Sulfonylurea Inhibitor Research Articles

Novel chemotype NLRP3 inhibitors that target the CRID3-binding pocket with high potency.

The NLRP3 inflammasome plays a central role in various human diseases. Despite significant interest, most clinical-grade NLRP3 inhibitors are derived from sulfonylurea inhibitor CRID3 (also called MCC950). Here, we describe a novel chemical class of NLRP3-inhibiting compounds (NIC) that exhibit potent and selective NLRP3 inflammasome inhibition in human monocytes and mouse macrophages. BRET assays demonstrate that they physically interact with NLRP3. Structural modeling further reveals they occupy the same binding site of CRID3 but in a critically different conformation. Furthermore, we show that NIC-11 and NIC-12 lack the off-target activity of CRID3 against the enzymatic activity of carbonic anhydrases I and II. NIC-12 selectively reduces circulating IL-1ß levels in the LPS-endotoxemia model in mice and inhibits NLRP3 inflammasome activation in CAPS patient monocytes and mouse macrophages with about tenfold increased potency compared with CRID3. Altogether, this study unveils a new chemical class of highly potent and selective NLRP3-targeted inhibitors with a well-defined molecular mechanism to complement existing CRID3-based NLRP3 inhibitors in pharmacological studies and serve as novel chemical leads for the development of NLRP3-targeted therapies.

High Activity and Easily Hydrolyzable Sulfonylurea Inhibitor Design Based on Density Functional Theory Calculations

To find new sulfonylurea inhibitors with high efficacy and fast hydrolysis degradation rate, a few compounds were first designed based on the commercial product Chlorimuron-Ethyl (CE) by estimating the binding interaction between the inhibitor and the Acetohydroxyacid Synthase (AHAS) using the quantum mechanical approach. Meanwhile, the activation energy barriers of hydrolysis for the sulfonylurea inhibitors with the amino and nitro groups onto para position of the benzene ring were calculated. Based on the calculated binding interaction energy and hydrolysis energy barrier, six new sulfonylurea inhibitors I1–I6 were designed and synthesized. By measuring the half-lives through hydrolysis degradation assay, it was indicated that the compounds I1–I3 with the introduction of an amino group at the fourth position of benzene ring show much faster degradation rate than those compounds with nitro groups, which is in a good agreement with the calculated results for hydrolysis barrier. The herbicide activity tests show that the compounds I1 and I2 remained excellent herbicidal activity on both broadleaf weeds with soil treatment at a concentration about 150[Formula: see text]mg/l. Due to their short half-lives of chemical hydrolysis and high herbicidal activities, compounds I1 and I2 could be potential herbicidal candidates in the future, which are helpful for the sustainable development of the environment and ecology.

Glibenclamide attenuates 2,5-hexanedione-induced neurotoxicity in the spinal cord of rats through mitigation of NLRP3 inflammasome activation, neuroinflammation and oxidative stress

Chronic exposure to n-hexane, a widely used solvent in industry, causes sensorimotor neuropathy, which is mainly mediated by its toxic metabolite, 2,5-hexanedione (HD). However, the mechanisms remain unclear. This study is designed to investigate whether nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is involved in HD-induced neurotoxicity. Results showed that HD intoxication significantly elevated NLRP3 expression, caspase-1 activation and interleukin-1β (IL-1β) maturation in the spinal cord of rats, indicating NLRP3 inflammasome activation. Glibenclamide, a sulfonylurea inhibitor of NLRP3 inflammasome, reduced HD-induced NLRP3 inflammasome activation, which was associated with mitigated gasdermin D (GSDMD) cleavage, neurofilament protein L (NF-L) reduction and demyelination as well as axon degeneration in the spinal cord of rats. Subsequently, we found that inhibition of NLRP3 inflammasome by glibenclamide suppressed microglial activation and M1 polarization and simultaneously recovered M2 polarization in HD-intoxicated rats. Furthermore, glibenclamide treatment reduced the contents of malondialdehyde (MDA) as well as elevated glutathione (GSH) levels and total-antioxidative capacity in the spinal cord of HD-intoxicated rats, indicating attenuated oxidative stress. Collectively, our findings suggested that NLRP3 inflammasome activation contributed to HD-induced neurotoxicity by enhancing microglial M1 polarization and oxidative damage. Inhibition of NLRP3 inflammasome by glibenclamide might a potential avenue to combat n-hexane-induced neuropathy.

Integrin CD11b mediates locus coeruleus noradrenergic neurodegeneration in a mouse Parkinson\u2019s disease model

BackgroundThe loss of locus coeruleus noradrenergic (LC/NE) neurons in the brainstem is reported in multiple neurodegenerative disorders, including Parkinson’s disease (PD). However, the mechanisms remain unclear. Strong evidence suggested that microglia-mediated neuroinflammation contributes to neurodegeneration in PD. We recently recognized integrin CD11b, the α-chain of macrophage antigen complex-1 (Mac-1, also called CR3), as a key regulator for microglial activation. However, whether CD11b is involved in LC/NE neurodegeneration in PD remains to be investigated.MethodsLC/NE neurodegeneration and microglial activation were compared between wild type (WT) and CD11b KO mice after treated with paraquat and maneb, two pesticides that widely used to create PD model. The role of NLRP3 inflammasome in CD11b-mediated microglial dysfunction and LC/NE neurodegeneration was further explored. LC/NE neurodegeneration, microglial phenotype, and NLRP3 inflammasome activation were determined by using Western blot, immunohistochemistry, and RT-PCR technologies.ResultsParaquat and maneb co-exposure elevated the expressions of CD11b in the brainstem of mice, and CD11b knockout significantly reduced LC/NE neurodegeneration induced by paraquat and maneb. Mitigated microglial activation and gene expressions of proinflammatory cytokines were also observed in paraquat and maneb-treated CD11b−/− mice. Mechanistically, CD11b-mediated NLRP3 inflammasome activation contributes to paraquat and maneb-induced LC/NE neurodegeneration. Compared with WT controls, CD11b deficiency reduced paraquat and maneb-induced NLRP3 expression, caspase-1 activation, and interleukin-1β production in mice. Furthermore, inhibition of NLRP3 inflammasome by glybenclamide, a sulfonylurea inhibitor of NLRP3 inflammasome, was found to be able to suppress microglial proinflammatory activation and nuclear factor-κB activation induced by paraquat and maneb. Moreover, reduced reactive oxygen species production, NADPH oxidase, and inducible nitric oxide synthase expressions as well as 4-hydroxynonenal and malondialdehyde levels were detected in combined glybenclamide and paraquat and maneb-treated mice compared with paraquat and maneb alone group. Finally, we found that glybenclamide treatment ameliorated LC/NE neurodegeneration and α-synuclein aggregation in paraquat and maneb-treated mice.ConclusionOur findings suggested that CD11b mediates LC/NE neurodegeneration through NLRP3 inflammation-dependent microglial proinflammatory activation in a two pesticide-induced mouse PD model, providing a novel insight into the immune pathogenesis of LC/NE neuronal damage in related disorders.

Computational Identification of Novel Kir6 Channel Inhibitors.

KATP channels consist of four Kir6.x pore–forming subunits and four regulatory sulfonylurea receptor (SUR) subunits. These channels couple the metabolic state of the cell to membrane excitability and play a key role in physiological processes such as insulin secretion in the pancreas, protection of cardiac muscle during ischemia and hypoxic vasodilation of arterial smooth muscle cells. Abnormal channel function resulting from inherited gain or loss-of-function mutations in either the Kir6.x and/or SUR subunits are associated with severe diseases such as neonatal diabetes, congenital hyperinsulinism, or Cantú syndrome (CS). CS is an ultra-rare genetic autosomal dominant disorder, caused by dominant gain-of-function mutations in SUR2A or Kir6.1 subunits. No specific pharmacotherapeutic treatment options are currently available for CS. Kir6 specific inhibitors could be beneficial for the development of novel drug therapies for CS, particular for mutations, which lack high affinity for sulfonylurea inhibitor glibenclamide. By applying a combination of computational methods including atomistic MD simulations, free energy calculations and pharmacophore modeling, we identified several novel Kir6.1 inhibitors, which might be possible candidates for drug repurposing. The in silico predictions were confirmed using inside/out patch-clamp analysis. Importantly, Cantú mutation C166S in Kir6.2 (equivalent to C176S in Kir6.1) and S1020P in SUR2A, retained high affinity toward the novel inhibitors. Summarizing, the inhibitors identified in this study might provide a starting point toward developing novel therapies for Cantú disease.

Conserved functional consequences of disease-associated mutations in the slide helix of Kir6.1 and Kir6.2 subunits of the ATP-sensitive potassium channel

Cantu syndrome (CS) is a condition characterized by a range of anatomical defects, including cardiomegaly, hyperflexibility of the joints, hypertrichosis, and craniofacial dysmorphology. CS is associated with multiple missense mutations in the genes encoding the regulatory sulfonylurea receptor 2 (SUR2) subunits of the ATP-sensitive K+ (KATP) channel as well as two mutations (V65M and C176S) in the Kir6.1 (KCNJ8) subunit. Previous analysis of leucine and alanine substitutions at the Val-65-equivalent site (Val-64) in Kir6.2 indicated no major effects on channel function. In this study, we characterized the effects of both valine-to-methionine and valine-to-leucine substitutions at this position in both Kir6.1 and Kir6.2 using ion flux and patch clamp techniques. We report that methionine substitution, but not leucine substitution, results in increased open state stability and hence significantly reduced ATP sensitivity and a marked increase of channel activity in the intact cell irrespective of the identity of the coassembled SUR subunit. Sulfonylurea inhibitors, such as glibenclamide, are potential therapies for CS. However, as a consequence of the increased open state stability, both Kir6.1(V65M) and Kir6.2(V64M) mutations essentially abolish high-affinity sensitivity to the KATP blocker glibenclamide in both intact cells and excised patches. This raises the possibility that, at least for some CS mutations, sulfonylurea therapy may not prove to be successful and highlights the need for detailed pharmacogenomic analyses of CS mutations.

Glibenclamide: A second wind for refractory hyperkalemia.

Sir, In the clinical case reported by Sodhi et al.[1] the authors have discussed heparin-induced hyperkalemia, a less known problem,[2] and expressed the nonresponsiveness to conventional antihyperkalemic measures. Under such circumstances, we would like to recall the role of sulfonylurea inhibitor glibenclamide[3] in the reversal of refractory hyperkalemia. This is a novel approach that succeeded remarkably despite failure of all conventional treatments and it accords with interpretation of blockade of excessive KATP channel activity. The KATP channels are composed of two components, an inwardly rectifying potassium channel (Kir) pore subunit and the regulatory sulfonylurea-receptor (SUR). KATP channels are present in various tissues, including the pancreas, kidney, heart, skeletal muscle and vascular smooth muscle, and it opens in response to hypoxia, metabolic acidosis, and hypercapnia. However, glibenclamide could close the KATP channels after binding to SURs.[4] Thereby, it prevents the exit of potassium from intracellular to extracellular. Based on the aforementioned mechanisms, one may speculate that glibenclamide may be an alternative for those with refectory hyperkalemia. When no other alternatives are available, naturally one may try to use a well-established and time honored therapeutic molecule glibenclamide. However, more studies/reports are warranted to establish the usefulness of glibenclamide in refractory hyperkalemia. Another aspect is that cardioversion may not be effective in the presence of hyperkalemia. Despite all, the issues related to refractory hyperkalemia are less discussed and rarely considered in education and practice. Therefore, practitioners and emergency physicians shall be made aware of off label use of glibenclamide, while handling a case of refectory refractory hyperkalemia.

Refractory hyperkalemia related to heparin abuse.

Sir, We read with interest the case report of refractory hyperkalemia due to heparin abuse.[1] An effect of heparin is hypoaldosteronism with hyperkalemia caused by direct inhibition of aldosterone biosynthesis and by inhibition of angiotensin-II with secondary hypoaldosteronism.[2,3] In present case hyperkalemia persisted irrespective of calcium gluconate, dextrose insulin, fludrocortisone, salbutamol nebulizers and slow low efficiency renal dialysis.[1] Patient had acidosis, hypotension, septicemia and died of cardiac arrest.[1] The potassium-adenosine triphosphate (K-ATP) is a poor inwardly rectifying channel consisting of pore-forming and sulfonylurea - receptor subunit. The pores confer ATP inhibition while the sulfonylurea receptor is the primary target for sulfonylureas, K-ATP channel openers, and nucleoside diaphosphates.[2] Hypoxia, metabolic acidosis and hypercapnia activates the K-ATP channels, resulting in vasodilatation of coronary, mesenteric, renal and smooth muscle bed and increases potassium efflux and modulates many of the kidney transport functions and maintains external potassium balance.[3] The potency of sulfonylurea drugs in antagonizing vasorelaxant action of K-ATP channel stimulation after the sepsis or endotoxin is well recognized in the laboratory model.[2] Reversal of the life threatening complications of hyperkalemia, vasodilator shock and severe bradycardia by the sulfonylurea inhibitor glibenclamide is a novel approach to the treatment of refractory hyperkalemia.[4] In present case refractory hyperkalemia would have been rectified by glibenclamide.[1,4] The authors working at critical care at tertiary care institute thus could have tried glibenclamide in their case for better outcome.[1,4]

DFT Study of Binding Energies Between Acetohydroxyacid Synthase and Its Sulfonylurea Inhibitors: An Application of Quantum Pseudoreceptor Model

The quantum mechanical interaction energy between the Acetohydroxyacid synthase (AHAS) and its sulfonylurea inhibitors were calculated with an efficient density functional theory (DFT) and a pseudoreceptor model composed of the amino acids surrounding the ligands. The results show that the calculated quantum mechanical interaction energies correlate well with experimental free energies with the correlation coefficients of 0.92 for six sulfonylurea inhibitors and the standard deviation of 0.83kcal/mol. In comparison with the force field method, the binding free energies were estimated by AutoDock 4.2 program with the correlation coefficient of 0.76 and the standard deviation of 1.40kcal/mol. It indicates that the binding between the AHAS and herbicides can be well characterized by quantum pseudoreceptor model. Based on the quantum mechanical interaction energies, some AHAS inhibitors with high binding affinity were designed by introducing a hydroxyl group at the para position of aromatic ring and on the sulfonylurea bridge respectively. AMS subject classifications: 92E10, 92C05 * Corresponding author. Email address: wangml@szu.edu.cn (M.-L. Wang) M. Chen et. al / Commun. Comput. Chem., 1 (2013), pp. 72-87 73

Glibenclamide Reduces Hippocampal Injury and Preserves Rapid Spatial Learning in a Model of Traumatic Brain Injury

Cognitive disturbances after traumatic brain injury (TBI) are frequent, even when neuroimaging shows no overt hemorrhagic or other abnormality. Sulfonylurea receptor 1 (SUR1) plays a key role in various forms of CNS injury, but its role in hippocampal dysfunction after mild to moderate TBI is unknown. To assess the hypothesis that postinjury SUR1 upregulation in the hippocampus is associated with a later disturbance in learning, we studied a rat model of cortical impact TBI calibrated to avoid primary and secondary hemorrhage in the underlying hippocampus. The transcription factor, specificity protein 1, which regulates expression of SUR1 and caspase-3, was activated in the hippocampus 15 minutes after injury. Upregulation of SUR1 protein and of Abcc8 (which encodes SUR1) messenger RNA was evident by 6 hours. To assess the role of SUR1, injured rats were administered vehicle or a low dose of the specific sulfonylurea inhibitor glibenclamide for 1 week. At 2 weeks, the increase in activated caspase-3 in the hilus of glibenclamide-treated rats was half of that in vehicle-treated rats. Testing for rapid learning in a Morris water maze at 4 weeks showed significantly better performance in glibenclamide-treated rats; performance inversely correlated with Fluoro-Jade staining for degenerated neurons in the hilus. We conclude that glibenclamide may have long-term protective effects on the hippocampus after mild-to-moderate TBI.

Insight into herbicide resistance of W574L mutant Arabidopsis thaliana acetohydroxyacid synthase: molecular dynamics simulations and binding free energy calculations

Acetohydroxyacid synthase (AHAS) is the target enzyme of several classes of herbicides, such as sulfonylureas and imidazolinones. Now many mutant AHASs with herbicide resistance have emerged along with extensive use of herbicides, therefore it is imperative to understand the detailed interaction mechanism and resistance mechanism so as to develop new potent inhibitors for wild-type or resistant AHAS. With the aid of available crystal structures of the Arabidopsis thaliana (At) AHAS-inhibitor complex, molecular dynamics (MD) simulations were used to investigate the interaction and resistance mechanism directly and dynamically at the atomic level. Nanosecond-level MD simulations were performed on six systems consisting of wild-type or W574L mutant AtAHAS in the complex with three sulfonylurea inhibitors, separately, and binding free energy was calculated for each system using the MM-GBSA method. Comprehensive analyses from structural and energetic aspects confirmed the importance of residue W574, and also indicated that W574L mutation might alert the structural charactersistic of the substrate access channel and decrease the binding affinity of inhibitors, which cooperatively weaken the effective channel-blocked effect and finally result in weaker inhibitory effect of inhibitor and corresponding herbicide resistance of W574L mutant. To our knowledge, it is the first report about MD simulations study on the AHAS-related system, which will pave the way to study the interactions between herbicides and wild-type or mutant AHAS dynamically, and decipher the resistance mechanism at the atomic level for better designing new potent anti-resistance herbicides.

Molecular modelling of Mycobacterium tuberculosis acetolactate synthase catalytic subunit and its molecular docking study with inhibitors

Mycobacterium tuberculosis is a leading cause of infectious disease in the world today. This outlook is aggravated by a growing number of M. tuberculosis infections in individuals who are immunocompromised as a result of HIV infections. Thus, new and more potent anti-TB agents are necessary. Therefore, acetolactate synthase (mtALS) was selected as a target enzyme to combat M. tuberculosis. In this work, the three-dimensional molecular model of the hypothetical structure for the ALS catalytic subunit of M. tuberculosis was elucidated by homology modelling. In addition, the orientations and binding affinities of sulfonylurea inhibitors with the new structure was investigated. Our findings could be helpful for the design of new, more potent mtAHAS inhibitors.

Reducing Cytotoxic Edema after Cerebral Injury by Targeting the NCCa-ATP Channel

Cerebral edema complicates the management of a wide range of afflictions of the central nervous system; indeed, our efforts to limit its damaging effects often supersede treatment of the underlying pathophysiology. While the commonly used treatment regimens of osmotic diuresis are well-described, and other options like decompressive hemicraniectomy in extreme cases continue to be debated, what's clear is that there is significant room for additional weaponry in our therapeutic arsenal. In April's Nature Medicine (12:433–40, 2006) Simard et al. give us a glimpse of how we may attack this pathologic entity in the coming years, extending their previous body of work in which they have characterized the nonselective NCCa-ATP channel (J Neurosci. 23:8568–8577, 2003), so named as it requires Ca2+ for opening and is activated by depletion of intracellular ATP; SUR1 forms its regulatory subunit and confers sensitivity to sulfonylurea inhibitors such as glibenclamide. Having demonstrated that opening of NCCa-ATP channels causes depolarization and membrane blebbing characteristic of cytotoxic edema, the authors hypothesized that newly-expressed NCCa-ATP channels could constitute a mechanism for Na+ flux required for edema formation. The NCCa-ATP channel was first discovered in hypoxic gliotic tissues; this, coupled with its activation by low intracellular AT P, suggested that other causes of hypoxia may trigger channel expression. As such, the authors investigated NCCa-ATP and SUR1 expression in a rat model of middle cerebral artery occlusion (MCAO), showing first that SUR1 is upregulated in the infarct core and later in the peri-infarct regions (Figure 1) in astrocytes, neurons, and endothelial cells; moreover, NCCa-ATP channels were upregulated in astrocytes and neurons, while KATP channels—with which SUR1 is also associated—were not, suggesting a specificity for NCCa-ATP channels in ischemia. They then showed that inhibiting the channel with glibenclamide reduced ion flow through this channel and prevented cell death. Lastly, they demonstrated the efficacy of subcutaneously- delivered glibenclamide at reducing brain water and infarct volume after stroke and extending animal survival in their rodent MCAO model (Figure 2). Taken together, they show increased expression of the NCCa-ATP channel and its regulatory subunit after stroke; that glibenclamide blocks conductance through this channel; and that blocking the channel in vivo after stroke limits the extent of cerebral edema, reduces infarct volume and spares the cortical ribbon, and improves animal survival.Figure 1: Increased SUR1 expression after middle cerebral artery occlusion.Figure 2: Reduced infarct volume in glib enclamide-treated animal (right) compared to control (left). Note cortical sparing.The therapeutic implications of this work are clear, particularly since glibenclamide has been used safely for decades in the treatment of NIDDM. Limiting cerebral edema by targeting the NCCa-ATP channel will have benefit not only in stroke but also in trauma and in a range of other conditions in which cerebral edema contributes to morbidity and mortality. IAN F. DUNN, M.D. ROBERT M. FRIEDLANDER, M.D., M.A. BASIC SCIENCE RESEARCH

State-dependent Inhibition of the Mitochondrial KATP Channel by Glyburide and 5-Hydroxydecanoate

The mitochondrial KATP channel (mitoKATP) is hypothesized to be the receptor for the cardioprotective effects of K+ channel openers (KCO) and for the blocking of cardioprotection by glyburide and 5-hydroxydecanoate (5-HD). Studies on glyburide have indicated that this drug is inactive in isolated mitochondria. No studies of the effects of 5-HD on isolated mitochondria have been reported. This paper examines the effects of glyburide and 5-HD on K+ flux in isolated, respiring mitochondria. We show that mitoKATP is completely insensitive to glyburide and 5-HD under the experimental conditions in which the open state of the channel is induced by the absence of ATP and Mg2+. On the other hand, mitoKATP became highly sensitive to glyburide and 5-HD when the open state was induced by Mg2+, ATP, and a physiological opener, such as GTP, or a pharmacological opener, such as diazoxide. In these open states, glyburide (K1/2 values 1-6 microM) and 5-HD (K1/2 values 45-75 microM) inhibited specific, mitoKATP-mediated K+ flux in both heart and liver mitochondria from rat. These results are consistent with a role for mitoKATP in cardioprotection and show that different open states of mitoKATP, although catalyzing identical K+ fluxes, exhibit very different susceptibilities to channel inhibitors.

Quantum molecular similarity via momentum-space indices

A method for assessing quantum molecular similarity has been developed based on the momentum‐space representation. The principles of the method are summarized and the results of two applications are presented. The approach emphasizes the variation of the outer valence electron density much more than the bonding topology, it avoids several problems associated with more conventional approaches based on the position‐space representation, and it can now be applied to extended series of large molecules. The first of the applications involves different sulfonylurea inhibitors of the enzyme acyl‐CoA : cholesterol acyltransferase (ACAT). The second is concerned with the relative toxicity of a number of anti‐HIV phospholipids. Further experimental work is suggested in both cases.

Inhibitors of acyl-coa:cholesterol acyltransferase (ACAT). 15. sulfonylurea inhibitors with excellent hypocholesterolemic activity in vivo.

A series of sulfonylureas were prepared and tested for the ability to inhibit the enzyme acyl-CoA: cholesterol acyltransferase (ACAT) in vitro and lower plasma cholesterol in cholesterol-fed rats in vivo. Although compounds from this series were generally weak inhibitors of ACAT in vitro, several displayed excellent hypocholesterolemic activity in vivo.

Putative pre- and postsynaptic ATP-sensitive potassium channels in the rat substantia nigra in vitro

Pre- and postsynaptic adenosine 5'-triphosphate-sensitive potassium (ATP-K+) currents were studied using whole-cell recordings from substantia nigra zona compacta "principal" neurons in midbrain slices. The GABAA and GABAB receptor-mediated synaptic potentials were unaffected by the ATP-K+ channel inhibitor glibenclamide (30 microM) or by the opener diazoxide (500 microM), indicating that ATP-K+ channels on GABA-ergic terminals are not active, nor can they be activated pharmacologically, under control conditions. However, application of a glucose-free solution to reduce intracellular ATP levels caused a reduction of the GABAB IPSP in all neurons. This was substantially reversed by the sulfonylurea inhibitor tolbutamide (300 microM) in 50% of the neurons tested. The reduction of the GABAB IPSP was a presynaptic effect since postsynaptic hyperpolarizations induced by the GABAB receptor agonist baclofen (10 microM) were unaffected by glucose-free solutions. Diazoxide (500 microM) induced a slowly developing hyperpolarization or outward current in 64% of principal neurons, which was tolbutamide- (100-300 microM) or glibenclamide- (30 microM) sensitive. In contrast, the GABAB receptor agonist baclofen (30 microM) induced a rapid hyperpolarization or outward current in all neurons tested that was unaffected by tolbutamide (300 microM). Although both the diazoxide-induced current and the baclofen-induced current were inhibited by Ba2+ (300 microM), the currents elicited by diazoxide and baclofen summated. The reversal potential for the diazoxide-induced current was also less negative than that for baclofen, which was close to EK. In the presence of intracellular cesium, diazoxide induced a tolbutamide-sensitive inward current in a proportion of neurons, indicating that it has other actions in addition to activating a potassium current. Our results suggest that functional ATP-K+ channels exist both pre- and postsynaptically in the SN, where they modulate the activity of principal neurons. They are different to the potassium channels activated by the GABAB receptor agonist baclofen.