior of the prototype inhibitor and drug tranexamic acid (TXA). Finally, we demonstrate proof of concept for effective counter-screening of lead series compounds towards unwanted GABAA receptor activities. No activity was observed for a previously identified PBI candidate drug, AZD6564, whereas a discontinued analog, AZ13267257, could be characterized as a potent GABAA receptor agonist.

The endogenous agonist, ß-alanine, activates glycine receptors in rat spinal dorsal neurons.

ß-alanine is a structural analog of glycine and γ-aminobutyric acid (GABA) and is thought to be involved in the modulation of nociceptive information at the spinal cord. However, it is not known whether ß-alanine exerts its effect in substantia gelatinosa (SG) neurons of the spinal dorsal horn, where glycine and GABA play an important role in regulating nociceptive transmission from the periphery. Here, we investigated the effects of ß-alanine on inhibitory synaptic transmission in adult rat SG neurons using whole-cell patch-clamp. ß-alanine dose-dependently induced outward currents in SG neurons. Current-voltage plots revealed a reversal potential at approximately -70 mV, which was close to the equilibrium potential of Cl-. Pharmacological analysis revealed that ß-alanine activates glycine receptors, but not GABAA receptors. These results suggest that ß-alanine hyperpolarizes the membrane potential of SG neurons by activating Cl- channels through glycine receptors. Our findings raise the possibility that ß-alanine may modulate pain sensation through glycine receptors.

Single Channel Analysis of Isoflurane and Ethanol Enhancement of Taurine-Activated Glycine Receptors.

The amino acid taurine is an endogenous ligand acting on glycine receptors (GlyRs), which is released by astrocytes in many brain regions, such as the nucleus accumbens and prefrontal cortex. Taurine is a partial agonist with an efficacy significantly lower than that of glycine. Allosteric modulators such as ethanol and isoflurane produce leftward shifts of glycine concentration-response curves but have no effects at saturating glycine concentrations. In contrast, in whole-cell electrophysiology studies these modulators increase the effects of saturating taurine concentrations. A number of possible mechanisms may explain these enhancing effects, including modulator effects on conductance, channel open times, or channel closed times. We used outside-out patch-clamp single channel electrophysiology to investigate the mechanism of action of 200 mM ethanol and 0.55 mM isoflurane in enhancing the effects of a saturating concentration of taurine. Neither modulator enhanced taurine-mediated conductance. Isoflurane increased the probability of channel opening. Isoflurane also increased the lifetimes of the two shortest open dwell times while both agents decreased the likelihood of occurrence of the longest-lived intracluster channel-closing events. The mechanism of enhancement of GlyR functioning by these modulators is dependent on the efficacy of the agonist activating the receptor and the concentration of agonist tested.

Probing the Structural Mechanism of Partial Agonism in Glycine Receptors Using the Fluorescent Artificial Amino Acid, ANAP.

The efficacy of an agonist at a pentameric ligand-gated ion channel is determined by the rate at which it induces a conformational change from the resting closed state to a preopen ("flip") state. If the ability of an agonist to promote this isomerization is sufficiently low, then it becomes a partial agonist. As partial agonists at pentameric ligand-gated ion channels show considerable promise as therapeutics, understanding the structural basis of the resting-flip-state isomerization may provide insight into therapeutic design. Accordingly, we sought to identify structural correlates of the resting-flip conformational change in the glycine receptor chloride channel. We used nonsense suppression to introduce the small, fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP), into specific sites in the extracellular and transmembrane domains. Then, under voltage-clamp conditions in Xenopus oocytes, we simultaneously quantified current and fluorescence responses induced by structurally similar agonists with high, medium, and low efficacies (glycine, ß-alanine, and taurine, respectively). Analyzing results from nine ANAP-incorporated sites, we show that glycine receptor activation by agonists with graded efficacies manifests structurally as correspondingly graded movements of the ß1-ß2 loop, the ß8-ß9 loop, and the Cys-loop from the extracellular domain and the TM2-TM3 linker in the transmembrane domain. We infer that the resting-flip transition involves an efficacy-dependent molecular reorganization at the extracellular-transmembrane domain interface that primes receptors for efficacious opening.

Interactions between Zinc and Allosteric Modulators of the Glycine Receptor.

The glycine receptor is a pentameric ligand-gated ion channel that is involved in fast inhibitory neurotransmission in the central nervous system. Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nanomolar to low-micromolar concentrations and inhibiting its effects at higher concentrations. Low-nanomolar concentrations of contaminating zinc in electrophysiological buffers are capable of synergistically enhancing receptor modulation by other compounds, such as ethanol. This suggests that, unless accounted for, previous studies of glycine receptor modulation were measuring the effects of modulator plus comodulation by zinc on receptor function. Since zinc is present in vivo at a variety of concentrations, it will influence glycine receptor modulation by other pharmacologic agents. We investigated the utility of previously described "zinc-enhancement-insensitive" α1 glycine receptor mutants D80A, D80G, and W170S to probe for interactions between zinc and other allosteric modulators at the glycine receptor. We found that only the W170S mutation conferred complete abolishment of zinc enhancement across a variety of agonist and zinc concentrations. Using α1 W170S receptors, we established that, in addition to ethanol, zinc interacts with inhalants, but not volatile anesthetics, to synergistically enhance channel function. Additionally, we determined that this interaction is abolished at higher zinc concentrations when receptor-enhancing binding sites are saturated, suggesting a mechanism by which modulators such as ethanol and inhalants are capable of increasing receptor affinity for zinc, in addition to enhancing channel function on their own.