How ATP Inhibits the Open KATP Channel

ATP-sensitive potassium (KATP) channels are composed of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits. Binding of ATP to Kir6.2 leads to inhibition of channel activity. Because there are four subunits and thus four ATP-binding sites, four binding events are possible. ATP binds to both the open and closed states of the channel and produces a decrease in the mean open time, a reduction in the mean burst duration, and an increase in the frequency and duration of the interburst closed states. Here, we investigate the mechanism of interaction of ATP with the open state of the channel by analyzing the single-channel kinetics of concatenated Kir6.2 tetramers containing from zero to four mutated Kir6.2 subunits that possess an impaired ATP-binding site. We show that the ATP-dependent decrease in the mean burst duration is well described by a Monod-Wyman-Changeux model in which channel closing is produced by all four subunits acting in a single concerted step. The data are inconsistent with a Hodgkin-Huxley model (four independent steps) or a dimer model (two independent dimers). When the channel is open, ATP binds to a single ATP-binding site with a dissociation constant of 300 μM

Mutations within the P-Loop of Kir6.2 Modulate the Intraburst Kinetics of the Atp-Sensitive Potassium Channel

The ATP-sensitive potassium (KATP) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 KATP channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (τo) and the short closed time (τC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer τo, shorter τC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the KATP channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings

New insights into the tissue specificity of sulphonylureas

Pochodne sulfonylomocznika, podawane chorym na cukrzycę typu 2, stymulują wydzielanie insuliny poprzez zamknięcie ATP-zależnych kanałów potasowych (KATP) w błonie komórkowej komórek b trzustki. Leki te wiążą się z podjednostką kanału potasowego, będącą receptorem dla pochodnych sulfonylomocznika (SUR 1). Kanały KATP są zbudowane z 2 różnych typów podjednostek: podjednostki tworzącej światło kanału (zwykle Kir 6.2) oraz receptora dla pochodnych sulfonylomocznika (SUR), które wspólnie tworzą heteromeryczny kompleks 4:4. Obecnie znanych jest kilka izoform podjednostek receptora SUR, które występują w kanałach KATP w różnych tkankach: kanały KATP w komórkach b trzustki zawierają podjednostkę SUR 1, kardiomiocyty - podjednostkę SUR 2A, a komórki mięśni gładkich - podjednostkę SUR 2B. Wrażliwość kanałów KATP na pochodne sulfonylomocznika zależy od typu podjednostki SUR. Gliklazyd i tolbutamid z dużym powinowactwem hamują przewodnictwo w kanałach komórek b trzustki, lecz nie w kanałach KATP kardiomiocytów i komórek mięśni gładkich. W przeciwieństwie do tych leków, glibenklamid i glimepiryd blokują wszystkie 3 typy kanałów KATP z podobną siłą. Pochodne sulfonylomocznika różnią się również odwracalnością wiązania z receptorami -tolbutamid i gliklazyd zamykają wszystkie typy kanałów KATP w sposób odwracalny, podczas gdy glibenklamid i glimepiryd powodują wprawdzie odwracalną blokadę kanałów sercowych, ale nie kanałów KATP w komórkach b trzustki. Wrażliwość kanałów KATP na pochodne sulfonylomocznika reguluje znajdujący się wewnątrz komórki MgADP, który nasila zamykanie kanałów KATP wywołane przez pochodne sulfonylomocznika w komórkach b, a osłabia tę blokadę w kardiomiocytach. W niniejszej pracy przedstawiono najnowsze osiągnięcia dotyczące mechanizmów działania pochodnych sulfonylomocznika na kanały KATP oraz omówiono ich konsekwencje w przypadku stosowania tej grupy leków w terapii cukrzycy typu 2.Sulphonylureas stimulate insulin secretion in type-2 diabetic patients by closing ATP-sensitive (KATP) potassium channels in the plasma membrane of pancreatic b-cells. This effect is mediated by binding of the drug to the sulphonylurea receptor (SUR 1) subunit of the channel. KATP channels are formed of two different types of subunit: a pore-forming subunit (usually Kir 6.2) and a sulphonylurea receptor subunit (SUR), which associate in a 4:4 heteromeric complex. Several different isoforms of SUR are known and KATP channels in different tissues possess different types of SUR subunit (SUR 1 in b-cells, SUR 2A in heart, and SUR 2B in smooth muscle). The sulphonylurea-sensitivity of KATP channels varies with the type of SUR subunit: thus, gliclazide and tolbutamide potently block the b-cell, but not the cardiac or smooth muscle types of KATP channel. In contrast, glibenclamide and glimepiride block all three types of KATP channel with similar potency

Evaluating inositol phospholipid interactions with inward rectifier potassium channels and characterising their role in disease

Membrane proteins are frequently modulated by specific protein-lipid interactions. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised. Here, we apply a coarse-grained molecular dynamics free energy perturbation (CG-FEP) protocol to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step approach, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid. Furthermore, by perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K increases PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. We show good agreement with electrophysiological data where E179K exhibits a reduction in neomycin sensitivity, implying that PIP2 binds more tightly E179K channels. This illustrates the application of CG-FEP to compare affinities between lipid species, and for annotating amino acid residues

Dysregulation of Glucagon Secretion by Hyperglycemia-Induced Sodium-Dependent Reduction of ATP Production

© 2018 The Author(s). Published by Elsevier Inc.Diabetes is a bihormonal disorder resulting from combined insulin and glucagon secretion defects. Mice lacking fumarase (Fh1) in their β cells (Fh1βKO mice) develop progressive hyperglycemia and dysregulated glucagon secretion similar to that seen in diabetic patients (too much at high glucose and too little at low glucose). The glucagon secretion defects are corrected by low concentrations of tolbutamide and prevented by the sodium-glucose transport (SGLT) inhibitor phlorizin. These data link hyperglycemia, intracellular Na+ accumulation, and acidification to impaired mitochondrial metabolism, reduced ATP production, and dysregulated glucagon secretion. Protein succination, reflecting reduced activity of fumarase, is observed in α cells from hyperglycemic Fh1βKO and β-V59M gain-of-function KATP channel mice, diabetic Goto-Kakizaki rats, and patients with type 2 diabetes. Succination is also observed in renal tubular cells and cardiomyocytes from hyperglycemic Fh1βKO mice, suggesting that the model can be extended to other SGLT-expressing cells and may explain part of the spectrum of diabetic complications.Peer reviewe

New insights into KATP channel gene mutations and neonatal diabetes mellitus

The ATP-sensitive potassium channel (KATP channel) couples blood levels of glucose to insulin secretion from pancreatic β-cells. KATP channel closure triggers a cascade of events that results in insulin release. Metabolically generated changes in the intracellular concentrations of adenosine nucleotides are integral to this regulation, with ATP and ADP closing the channel and MgATP and MgADP increasing channel activity. Activating mutations in the genes encoding either of the two types of KATP channel subunit (Kir6.2 and SUR1) result in neonatal diabetes mellitus, whereas loss-of-function mutations cause hyperinsulinaemic hypoglycaemia of infancy. Sulfonylurea and glinide drugs, which bind to SUR1, close the channel through a pathway independent of ATP and are now the primary therapy for neonatal diabetes mellitus caused by mutations in the genes encoding KATP channel subunits. Insight into the molecular details of drug and nucleotide regulation of channel activity has been illuminated by cryo-electron microscopy structures that reveal the atomic-level organization of the KATP channel complex. Here we review how these structures aid our understanding of how the various mutations in the genes encoding Kir6.2 (KCNJ11) and SUR1 (ABCC8) lead to a reduction in ATP inhibition and thereby neonatal diabetes mellitus. We also provide an update on known mutations and sulfonylurea therapy in neonatal diabetes mellitus

Gain-of-Function Mutations in the K_ATP Channel (KCNJ11) Impair Coordinated Hand-Eye Tracking

Background: Gain-of-function mutations in the ATP-sensitive potassium channel can cause permanent neonatal diabetes mellitus (PNDM) or neonatal diabetes accompanied by a constellation of neurological symptoms (iDEND syndrome). Studies of a mouse model of iDEND syndrome revealed that cerebellar Purkinje cell electrical activity was impaired and that the mice exhibited poor motor coordination. In this study, we probed the hand-eye coordination of PNDM and iDEND patients using visual tracking tasks to see if poor motor coordination is also a feature of the human disease.Methods: Control participants (n = 14), patients with iDEND syndrome (n = 6 or 7), and patients with PNDM (n = 7) completed three computer-based tasks in which a moving target was tracked with a joystick-controlled cursor. Patients with PNDM and iDEND were being treated with sulphonylurea drugs at the time of testing.Results: No differences were seen between PNDM patients and controls. Patients with iDEND syndrome were significantly less accurate than controls in two of the three tasks. The greatest differences were seen when iDEND patients tracked blanked targets, i.e. when predictive tracking was required. In this task, iDEND patients incurred more discrepancy errors (p = 0.009) and more velocity errors (p  = 0.009) than controls.Conclusions: These results identify impaired hand-eye coordination as a new clinical feature of iDEND. The aetiology of this feature is likely to involve cerebellar dysfunction. The data further suggest that sulphonylurea doses that control the diabetes of these patients may be insufficient to fully correct their neurological symptoms.</p

CLEAR-AI: empowering people living with dementia and their carers to understand and reduce distress

People living with dementia sometimes present with behaviours that carers find difficult to understand and manage. These include aggression, pacing, vocalising, exit-seeking and sexually inappropriate behaviour. They can be present in up to 70% of people living with dementia and often present because of misunderstanding or because of the distress the person experiences trying to cope with the daily challenges of living with their illness. These behaviours increase the risk that a person will move from their home to a care home. CLEAR Dementia Care© helps carers to understand behaviour in the context of the person and their environment, identify unmet needs and respond in ways to reduce distress. We present the pilot of “CLEAR-AI”, an artificial intelligence (AI) powered platform that interprets data from a range of connected smart sensors, apps and devices to model the person with dementia’s daily routines. Analysis of the data and training the AI model enables the platform to identify the triggers that precede distress episodes and to recognise when episodes occur in the context of previous activities in the day. Using these models, and with CLEAR’s assessment as baseline, we can initiate interventions into daily schedules that reduce or mitigate distress where it is likely to arise. The goal is to reduce carer burden and enable the person to live at home with as much independence as possible for as long as possible. Our consortium brings together people living with dementia and their carers, commissioners of digital social care, specialists in dementia care, AI and digital solutions. The co-design approach ensures that we are led by stakeholders’ needs to improve quality of life