ATP binding without hydrolysis switches sulfonylurea receptor 1 (SUR1) to outward-facing conformations that activate KATP channels.

Neuroendocrine-type ATP-sensitive K+ (KATP) 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, KD , values for the conformation-sensitive channel antagonist [3H]glibenclamide ([3H]GBM), indicating that ATP can act as a pure agonist. Assembly with Kir6.2 reduced SUR1's KD for [3H]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 KD for [3H]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 KATP channels toward open states, consistent with SUR1 variants with lower KD values causing neonatal diabetes, whereas increased KD values cause congenital hyperinsulinism.

Possible New Strategies for the Treatment of Congenital Hyperinsulinism.

Objective: Congenital hyperinsulinism (CHI) is a rare disease characterized by persistent hypoglycemia as a result of inappropriate insulin secretion, which can lead to irreversible neurological defects in infants. Poor efficacy and strong adverse effects of the current medications impede successful treatment. The aim of the study was to investigate new approaches to silence ß-cells and thus attenuate insulin secretion. Research Design and Methods: In the scope of our research, we tested substances more selective and more potent than the gold standard diazoxide that also interact with neuroendocrine ATP-sensitive K+ (KATP) channels. Additionally, KATP channel-independent targets as Ca2+-activated K+ channels of intermediate conductance (KCa3.1) and L-type Ca2+ channels were investigated. Experiments were performed using human islet cell clusters isolated from tissue of CHI patients (histologically classified as pathological) and islet cell clusters obtained from C57BL/6N (WT) or SUR1 knockout (SUR1-/-) mice. The cytosolic Ca2+ concentration ([Ca2+]c) was used as a parameter for the pathway regulated by electrical activity and was determined by fura-2 fluorescence. The mitochondrial membrane potential (ΔΨ) was determined by rhodamine 123 fluorescence and single channel currents were measured by the patch-clamp technique. Results: The selective KATP channel opener NN414 (5 µM) diminished [Ca2+]c in isolated human CHI islet cell clusters and WT mouse islet cell clusters stimulated with 10 mM glucose. In islet cell clusters lacking functional KATP channels (SUR1-/-) the drug was without effect. VU0071063 (30 µM), another KATP channel opener considered to be selective, lowered [Ca2+]c in human CHI islet cell clusters. The compound was also effective in islet cell clusters from SUR1-/- mice, showing that [Ca2+]c is influenced by additional effects besides KATP channels. Contrasting to NN414, the drug depolarized ΔΨ in murine islet cell clusters pointing to severe interference with mitochondrial metabolism. An opener of KCa3.1 channels, DCEBIO (100 µM), significantly decreased [Ca2+]c in SUR1-/- and human CHI islet cell clusters. To target L-type Ca2+ channels we tested two already approved drugs, dextromethorphan (DXM) and simvastatin. DXM (100 µM) efficiently diminished [Ca2+]c in stimulated human CHI islet cell clusters as well as in stimulated SUR1-/- islet cell clusters. Similar effects on [Ca2+]c were observed in experiments with simvastatin (7.2 µM). Conclusions: NN414 seems to provide a good alternative to the currently used KATP channel opener diazoxide. Targeting KCa3.1 channels by channel openers or L-type Ca2+ channels by DXM or simvastatin might be valuable approaches for treatment of CHI caused by mutations of KATP channels not sensitive to KATP channel openers.

ATP mediates a negative autocrine signal on stimulus-secretion coupling in mouse pancreatic ß-cells.

PURPOSE: The role of ATP, which is secreted by pancreatic ß-cells, is still a matter of debate. It has been postulated that extracellular ATP acts as a positive auto- or paracrine signal in ß-cells amplifying insulin secretion. However, there is rising evidence that extracellular ATP may also mediate a negative signal. METHODS: We evaluated whether extracellular ATP interferes with the Ca2+-mediated negative feedback mechanism that regulates oscillatory activity of ß-cells. RESULTS: To experimentally uncover the Ca2+-induced feedback we applied a high extracellular Ca2+ concentration. Under this condition ATP (100 µM) inhibited glucose-evoked oscillations of electrical activity and hyperpolarized the membrane potential. Furthermore, ATP acutely increased the interburst phase of Ca2+ oscillations and reduced the current through L-type Ca2+ channels. Accordingly, ATP (500 µM) decreased glucose-induced insulin secretion. The ATP effect was not mimicked by AMP, ADP, or adenosine. The use of specific agonists and antagonists and mice deficient of large conductance Ca2+-dependent K+ channels revealed that P2X, but not P2Y receptors, and Ca2+-dependent K+ channels are involved in the underlying signaling cascade induced by ATP. The effectiveness of ATP to interfere with parameters of stimulus-secretion coupling is markedly reduced at low extracellular Ca2+ concentration. CONCLUSION: It is suggested that extracellular ATP which is co-secreted with insulin in a pulsatile manner during glucose-stimulated exocytosis provides a negative feedback signal driving ß-cell oscillations in co-operation with Ca2+ and other signals.

Atrial Natriuretic Peptide Affects Stimulus-Secretion Coupling of Pancreatic ß-Cells.

Atrial natriuretic peptide (ANP) influences glucose homeostasis and possibly acts as a link between the cardiovascular system and metabolism, especially in metabolic disorders like diabetes. The current study evaluated effects of ANP on ß-cell function by the use of a ß-cell-specific knockout of the ANP receptor with guanylate cyclase activity (ßGC-A-KO). ANP augmented insulin secretion at the threshold glucose concentration of 6 mmol/L and decreased KATP single-channel activity in ß-cells of control mice but not of ßGC-A-KO mice. In wild-type ß-cells but not ß-cells lacking functional KATP channels (SUR1-KO), ANP increased electrical activity, suggesting no involvement of other ion channels. At 6 mmol/L glucose, ANP readily elicited Ca2+ influx in control ß-cells. This effect was blunted in ß-cells of ßGC-A-KO mice, and the maximal cytosolic Ca2+ concentration was lower. Experiments with inhibitors of protein kinase G (PKG), protein kinase A (PKA), phosphodiesterase 3B (PDE3B), and a membrane-permeable cyclic guanosine monophosphate (cGMP) analog on KATP channel activity and insulin secretion point to participation of the cGMP/PKG and cAMP/PKA/Epac (exchange protein directly activated by cAMP) directly activated by cAMP Epac pathways in the effects of ANP on ß-cell function; the latter seems to prevail. Moreover, ANP potentiated the effect of glucagon-like peptide 1 (GLP-1) on glucose-induced insulin secretion, which could be caused by a cGMP-mediated inhibition of PDE3B, which in turn reduces cAMP degradation.

The LXR Ligand T0901317 Acutely Inhibits Insulin Secretion by Affecting Mitochondrial Metabolism.

The role of liver X receptor (LXR) in pancreatic ß-cell physiology and pathophysiology is still unclear. It has been postulated that chronic LXR activation in ß-cells induces lipotoxicity, a key step in the development of ß-cell dysfunction, which accompanies type 2 diabetes mellitus. In most of these studies, the LXR ligand T0901317 has been administered chronically in the micromolar range to study the significance of LXR activation. In the current study, we have evaluated acute effects of T0901317 on stimulus-secretion coupling of ß-cells. We found that 10 µM T0901317 completely suppressed oscillations of the cytosolic Ca2+ concentration induced by 15 mM glucose. Obviously, this effect was due to inhibition of mitochondrial metabolism. T0901317 markedly depolarized the mitochondrial membrane potential, thus inhibiting adenosine triphosphate (ATP) production and reducing the cytosolic ATP concentration. This led in turn to a huge increase in KATP current and hyperpolarization of the cell membrane potential. Eventually, T0901317 inhibited glucose-induced insulin secretion. These effects were rapid in on-set and not compatible with the activation of a nuclear receptor. In vivo, T0901317 acutely increased the blood glucose concentration after intraperitoneal application. In summary, these data clearly demonstrate that T0901317 exerts acute effects on stimulus-secretion coupling. This observation questions the chronic use of T0901317 and limits the interpretation of results obtained under these experimental conditions.