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Sulfonylureas, widely used as hypoglycemic agents in adults with type 2 diabetes, have neuroprotective effects in preclinical models of central nervous system injury, and in children with neuropsychomotor impairments linked to neonatal diabetes secondary to ATP-sensitive potassium channel mutations. In the human and rodent retina, we show that the glibenclamide-activated channel sulfonylurea receptor 1 (SUR1) is expressed in the retina and enriched in the macula; we also show that it colocalizes with the potassium channel Kir6.2, and with the cation channel transporter TRPM4. Glibenclamide (glyburide), administered at doses that did not decrease the glycemia, or injected directly into the eye, protected the structure and the function of the retina in various models of retinal injury that recapitulate the pathogenic neurodegenerative events in the diabetic retina. The downregulation of SUR1 using a siRNA suppressed the neuroprotective effects of glibenclamide on excitotoxic stress-induced cell death. The glibenclamide effects include the transcriptional regulation of antioxidant and neuroprotective genes. Ocular glibenclamide could be repurposed for diabetic retinopathy.

Aims/hypothesis Congenital hyperinsulinism caused by mutations in the K-ATP-channel-encoding genes (KATPHI) is a potentially life-threatening disorder of the pancreatic beta cells. No optimal medical treatment is available for patients with diazoxide-unresponsive diffuse KATPHI. Therefore, we aimed to create a model of KATPHI using patient induced pluripotent stem cell (iPSC)-derived islets. Methods We derived iPSCs from a patient carrying a homozygous ABCC8(V187D) mutation, which inactivates the sulfonylurea receptor 1 (SUR1) subunit of the K-ATP-channel. CRISPR-Cas9 mutation-corrected iPSCs were used as controls. Both were differentiated to stem cell-derived islet-like clusters (SC-islets) and implanted into NOD-SCID gamma mice. Results SUR1-mutant and -corrected iPSC lines both differentiated towards the endocrine lineage, but SUR1-mutant stem cells generated 32% more beta-like cells (SC-beta cells) (64.6% vs 49.0%, p = 0.02) and 26% fewer alpha-like cells (16.1% vs 21.8% p = 0.01). SUR1-mutant SC-beta cells were 61% more proliferative (1.23% vs 0.76%, p = 0.006), and this phenotype could be induced in SUR1-corrected cells with pharmacological K-ATP-channel inactivation. The SUR1-mutant SC-islets secreted 3.2-fold more insulin in low glucose conditions (0.0174% vs 0.0054%/min, p = 0.0021) and did not respond to K-ATP-channel-acting drugs in vitro. Mice carrying grafts of SUR1-mutant SC-islets presented with 38% lower fasting blood glucose (4.8 vs 7.7 mmol/l, p = 0.009) and their grafts failed to efficiently shut down insulin secretion during induced hypoglycaemia. Explanted SUR1-mutant grafts displayed an increase in SC-beta cell proportion and SC-beta cell nucleomegaly, which was independent of proliferation. Conclusions/interpretation We have created a model recapitulating the known pathophysiology of KATPHI both in vitro and in vivo. We have also identified a novel role for K-ATP-channel activity during human islet development. This model will enable further studies for the improved understanding and clinical management of KATPHI without the need for primary patient tissue.

Sulfonylurea receptor 1 (SUR1) is the regulatory subunit of ATP-sensitive potassium channels (KATP channels) and the receptor of antidiabetic drugs, such as glibenclamide, which induce insulin secretion in pancreatic β cells. However, the expression and role of SUR1 in cancer are unknown. In this study, we found that SUR1 expression was elevated in human non–small cell lung carcinoma (NSCLC) tissues and cell lines. SUR1 silencing suppressed the growth of NSCLC cells, while SUR1 overexpression promoted cell growth. Targeting SUR1 with glibenclamide suppressed cell growth, cell-cycle progression, epithelial–mesenchymal transition (EMT), and cell migration. Moreover, SUR1 directly interacted with p70S6K and upregulated p70S6K phosphorylation and activity. In addition, glibenclamide inhibited p70S6K, and overexpression of p70S6K partially reversed the growth-inhibiting effect of glibenclamide. Furthermore, glibenclamide upregulated the expression of the tumor suppressor Krüppel-like factor 4 (KLF4), and silencing KLF4 partially reversed the inhibitory effect of glibenclamide on cell growth, EMT, and migration. We found that SUR1 targeted p70S6K to downregulate KLF4 expression by enhancing DNA-methyltransferase 1–mediated methylation of the KLF4 promoter. Finally, in xenograft mouse models, SUR1 expression silencing or glibenclamide treatment inhibited the growth of A549 tumors, downregulated p70S6K activity, and upregulated KLF4 expression. These findings suggested that SUR1 expression was elevated in some NSCLC tissues and functioned as a tumor enhancer. Targeting SUR1 with glibenclamide inhibited NSCLC through downregulation of p70S6K activity and subsequent upregulation of the expression of the tumor suppressor gene KLF4. SUR1 can be developed as a new target for cancer therapy and glibenclamide has potential anticancer effects.

K(ATP) channels are metabolic sensors that translate intracellular ATP/ADP balance into membrane excitability. The molecular composition of K(ATP) includes an inward-rectifier potassium channel (Kir) and an ABC transporter–like sulfonylurea receptor (SUR). Although structures of K(ATP) have been determined in many conformations, in all cases, the pore in Kir is closed. Here, we describe human pancreatic K(ATP) (hK(ATP)) structures with an open pore at 3.1- to 4.0-Å resolution using single-particle cryo-electron microscopy (cryo-EM). Pore opening is associated with coordinated structural changes within the ATP-binding site and the channel gate in Kir. Conformational changes in SUR are also observed, resulting in an area reduction of contact surfaces between SUR and Kir. We also observe that pancreatic hK(ATP) exhibits the unique (among inward-rectifier channels) property of PIP(2)-independent opening, which appears to be correlated with a docked cytoplasmic domain in the absence of PIP(2).

ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.

Hinchazón celular; Edema; Traumatismo cerebral Cellular swelling; Edema; Traumatic brain injury Inflor cel·lular; Edema; Traumatisme cerebral Sulfonylurea receptor 1 (SUR1) is a member of the adenosine triphosphate (ATP)-binding cassette (ABC) protein superfamily, encoded by Abcc8, and is recognized as a key mediator of central nervous system (CNS) cellular swelling via the transient receptor potential melastatin 4 (TRPM4) channel. Discovered approximately 20 years ago, this channel is normally absent in the CNS but is transcriptionally upregulated after CNS injury. A comprehensive review on the pathophysiology and role of SUR1 in the CNS was published in 2012. Since then, the breadth and depth of understanding of the involvement of this channel in secondary injury has undergone exponential growth: SUR1-TRPM4 inhibition has been shown to decrease cerebral edema and hemorrhage progression in multiple preclinical models as well as in early clinical studies across a range of CNS diseases including ischemic stroke, traumatic brain injury, cardiac arrest, subarachnoid hemorrhage, spinal cord injury, intracerebral hemorrhage, multiple sclerosis, encephalitis, neuromalignancies, pain, liver failure, status epilepticus, retinopathies and HIV-associated neurocognitive disorder. Given these substantial developments, combined with the timeliness of ongoing clinical trials of SUR1 inhibition, now, another decade later, we review advances pertaining to SUR1-TRPM4 pathobiology in this spectrum of CNS disease—providing an overview of the journey from patch-clamp experiments to phase III trials. No funding directly supported the writing of this review. R.M.J. is supported by grants from the National Institute of Neurological Disorders and Stroke (NINDS) (K23NS101036; R01NS115815), and the Barrow Neurological Foundation. J.M.S. is supported by grants from the Department of Veterans Affairs (I01RX003060; 1I01BX004652), the Department of Defense (SC170199), the National Heart, Lung and Blood Institute (R01HL082517) and the NINDS (R01NS102589; R01NS105633).

Sulfonylurea receptor-1 (SUR1) is recognized increasingly as a key contributor to cerebral edema, hemorrhage progression, and possibly neuronal death in multiple forms of acute brain injury. SUR1 inhibition may be protective and is actively undergoing evaluation in Phase-2/3 trials of traumatic brain injury (TBI) and stroke. In adult TBI, SUR1 expression is associated with intracranial hypertension and contusion expansion; its role in pediatric TBI remains unexplored. We tested 61 cerebrospinal fluid (CSF) samples from 16 pediatric patients with severe TBI enrolled in the multicenter Phase-3 randomized controlled “Cool Kids” trial and seven non-brain injured pediatric controls for SUR1 expression by enzyme-linked immunosorbent assay. Linear mixed models evaluated associations between mean SUR1 and intracranial pressure (ICP) over the first seven days and pediatric Glasgow Outcome Scale-Extended (GOS-E Peds) over the initial year after injury. SUR1 was undetectable in control CSF and increased versus control in nine of 16 patients with TBI. Mean SUR1 was not associated with age, sex, or therapeutic hypothermia. Each 1-point increase in initial Glasgow Coma Score was associated with a 1.68 ng/mL decrease in CSF SUR1. The CSF SUR1 was associated with increased ICP over seven days (b = 0.73, p = 0.004) and worse (higher) GOS-E Peds score (b = 0.24, p = 0.004). In this exploratory pediatric study, CSF SUR1 was undetectable in controls and variably elevated in severe TBI. Mean CSF SUR1 concentration was associated with ICP and outcome. These findings are distinct from our previous report in adults with severe TBI, where SUR1 was detected universally. SUR1 may be a viable therapeutic target in a subset of pediatric TBI, and further study is warranted.

BACKGROUND: Gain-of-function of ATP-sensitive K+ (KATP ) channels because of mutations in the genes encoding SUR1 (ABCC8) or Kir6.2 (KCNJ11) is a major cause of neonatal diabetes mellitus (NDM). Our aim is to determine molecular defects in KATP channels caused by ABCC8 mutations in Asian Indian children with NDM by in vitro functional studies. METHODS: Wild-type (WT; NM_000352.4) or mutant sulfonylurea receptor 1 (SUR1) and Kir6.2 were co-expressed in COSm6 cells. Biogenesis efficiency and surface expression of mutant channels were assessed by immunoblotting and immunostaining. The response of mutant channels to cytoplasmic ATP and ADP was assessed by inside-out patch-clamp recordings. The response of mutant channels to known KATP inhibitors in intact cells were determined by 86 Rb efflux assays. RESULTS: Five SUR1 missense mutations, D212Y, P254S, R653Q, R992C, and Q1224H, were studied and showed increased activity in MgATP/MgADP. Two of the mutants, D212Y and P254S, also showed reduced response to ATP4- inhibition, as well as markedly reduced surface expression. Moreover, all five mutants were inhibited by the KATP channel inhibitors glibenclamide and carbamazepine. CONCLUSIONS: The study shows the mechanisms by which five SUR1 mutations identified in Asian Indian NDM patients affect KATP channel function to cause the disease. The reduced ATP4- sensitivity caused by the D212Y and P254S mutations in the L0 of SUR1 provides novel insight into the role of L0 in channel inhibition by ATP. The results also explain why sulfonylurea therapy is effective in two patients and inform how it should be effective for the other three patients.

Neuroendocrine-type ATP-sensitive K(+) (K(ATP)) channels are metabolite sensors coupling membrane potential with metabolism, thereby linking insulin secretion to plasma glucose levels. They are octameric complexes, (SUR1/Kir6.2)(4), comprising sulfonylurea receptor 1 (SUR1 or ABCC8) and a K(+)-selective inward rectifier (Kir6.2 or KCNJ11). Interactions between nucleotide-, agonist-, and antagonist-binding sites affect channel activity allosterically. Although it is hypothesized that opening these channels requires SUR1-mediated MgATP hydrolysis, we show here that ATP binding to SUR1, without hydrolysis, opens channels when nucleotide antagonism on Kir6.2 is minimized and SUR1 mutants with increased ATP affinities are used. We found that ATP binding is sufficient to switch SUR1 alone between inward- or outward-facing conformations with low or high dissociation constant, K(D), values for the conformation-sensitive channel antagonist [(3)H]glibenclamide ([(3)H]GBM), indicating that ATP can act as a pure agonist. Assembly with Kir6.2 reduced SUR1's K(D) for [(3)H]GBM. This reduction required the Kir N terminus (KNtp), consistent with KNtp occupying a “transport cavity,” thus positioning it to link ATP-induced SUR1 conformational changes to channel gating. Moreover, ATP/GBM site coupling was constrained in WT SUR1/WT Kir6.2 channels; ATP-bound channels had a lower K(D) for [(3)H]GBM than ATP-bound SUR1. This constraint was largely eliminated by the Q1179R neonatal diabetes-associated mutation in helix 15, suggesting that a “swapped” helix pair, 15 and 16, is part of a structural pathway connecting the ATP/GBM sites. Our results suggest that ATP binding to SUR1 biases K(ATP) channels toward open states, consistent with SUR1 variants with lower K(D) values causing neonatal diabetes, whereas increased K(D) values cause congenital hyperinsulinism.

Linking two pharmacophores that bind different cell surface receptors into a single molecule can enhance cell-targeting specificity to cells that express the complementary receptor pair. In this report, we developed and tested a synthetic multivalent ligand consisting of glucagon-like peptide-1 (GLP-1) linked to glibenclamide (Glb) (GLP-1/Glb) for signaling efficacy in β-cells. Expression of receptors for these ligands, as a combination, is relatively specific to the β-cell in the pancreas. The multivalent GLP-1/Glb increased both intracellular cAMP and Ca2+, although Ca2+ responses were significantly depressed compared with the monomeric Glb. Moreover, GLP-1/Glb increased glucose-stimulated insulin secretion in a dose-dependent manner. However, unlike the combined monomers, GLP-1/Glb did not augment insulin secretion at nonstimulatory glucose concentrations in INS 832/13 β-cells or human islets of Langerhans. These data suggest that linking two binding elements, such as GLP-1 and Glb, into a single bivalent ligand can provide a unique functional agent targeted to β-cells.

ATP-sensitive potassium (K-ATP) channels express in the central nervous system extensively which coupling cell metabolism and cellular electrical activity. K-ATP channels in mature substantia nigra (SN) dopaminergic (DA) neurons are composed of inwardly rectifying potassium channel (Kir) subunit 6.2 and sulfonylurea receptor 1 (SUR1). Our previous study revealed that regulating K-ATP channel exerts the protective effect on DA neurons in a mouse model of Parkinson's disease (PD). However, the detailed mechanism underlying the role of Kir6.2/K-ATP remains unclear. In the present study, we found the deletion of Kir6.2 dramatically alleviated PD-like motor dysfunction of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) PD model. We further found that Kir6.2 knockout selectively restored the reduction of both DA neuronal number and dopamine transmitter level in the nigrostriatal of MPTP-treated PD mice. To gain some understanding on the molecular basis of this effect, we focused on the regulation of Kir6.2 deletion on iron metabolism which is tightly associated with DA neuron damage. We found that Kir6.2 knockout suppressed the excessive iron accumulation in MPTP-treated mouse midbrain and inhibited the upregulation of ferritin light chain (FTL), which is a main intracellular iron storage protein. We probed further and found out that the deletion of Kir6.2 inhibited the excessive production of FTL via IRP-IRE regulatory system, and thereby protecting SN DA neurons against MPTP challenge. Our findings suggest that Kir6.2 plays a crucial role in the pathogenesis of PD and regulating Kir6.2/K-ATP channel may be a promising strategy for PD treatment.

Objective Transient neonatal diabetes mellitus (TNDM) is caused by activating mutations in ABCC8 and KCNJ11 genes (KATP/TNDM) or by chromosome 6q24 abnormalities (6q24/TNDM). We wanted to assess whether these different genetic aetiologies result in distinct clinical features. Design Retrospective analysis of the Italian data set of patients with TNDM. Methods Clinical features and treatment of 22 KATP/TNDM patients and 12 6q24/TNDM patients were compared. Results Fourteen KATP/TNDM probands had a carrier parent with abnormal glucose values, four patients with 6q24 showed macroglossia and/or umbilical hernia. Median age at diabetes onset and birth weight were lower in patients with 6q24 (1 week; −2.27 SD) than those with KATP mutations (4.0 weeks; −1.04 SD) (P = 0.009 and P = 0.007, respectively). Median time to remission was longer in KATP/TNDM than 6q24/TNDM (21.5 weeks vs 12 weeks) (P = 0.002). Two KATP/TNDM patients entered diabetes remission without pharmacological therapy. A proband with the ABCC8/L225P variant previously associated with permanent neonatal diabetes entered 7-year long remission after 1 year of sulfonylurea therapy. Seven diabetic individuals with KATP mutations were successfully treated with sulfonylurea monotherapy; four cases with relapsing 6q24/TNDM were treated with insulin, metformin or combination therapy. Conclusions If TNDM is suspected, KATP genes should be analyzed first with the exception of patients with macroglossia and/or umbilical hernia. Remission of diabetes without pharmacological therapy should not preclude genetic analysis. Early treatment with sulfonylurea may induce long-lasting remission of diabetes in patients with KATP mutations associated with PNDM. Adult patients carrying KATP/TNDM mutations respond favourably to sulfonylurea monotherapy.

ATP-sensitive potassium (K(ATP)) channels are multimeric protein complexes made of four inward rectifying potassium channel (Kir6.x) subunits and four ABC protein sulfonylurea receptor (SURx) subunits. Kir6.x subunits form the potassium ion conducting pore of the channel, and SURx functions to regulate Kir6.x. Kir6.x and SURx are uniquely dependent on each other for expression and function. In pancreatic β-cells, channels comprising SUR1 and Kir6.2 mediate glucose-stimulated insulin secretion and are the targets of anti-diabetic sulfonylureas. Mutations in genes encoding SUR1 or Kir6.2 are linked to insulin secretion disorders, with loss- or gain-of-function mutations causing congenital hyperinsulinism or neonatal diabetes mellitus, respectively. Defects in the K(ATP) channel in other tissues underlie human diseases of the cardiovascular and nervous systems. Key to understanding how channels are regulated by physiological and pharmacological ligands and how mutations disrupt channel assembly or gating to cause disease is the ability to observe structural changes associated with subunit interactions and ligand binding. While recent advances in the structural method of single-particle cryo-electron microscopy (cryoEM) offers direct visualization of channel structures, success of obtaining high-resolution structures is dependent on highly concentrated, homogeneous K(ATP) channel particles. In this chapter, we describe a method for expressing K(ATP) channels in mammalian cell culture, solubilizing the channel in detergent micelles and purifying K(ATP) channels using an affinity tag to the SURx subunit for cryoEM structural studies.

A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K(+) (K(ATP)) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the K(ATP) channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys(166) prevented these effects. Clinical variants in KCNJ11 that affect Cys(166) had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP(2). Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys(166) enhanced the PIP(2) sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of K(ATP) channels by α1-adrenoreceptor stimulation. The Cys(166) residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.

When offered glucose and fructose solutions, rodents consume more glucose solution because it produces stronger postoral reinforcement. Intake of these sugars also conditions a higher avidity for glucose relative to fructose. We asked which chemosensory cue mediates the learned avidity for glucose. We subjected mice to 18 days of sugar training, offering them 0.3, 0.6, and 1 M glucose and fructose solutions. Before and after training, we measured avidity for 0.3 and 0.6 M glucose and fructose in brief-access lick tests. First, we replicated prior work in C57BL/6 mice. Before training, the mice licked at a slightly higher rate for 0.6 M fructose; after training, they licked at a higher rate for 0.6 M glucose. Second, we assessed the necessity of the glucose-specific ATP-sensitive K+(KATP) taste pathway for the learned avidity for glucose, using mice with a nonfunctional KATPchannel [regulatory sulfonylurea receptor (SUR1) knockout (KO) mice]. Before training, SUR1 KO and wild-type mice licked at similar rates for 0.6 M glucose and fructose; after training, both strains licked at a higher rate for 0.6 M glucose, indicating that the KATPpathway is not necessary for the learned discrimination. Third, we investigated the necessity of olfaction by comparing sham-treated and anosmic mice. The mice were made anosmic by olfactory bulbectomy or ZnSO4treatment. Before training, sham-treated and anosmic mice licked at similar rates for 0.6 M glucose and fructose; after training, sham-treated mice licked at a higher rate for 0.6 M glucose, whereas anosmic mice licked at similar rates for both sugars. This demonstrates that olfaction contributes significantly to the learned avidity for glucose.

Cerebral edema and contusion expansion are major determinants of morbidity and mortality after TBI. Current treatment options are reactive, suboptimal and associated with significant side effects. First discovered in models of focal cerebral ischemia, there is increasing evidence that the sulfonylurea receptor 1 (SUR1)—Transient receptor potential melastatin 4 (TRPM4) channel plays a key role in these critical secondary injury processes after TBI. Targeted SUR1-TRPM4 channel inhibition with glibenclamide has been shown to reduce edema and progression of hemorrhage, particularly in preclinical models of contusional TBI. Results from small clinical trials evaluating glibenclamide in TBI have been encouraging. A Phase-2 study evaluating the safety and efficacy of intravenous glibenclamide (BIIB093) in brain contusion is actively enrolling subjects. In this comprehensive narrative review, we summarize the molecular basis of SUR1-TRPM4 related pathology and discuss TBI-specific expression patterns, biomarker potential, genetic variation, preclinical experiments, and clinical studies evaluating the utility of treatment with glibenclamide in this disease.

Glyburide (also known as glibenclamide) is a second-generation sulfonylurea drug that inhibits sulfonylurea receptor 1 (Sur1) at nanomolar concentrations. Long used to target KATP (Sur1–Kir6.2) channels for the treatment of diabetes mellitus type 2, glyburide was recently repurposed to target Sur1–transient receptor potential melastatin 4 (Trpm4) channels in acute central nervous system injury. Discovered nearly two decades ago, SUR1–TRPM4 has emerged as a critical target in stroke, specifically in large hemispheric infarction, which is characterized by edema formation and life-threatening brain swelling. Following ischemia, SUR1–TRPM4 channels are transcriptionally upregulated in all cells of the neurovascular unit, including neurons, astrocytes, microglia, oligodendrocytes and microvascular endothelial cells. Work by several independent laboratories has linked SUR1–TRPM4 to edema formation, with blockade by glyburide reducing brain swelling and death in preclinical models. Recent work showed that, following ischemia, SUR1–TRPM4 co-assembles with aquaporin-4 to mediate cellular swelling of astrocytes, which contributes to brain swelling. Additionally, recent work linked SUR1–TRPM4 to secretion of matrix metalloproteinase-9 (MMP-9) induced by recombinant tissue plasminogen activator in activated brain endothelial cells, with blockade of SUR1–TRPM4 by glyburide reducing MMP-9 and hemorrhagic transformation in preclinical models with recombinant tissue plasminogen activator. The recently completed GAMES (Glyburide Advantage in Malignant Edema and Stroke) clinical trials on patients with large hemispheric infarctions treated with intravenous glyburide (RP-1127) revealed promising findings with regard to brain swelling (midline shift), MMP-9, functional outcomes and mortality. Here, we review key elements of the basic science, preclinical experiments and clinical studies, both retrospective and prospective, on glyburide in focal cerebral ischemia and stroke.

Puljung reviews recent cryo-EM KATP channel structures and proposes a mechanism by which ligand binding results in channel opening., Adenosine triphosphate (ATP)–sensitive K+ (KATP) channels are molecular sensors of cell metabolism. These hetero-octameric channels, comprising four inward rectifier K+ channel subunits (Kir6.1 or Kir6.2) and four sulfonylurea receptor (SUR1 or SUR2A/B) subunits, detect metabolic changes via three classes of intracellular adenine nucleotide (ATP/ADP) binding site. One site, located on the Kir subunit, causes inhibition of the channel when ATP or ADP is bound. The other two sites, located on the SUR subunit, excite the channel when bound to Mg nucleotides. In pancreatic β cells, an increase in extracellular glucose causes a change in oxidative metabolism and thus turnover of adenine nucleotides in the cytoplasm. This leads to the closure of KATP channels, which depolarizes the plasma membrane and permits Ca2+ influx and insulin secretion. Many of the molecular details regarding the assembly of the KATP complex, and how changes in nucleotide concentrations affect gating, have recently been uncovered by several single-particle cryo-electron microscopy structures of the pancreatic KATP channel (Kir6.2/SUR1) at near-atomic resolution. Here, the author discusses the detailed picture of excitatory and inhibitory ligand binding to KATP that these structures present and suggests a possible mechanism by which channel activation may proceed from the ligand-binding domains of SUR to the channel pore.

ATP-sensitive potassium (KATP) channels couple intracellular metabolism to the electrical activity by regulating K+ flux across the plasma membrane, thus playing an important role in both normal and pathophysiology. To understand the mechanism of ATP regulating biological ion channels, developing an ATP-responsive artificial nanochannel is an appealing but challenging topic because KATP channel is a heteromultimer of two subunits (potassium channel subunit (Kir6.x) and sulfonylurea receptor (SUR)) and exhibit dynamic functions with adjustability and reversibility. Inspired by the structure of KATP channels, we designed a smart copolymer modified nanochannel that may address the challenge. In the tricomponent poly(N-isopropylacrylamide) (PNIPAAm, PNI)-based copolymer system, phenylthiourea was used to bind the phosphate units of nucleotides and phenylboronic acid was introduced to combine the pentose ring of the nucleoside unit. Besides, a –COOH group with electron-withdrawing property was added into the phenylthiourea units, which may promote the hydrogen-bond-donating ability of thiourea. Specially, the smart copolymer not only provided static binding sites for recognition but also translated the recognition of ATP into their dynamic conformational transitions by changing the hydrogen-bonding environments surrounding PNIPAAm chains, thus achieving the gating function of nanochannel, which resembled the integration and coordination of Kir6.x and SUR units in active KATP. The ATP-regulated ion channel exhibited excellent stability and reversibility. This study is the first example showing how to learn from nature to assemble the ATP-responsive artificial nanochannel and demonstrate the possible mechanism of ATP gating.

Peroxisome proliferator-activated receptor gamma (PPARγ) and sulfonylurea receptor (SUR) play crucial roles in management of type-2 diabetes mellitus. In this study, a series of novel quinazoline-4(3H)-one-sulfonylurea hybrids were designed and synthesized as dual PPARγ and SUR agonists. The synthesized compounds were evaluated for their in vivo anti-hyperglycemic activities against STZ-induced hyperglycemic rats. Four compounds (19a, 19d, 19f and 25g) demonstrated potent activities with reduction in blood glucose levels of 40.43, 46.42, 41.23 and 42.50 %, respectively. The most active ten compounds were further evaluated in vitro for their PPARγ binding affinities and insulin-secreting abilities. Compounds 19b, 19d, 19f, 25f and 25g exhibited the highest affinities against PPARγ with IC50 values of 0.371, 0.350, 0.369, 0.408 and 0.353µM, respectively. In addition, compounds 19d, 19f, and 25d showed the highest insulin-secreting activities with EC50 values of 0.97, 1.01 and 1.15µM, respectively. Furthermore, molecular docking and pharmacophore generation techniques were carried out to investigate binding patterns and fit values of the designed compounds with PPARγ and SUR, respectively. Also, two QSAR models were generated to explore the structural requirements controlling the different biological activities of the synthesized compounds against PPARγ and SUR.

Glibenclamide (GBC), a sulfonylurea receptor 1 blocker, emerges recently as a promising neuron protectant in various neurological disorders. This study aimed to determine whether GBC improves survival and neurological outcome of status epilepticus (SE). Male Sprague-Dawley rats successfully undergoing SE for 2.5 h (n = 134) were randomly assigned to GBC or vehicle group. Rats in the GBC group received a loading dose of 10 μg/kg of GBC, followed by 1.2 μg/6 h for 3 days, while same dose of vehicle was used as control. The 28-day survival rate in the GBC group (11/23) was significantly higher than that in the vehicle group (8/36). In addition, the frequency and duration of spontaneous recurrent seizures in SE rats were profoundly reduced by GBC but not by vehicle treatment. Moreover, cognitive impairment was observed in the SE rats at day 28, which was reversed by GBC treatment. Meanwhile, cerebral edema, as well as neuronal loss, was decreased in several brain areas in the GBC group. Additionally, on the molecular basis, the subunits of sulfonylurea receptor 1/transient receptor potential M4 (SUR1-TRPM4) heterodimer were both strongly upregulated after SE but partly suppressed by GBC treatment. Furthermore, gene knockdown of Trpm4 in SE rats reduced BBB disruption and neuronal loss, similar to the inhibitory effects with GBC treatment. Taken together, GBC treatment markedly improved survival and neurologic outcomes after SE. The salutary effects of GBC were correlated to the alleviation of cerebral edema and reduction in neurological injury via down-regulation of SUR1-TRPM4 channel.

The sulfonylurea receptor 1 (SUR1) protein forms the regulatory subunit in ATP sensitive K+ (KATP) channels in the pancreas. SUR proteins are members of the ATP binding cassette (ABC) superfamily of proteins. Binding and hydrolysis of MgATP at the SUR nucleotide binding domains (NBDs) lead to channel opening. Pancreatic KATP channels play an important role in insulin secretion. SUR1 mutations that result in increased levels of channel opening ultimately inhibit insulin secretion and lead to neonatal diabetes. In contrast, SUR1 mutations that disrupt trafficking and/or decrease gating of KATP channels cause congenital hyperinsulinism, where oversecretion of insulin occurs even in the presence of low glucose levels. Here, we present data on the effects of specific congenital hyperinsulinism-causing mutations (G716V, R842G, and K890T) located in different regions of the first nucleotide binding domain (NBD1). Nuclear magnetic resonance (NMR) and fluorescence data indicate that the K890T mutation affects resi...

Despite widespread clinical use in the treatment of type 2 diabetes, the impact of sulfonylurea therapy on cardiovascular outcomes remains uncertain. Studies of naturally occurring genetic variation can be used to anticipate the expected clinical consequences of a pharmacological therapy. A common missense variant in the gene encoding a component of the sulfonylurea receptor (ABCC8 p.A1369S) promotes closure of the target channel of sulfonylurea therapy and is associated with increased insulin secretion, thus mimicking the effects of sulfonylurea therapy. Using individual-level data from 120,286 participants in the UK Biobank and summary association results from four large-scale genome-wide association studies, we examined the impact of this variant on cardiometabolic traits, type 2 diabetes, and coronary heart disease. The p.A1369S variant was associated with a significantly lower risk of type 2 diabetes (odds ratio [OR] 0.93; 95% CI 0.91, 0.95; P = 1.2 × 10−11). The variant was associated with increased BMI (+0.062 kg/m2; 95% CI 0.037, 0.086; P = 8.1 × 10−7) but lower waist-to-hip ratio adjusted for BMI, a marker of abdominal fat distribution. Furthermore, p.A1369S was associated with a reduced risk of coronary heart disease (OR 0.98; 95% CI 0.96, 0.99; P = 5.9 × 10−4). These results suggest that, despite a known association with increased weight, long-term sulfonylurea therapy may reduce the risk of coronary heart disease.

Cerebral edema is a key poor prognosticator in traumatic brain injury. There are no biomarkers identifying patients at-risk, or guiding mechanistically-precise therapies. Sulfonylurea receptor-1-transient receptor potential cation channel M4 is upregulated only after brain injury, causing edema in animal studies. We hypothesized that sulfonylurea receptor-1 is measurable in human cerebrospinal fluid after severe traumatic brain injury and is an informative biomarker of edema and outcome.A total of 119 cerebrospinal fluid samples were collected from 28 severe traumatic brain injury patients. Samples were retrieved at 12, 24, 48, 72 hours and before external ventricular drain removal. Fifteen control samples were obtained from patients with normal pressure hydrocephalus. Sulfonylurea receptor- 1 was quantified by enzyme-linked immunosorbent assay. Outcomes included CT edema, intracranial pressure measurements, therapies targeting edema, and 3-month Glasgow Outcome Scale score.Sulfonylurea receptor-1 was present in all severe traumatic brain injury patients (mean = 3.54 ± 3.39 ng/mL, peak = 7.13 ± 6.09 ng/mL) but undetectable in all controls (p0.001). Mean and peak sulfonylurea receptor-1 was higher in patients with CT edema (4.96 ± 1.13 ng/mL vs 2.10 ± 0.34 ng/mL; p = 0.023). There was a temporal delay between peak sulfonylurea receptor-1 and peak intracranial pressure in 91.7% of patients with intracranial hypertension. There was no association between mean/peak sulfonylurea receptor-1 and mean/peak intracranial pressure, proportion of intracranial pressure greater than 20 mm Hg, use of edema-directed therapies, decompressive craniotomy, or 3-month Glasgow Outcome Scale. However, decreasing sulfonylurea receptor-1 trajectories between 48 and 72 hours were significantly associated with improved cerebral edema and clinical outcome. Area under the multivariate model receiver operating characteristic curve was 0.881.This is the first report quantifying human cerebrospinal fluid sulfonylurea receptor-1. Sulfonylurea receptor-1 was detected in severe traumatic brain injury, absent in controls, correlated with CT-edema and preceded peak intracranial pressure. Sulfonylurea receptor-1 trajectories between 48 and 72 hours were associated with outcome. Because a therapy inhibiting sulfonylurea receptor-1 is available, assessing cerebrospinal fluid sulfonylurea receptor-1 in larger studies is warranted to evaluate our exploratory findings regarding its diagnostic, and monitoring utility, as well as its potential to guide targeted therapies in traumatic brain injury and other diseases involving cerebral edema.

Benzoylphenylureas (BPUs) are used as synthetic insect growth regulators for inhibiting chitin synthesis. Merging insecticidal BPUs with photoswitchable azobenzene generated photoresponsive chitin synthesis inhibitors. A prepared azobenzene-benzoylphenylurea can be activated upon irradiation with UV light, and shows 6-fold and 2-fold activity difference to armyworm (Mythimna separata) and German cockroach (Blattella germanica) sulfonylurea receptors, respectively. This is the first example of a photoswitchable BPU insecticide. The generation of such a photoresponsive BPU insecticide allows for modulation of the insecticidal activity by light, and may facilitate the spatiotemporal control over the sulfonylurea receptor and the mechanistic study of this kind of insecticide.

Both iptakalim (Ipt) and natakalim (Nat) activate the SUR2B/Kir6.1 channel, an ATP-sensitive potassium channel (KATP) subtype, with high selectivity. In this study we investigated the therapeutic effects of Ipt and Nat against isoproterenol-induced chronic heart failure (ISO-CHF) in rats, and demonstrated a new therapeutic approach to the treatment of CHF through activation of the SUR2B/Kir6.1 channel in endothelial cells. In ISO-CHF rats, oral administration of Nat (1, 3, 9 mg·kg−1·d−1) or Ipt (3 mg·kg−1·d−1) for 60 days significantly improved cardiac dysfunction, reversed cardiac remodeling, significantly attenuated the pathological increases in BNP levels, and improved endothelial dysfunction by adjusting the balance between endothelin and NO systems. The therapeutic effects of Nat were prevented by the selective KATP blocker glibenclamine (Gli, 50 mg·kg−1·d−1), confirming that these effects were mediated through activation of the SUR2B/Kir6.1 channel in endothelial cells. The molecular mechanisms underlying the therapeutic effects of Nat were further addressed using proteomic methods. We identified 724 proteins in the plasma of ISO-CHF rats; 55 proteins were related to Nat. These differentially expressed proteins were mainly involved in single-organism processes and the regulation of biological quality relative to CHF, including proteasome (Psm) and ATP protein clusters. We screened out PRKAR2β, GAS6/eNOS/NO and NO/PKG/VASP pathways involved in the amelioration of CHF among the 24 enriched pathways. We further confirmed 6 protein candidates, including PRKAR2β, GAS6 and VASP, which were involved in the endothelial mechanisms, and ATP, TIMP3 and AGT, which contributed to its cardiovascular actions. This study demonstrates a new pharmacological approach to the treatment of CHF through activation of the SUR2B/Kir6.1 channel in endothelial cells, and that the eNOS/VASP pathways are involved in its signaling mechanisms.

Glucose-stimulated insulin secretion from pancreatic β-cells is controlled by ATP-regulated potassium (K(ATP)) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) subunits. The K(ATP) channel-opener diazoxide is FDA-approved for treating hyperinsulinism and hypoglycemia but suffers from off-target effects on vascular K(ATP) channels and other ion channels. The development of more specific openers would provide critically needed tool compounds for probing the therapeutic potential of Kir6.2/SUR1 activation. Here, we characterize a novel scaffold activator of Kir6.2/SUR1 that our group recently discovered in a high-throughput screen. Optimization efforts with medicinal chemistry identified key structural elements that are essential for VU0071063-dependent opening of Kir6.2/SUR1. VU0071063 has no effects on heterologously expressed Kir6.1/SUR2B channels or ductus arteriole tone, indicating it does not open vascular K(ATP) channels. VU0071063 induces hyperpolarization of β-cell membrane potential and inhibits insulin secretion more potently than diazoxide. VU0071063 exhibits metabolic and pharmacokinetic properties that are favorable for an in vivo probe and is brain penetrant. Administration of VU0071063 inhibits glucose-stimulated insulin secretion and glucose-lowering in mice. Taken together, these studies indicate that VU0071063 is a more potent and specific opener of Kir6.2/SUR1 than diazoxide and should be useful as an in vitro and in vivo tool compound for investigating the therapeutic potential of Kir6.2/SUR1 expressed in the pancreas and brain.

ATP-sensitive K+ (KATP) channels are predominantly expressed in the brain and consist of four identical inward-rectifier potassium ion channel subunits (Kir6.1 or Kir6.2) and four identical high-affinity sulfonylurea receptor subunits (SUR1, SUR2A, or SUR2B). We previously observed that chronic corticosterone-treated (CORT) mice exhibited enhanced anxiety-like behaviors and cued fear memory. In the present study, the protein and mRNA expression levels of Kir6.1, but not Kir6.2, were decreased in the lateral amygdala (LA) of CORT mice. Heterozygous Kir6.1-null (Kir6.1+/-) mice also showed enhanced tone (cued) fear memory and long-term potentiation (LTP) in the cortico-LA pathway compared to those in wild-type mice. However, LTP was not enhanced in the hippocampal CA1 regions of Kir6.1+/- mice. Consistent with increased cued fear memory, both Ca2+/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) activities were significantly elevated in the LAs of Kir6.1+/- mice after tone stimulation. Our results indicate that increased CaMKII and ERK activities may induce LTP in the LA in Kir6.1+/- mice, leading to aberrant cued fear memory. The changes in neural plasticity in the LA of Kir6.1+/- mice were associated with anxiety-like behaviors and may be related to the pathogenic mechanisms of anxiety disorders in human patients.

Background Sulfonylurea receptor 1 (SUR1) is primarily responsible for glucose regulation in normal conditions. Here, we sought to investigate the glucose metabolism characteristics of SUR1−/− rats. Methods The TALEN technique was used to construct a SUR1 gene deficiency rat model. Rats were grouped by SUR1 gene knockout or not and sex difference. Body weight; glucose metabolism indicators, including IPGTT, IPITT, glycogen contents and so on; and other molecule changes were examined. Results Insulin secretion was significantly inhibited by knocking out the SUR1 gene. SUR1−/− rats showed lower body weights compared to wild-type rats, and even SUR1−/− males weighed less than wild-type females. Upon SUR1 gene knockout, the rats showed a peculiar plasma glucose profile. During IPGTT, plasma glucose levels were significantly elevated in SUR1−/− rats at 15 min, which could be explained by SUR1 mainly working in the first phase of insulin secretion. Moreover, SUR1−/− male rats showed obviously impaired glucose tolerance than before and a better insulin sensitivity in the 12th week compared with females, which might be related with excess androgen secretion in adulthood. Increased glycogen content and GLUT4 expression and the inactivation of GSK3 were also observed in SUR1−/− rats, which suggested an enhancement of insulin sensitivity. Conclusions These results reconfirm the role of SUR1 in systemic glucose metabolism. More importantly, our SUR1−/− rat model might be applied in other fields, such as for exploring other hypoglycaemic functions of sulfonylureas. Electronic supplementary material The online version of this article (10.1186/s10020-018-0067-9) contains supplementary material, which is available to authorized users.

Contusió cerebral; Glibenclamida; Progressió hemorràgica de contusió Brain contusion; Glibenclamide; Hemorrhagic progression of contusion Contusión cerebral; Glibenclamida; Progresión hemorrágica de contusión In severe traumatic brain injury (TBI), contusions often are worsened by contusion expansion or hemorrhagic progression of contusion (HPC), which may double the original contusion volume and worsen outcome. In humans and rodents with contusion-TBI, sulfonylurea receptor 1 (SUR1) is upregulated in microvessels and astrocytes, and in rodent models, blockade of SUR1 with glibenclamide reduces HPC. SUR1 does not function by itself, but must co-assemble with either KIR6.2 or transient receptor potential cation channel subfamily M member 4 (TRPM4) to form KATP (SUR1-KIR6.2) or SUR1-TRPM4 channels, with the two having opposite effects on membrane potential. Both KIR6.2 and TRPM4 are reportedly upregulated in TBI, especially in astrocytes, but the identity and function of SUR1-regulated channels post-TBI is unknown. Here, we analyzed human and rat brain tissues after contusion-TBI to characterize SUR1, TRPM4, and KIR6.2 expression, and in the rat model, to examine the effects on HPC of inhibiting expression of the three subunits using intravenous antisense oligodeoxynucleotides (AS-ODN). Glial fibrillary acidic protein (GFAP) immunoreactivity was used to operationally define core versus penumbral tissues. In humans and rats, GFAP-negative core tissues contained microvessels that expressed SUR1 and TRPM4, whereas GFAP-positive penumbral tissues contained astrocytes that expressed all three subunits. Förster resonance energy transfer imaging demonstrated SUR1-TRPM4 heteromers in endothelium, and SUR1-TRPM4 and SUR1-KIR6.2 heteromers in astrocytes. In rats, glibenclamide as well as AS-ODN targeting SUR1 and TRPM4, but not KIR6.2, reduced HPC at 24 h post-TBI. Our findings demonstrate upregulation of SUR1-TRPM4 and KATP after contusion-TBI, identify SUR1-TRPM4 as the primary molecular mechanism that accounts for HPC, and indicate that SUR1-TRPM4 is a crucial target of glibenclamide. J.M.S is supported by grants from the Department of Veterans Affairs (I01BX002889), the Department of Defense (SCI170199), the National Heart, Lung and Blood Institute (R01HL082517) and the National Institute of Neurological Disorders and Stroke (NINDS) (R01NS060801; R01NS102589; R01NS105633); V.G. is supported by a grant from NINDS (NS061934).

ATP-sensitive potassium (KATP) channels link membrane excitability to metabolic state to regulate a series of biological activities including the vascular tone. However, their ability to influence hypertension is controversial. Here we aim to investigate possible alteration of KATP channel in vascular smooth muscles (VSMs) during hypertension development process. In this study, we used 16-week-old spontaneously hypertensive rats (SHRs), 49-week-old SHRs, and their age-matched Wistar-Kyoto rats to study the expression of VSM KATP subunits at the mRNA and protein level and the function of VSM KATP by observing the relaxation reactivity of isolated aorta rings to KATP modulators. We found that the expression of VSM KATP subunits Kir6.1 and sulfonylurea receptor (SUR2B) decreased during hypertension. Moreover, the expression of SUR2B and Kir6.1 in 49-week-old SHRs decreased much more than that in 16-week-old SHRs. Furthermore, the aorta rings of 49-week-old SHRs showed lower reactivity to diazoxide than 16-week-old SHRs. This study suggests that KATP channels in VSM subunits Kir6.1 and SUR2B contribute to modify the functionality of this channel in hypertension with age.

ATP-sensitive potassium (KATP) channels are a major drug target for the treatment of type-2 diabetes. KATP channels are ubiquitously expressed and link the metabolic state to electrical excitability. In pancreatic β-cells, KATP channels are crucial in the regulation of glucose-induced insulin secretion. Also, KATP channels are involved in the protection against neuronal seizures and ischaemic stress in the heart, brain and in the regulation of vascular smooth muscle tone. Functional KATP channels are hetero-octamers composed of two subunits, a pore forming Kir6, which is a member of the inwardly rectifying potassium channels family, and a regulatory sulphonylurea receptor (SUR). In response to nucleotides and pharmaceutical agonists and antagonists, SUR allosterically regulates channel gating. The allosteric communication pathways between these two heterologus proteins in KATP channels are still poorly understood. This review will highlight the therapeutic agents that target KATP channels and are used to treat diabetes and cardiovascular diseases.

The monitoring of diabetes mellitus, as it develops and becomes clinically evident, remains a major challenge for diagnostic imaging in clinical practice. Here we present a novel approach to beta-cell imaging by targeting the sulphonylurea receptor subtype 1 (SUR1), using multivalent derivatives of the anti-diabetic drug glibenclamide. Since glibenclamide has a high affinity for SUR1 but does not contain a suitable functional group to be linked to an imaging probe, we have synthesized 11 glibenclamide derivatives and evaluated their affinity to SUR1 in MIN6 cells. The most promising compound has been used to obtain multivalent glibenclamide-polyamidoamine (PAMAM) derivatives, containing up to 15 sulphonylurea moieties per dendrimer. The remaining functional groups on the dendrimers can consecutively be used for labeling with reporter groups for different imaging modalities, thus allowing for multifunctional imaging, and for the modification of pharmacokinetic properties. We synthesized fluorochrome-labeled multivalent probes, that demonstrate in cellular assays affinities to SUR1 in the nanomolar range, superior to native glibenclamide. The probes specifically label MIN6 cells, but not HeLa or PANC-1 cells which do not express SUR1. A very low cytotoxicity of the multivalent probes is demonstrated by the persistent release of insulin from MIN6 cells exposed to high glucose concentrations. Furthermore, the probes display positive labeling of beta-cells of primary mouse and human islet-cells ex vivo and of islets of Langerhans in vivo. The data document that multivalent probes based on glibenclamide derivatives provide a suitable platform for further developments of cell-specific probes, and can be adapted for multiple imaging modalities, including those that are now used in the clinics.

ATP-sensitive potassium (KATP) channels formed by a combination of SUR/Kir6.x subunits play a crucial role in protection against hypoxic or ischemic injuries resulting from cell metabolic disorders. In this study we investigated the effects of Na-azide, a metabolic inhibitor, on KATP channels expressed in Xenopus oocytes, and explored the structure basis for their sensitivity to cell metabolic disorders. Six subtypes of KATP channels (wild SUR1/Kir6.2, SUR2B/Kir6.2, SUR1/Kir6.1, SUR2B/Kir6.1, SUR2A/Kir6.2 and SUR2A/Kir6.1), as well as eleven subtypes of KATP channels with mutant subunits were expressed in Xenopus oocytes. KATP currents were recorded using a two-electrode voltage clamp recording technique. The drugs were applied through bath. Except SUR2A/Kir6.1, five subtypes of KATP channels were activated by Na-azide (3 mmol/L) with an order of the responses: SUR1/Kir6.2>SUR2B/Kir6.2>SUR1/Kir6.1>SUR2B/Kir6.1>SUR2A/Kir6.2, and the opening rate (t1/2) was SUR1/Kir6.x>SUR2B/Kir6.x>SUR2A/Kir6.2. Furthermore, Kir6.2, rather than Kir6.1, had intrinsic sensitivity to Na-azide, and the residues involved in ATP-binding (R50 and K185) or pH-sensing (H175) were associated with the sensitivity of the Kir6.2 subunit to Na-azide. Moreover, the residues (K707 and K1348) within the Walker A (WA) motifs of two nucleotide-binding domains (NBDs) were essential for SUR2B/Kir6.x (especially SUR2B/Kir6.1) channel activation by Na-azide, suggesting a key role for Mg-adenine nucleotide binding and/or hydrolysis in the SUR2B subunit. Among the six subtypes of KATP channels, SUR1/Kir6.2 is the most sensitive, whereas SUR2A/Kir6.1 is insensitive, to cell metabolic disorders. The Kir6.2 subunit, rather than the Kir6.1 subunit, has intrinsic sensitivity to cell metabolic disorders. The residues (K707 and K1348) within the WA motifs of SUR2B are important for the sensitivity of SUR2B/Kir6.x channels to cell metabolic disorders.

Post-traumatic brain contusions (PTBCs) are traditionally considered primary injuries and can increase in size, generate perilesional edema, cause mass effect, induce neurological deterioration, and cause death. Most patients experience a progressive increase in pericontusional edema, and nearly half, an increase in the hemorrhagic component itself. The underlying molecular pathophysiology of contusion-induced brain edema and hemorrhagic progression remains poorly understood. The aim of this study was to investigate sulfonylurea 1/transient receptor potential melastatin 4 (SUR1-TRPM4) ion channel SUR1 expression in various cell types (neurons, astrocytes, endothelial cells, microglia, macrophages, and neutrophils) of human brain contusions and whether SUR1 up-regulation was related to time postinjury. Double immunolabeling of SUR1 and cell-type– specific proteins was performed in 26 specimens from traumatic brain injury patients whose lesions were surgically evacuated. Three samples from limited brain resections performed for accessing extra-axial skull-base tumors or intraventricular lesions were controls. We found SUR1 was significantly overexpresed in all cell types and was especially prominent in neurons and endothelial cells (ECs). The temporal pattern depended on cell type: 1) In neurons, SUR1 increased within 48 h of injury and stabilized thereafter; 2) in ECs, there was no trend; 3) in glial cells and microglia/macrophages, a moderate increase was observed over time; and 4) in neutrophils, it decreased with time. Our results suggest that up-regulation of SUR1 in humans point to this channel as one of the important molecular players in the pathophysiology of PTBCs. Our findings reveal opportunities to act therapeutically on the mechanisms of growth of traumatic contusions and therefore reduce the number of patients with neurological deterioration and poor neurological outcomes.

Neonatal diabetes mellitus is a rare monogenic disease with incidence of 1/90,000 new-borns. A case of two months aged male infant with life threatening diabetic ketoacidosis is presented with novel ABCC8 gene mutation (p.F577L), successful transition from insulin to sulfonylurea and follow-up of three years.

The gene of ATP-binding cassette subfamily C member 8 (Abcc8) is cytogenetically located at 11p15.1 and encodes the sulfonylurea receptor (SUR1). SUR1 is a subunit of ATP-sensitive potassium channel (KAPT) in the β-cell regulating insulin secretion. Mutations of ABCC8 are responsible for congenital hyperinsulinism (CHI). Here we reported that an Abcc8 heterozygous mutant cell line was generated by CRISPR/Cas9 technique with 1bp insertion resulting in abnormal splicing on human embryonic stem cell line H1. The phenotypic characteristics of this cell line reveal defective KATP channel and diazoxide-responsive that provides ideal model for molecular pathology research and drug screening for CHI.

Cantu syndrome (CS) is characterized by multiple vascular and cardiac abnormalities including vascular dilation and tortuosity, systemic hypotension, and cardiomegaly. The disorder is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits. However, there is little understanding of the link between molecular dysfunction and the complex pathophysiology observed, and there is no known treatment, in large part due to the lack of appropriate preclinical disease models in which to test therapies. Notably, expression of Kir6.1 and SUR2 does not fully overlap, and the relative contribution of KATP GOF in various cardiovascular tissues remains to be elucidated. To investigate pathophysiologic mechanisms in CS we have used CRISPR/Cas9 engineering to introduce CS-associated SUR2[A478V] and Kir6.1[V65M] mutations to the equivalent endogenous loci in mice. Mirroring human CS, both of these animals exhibit low systemic blood pressure and dilated, compliant blood vessels, as well dramatic cardiac enlargement, the effects being more severe in V65M animals than in A478V animals. In both animals, whole-cell patch-clamp recordings reveal enhanced basal KATP conductance in vascular smooth muscle, explaining vasodilation and lower blood pressure, and demonstrating a cardinal role for smooth muscle KATP dysfunction in CS etiology. Echocardiography confirms in situ cardiac enlargement and increased cardiac output in both animals. Patch-clamp recordings reveal reduced ATP sensitivity of ventricular myocyte KATP channels in A478V, but normal ATP sensitivity in V65M, suggesting that cardiac remodeling occurs secondary to KATP overactivity outside of the heart. These SUR2[A478V] and Kir6.1[V65M] animals thus reiterate the key cardiovascular features seen in human CS. They establish the molecular basis of the pathophysiological consequences of reduced smooth muscle excitability resulting from SUR2/Kir6.1–dependent KATP GOF, and provide a validated animal model in which to examine potential therapeutic approaches to treating CS.

ABCC8 gene mutations with different inheritance patterns have been well described to cause transient and permanent forms of neonatal diabetes with onset of hyperglycemia commonly before the age of 6 months, and rare cases between 6 and 12 months. However, recent analyses have also demonstrated ABCC8 gene mutations in patients with monogenic diabetes (maturity onset diabetes of the young, MODY), with milder clinical phenotypes and later onset of hyperglycemia. We report two siblings with diabetes mellitus due to a novel homozygous p.(Phe1068Ile) (c.3202T>A) missense mutation of the ABCC8 gene, but significantly different phenotypes. The index case was diagnosed with diabetes due to an incidental finding of hyperglycemia at the age of 3 years, while her younger sibling presented with severe hyperglycemia and hyperosmolar dehydration at the age of 10 weeks. The possibility of a significant discordance in the correlation between genotype and phenotype needs to be taken into account when ABCC8 mutation dependent diabetes occurs within the same family. Genetic screening in children with diabetes from consanguineous family needs consideration, especially in case of negative autoantibodies and early onset of hyperglycemia.

Background Hemorrhagic transformation is a major complication of ischemic stroke, is linked to matrix metalloproteinase-9 (MMP-9), and is exacerbated by tissue plasminogen activator (tPA). Cerebral ischemia/reperfusion is characterized by SUR1-TRPM4 (sulfonylurea receptor 1—transient receptor potential melastatin 4) channel upregulation in microvascular endothelium. In humans and rodents with cerebral ischemia/reperfusion (I/R), the SUR1 antagonist, glibenclamide, reduces hemorrhagic transformation and plasma MMP-9, but the mechanism is unknown. We hypothesized that tPA induces protease activated receptor 1 (PAR1)-mediated, Ca2+-dependent phasic secretion of MMP-9 from activated brain endothelium, and that SUR1-TRPM4 is required for this process. Methods Cerebral I/R, of 2 and 4 hours duration, respectively, was obtained using conventional middle cerebral artery occlusion. Immunolabeling was used to quantify p65 nuclear translocation. Murine and human brain endothelial cells (BEC) were studied in vitro, without and with NF-κB activation, using immunoblot, zymography and ELISA, patch clamp electrophysiology, and calcium imaging. Genetic and pharmacological manipulations were used to identify signaling pathways. Results Cerebral I/R caused prominent nuclear translocation of p65 in microvascular endothelium. NF-κB-activation of BEC caused de novo expression of SUR1-TRPM4 channels. In NF-κB-activated BEC: (i) tPA caused opening of SUR1-TRPM4 channels in a plasmin-, PAR1-, TRPC3- and Ca2+-dependent manner; (ii) tPA caused PAR1-dependent secretion of MMP-9; (iii) tonic secretion of MMP-9 by activated BEC was not influenced by SUR1 inhibition; (iv) phasic secretion of MMP-9 induced by tPA or the PAR1-agonist, TFLLR, required functional SUR1-TRPM4 channels, with inhibition of SUR1 decreasing tPA-induced MMP-9 secretion. Conclusions tPA induces PAR1-mediated, SUR1-TRPM4-dependent, phasic secretion of MMP-9 from activated brain endothelium.

ATP-sensitive potassium channels (KATP) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic KATP channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic KATP channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 (inward-rectifying potassium channel 6.2) N-terminus participates in the coupling between the peripheral SUR1 (sulfonylurea receptor 1) subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit. Electronic supplementary material The online version of this article (10.1007/s13238-018-0530-y) contains supplementary material, which is available to authorized users.

Despite being highly toxic, carbon monoxide (CO) is also an essential intracellular signalling molecule. The mechanisms of CO-dependent cell signalling are poorly defined, but are likely to involve interactions with heme proteins. One such role for CO is in ion channel regulation. Here, we examine the interaction of CO with KATP channels. We find that CO activates KATP channels and that heme binding to a CXXHX16H motif on the SUR2A receptor is required for the CO-dependent increase in channel activity. Spectroscopic and kinetic data were used to quantify the interaction of CO with the ferrous heme-SUR2A complex. The results are significant because they directly connect CO-dependent regulation to a heme-binding event on the channel. We use this information to present molecular-level insight into the dynamic processes that control the interactions of CO with a heme-regulated channel protein, and we present a structural framework for understanding the complex interplay between heme and CO in ion channel regulation., Despite being highly toxic, carbon monoxide (CO) is also essential as an intracellular signalling molecule, but CO-dependent signalling is poorly understood. Here, authors employ spectroscopic and electrophysiology methods and find that CO activates KATP channels via SUR2A, a heme-regulated receptor.