Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP(2)) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL(−)) with a distinct second site is required for high PIP(2) sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP(2) sensitivity, even in the absence of PL(−). Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP(2) (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL(−) binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP(2) site and explaining the positive allostery between PL(−) binding and PIP(2) sensitivity

Mutations at the same residue (R50) of Kir6.2 (KCNJ11) that cause neonatal diabetes produce different functional effects

Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive K(+) channel (K(ATP) channel), are a common cause of neonatal diabetes. We identified a novel KCNJ11 mutation, R50Q, that causes permanent neonatal diabetes (PNDM) without neurological problems. We investigated the functional effects this mutation and another at the same residue (R50P) that led to PNDM in association with developmental delay. Wild-type or mutant Kir6.2/SUR1 channels were examined by heterologous expression in Xenopus oocytes. Both mutations increased resting whole-cell currents through homomeric and heterozygous K(ATP) channels by reducing channel inhibition by ATP, an effect that was larger in the presence of Mg(2+). However the magnitude of the reduction in ATP sensitivity (and the increase in the whole-cell current) was substantially larger for the R50P mutation. This is consistent with the more severe phenotype. Single-R50P channel kinetics (in the absence of ATP) did not differ from wild type, indicating that the mutation primarily affects ATP binding and/or transduction. This supports the idea that R50 lies in the ATP-binding site of Kir6.2. The sulfonylurea tolbutamide blocked heterozygous R50Q (89%) and R50P (84%) channels only slightly less than wild-type channels (98%), suggesting that sulfonylurea therapy may be of benefit for patients with either mutation

Binding of sulphonylureas to plasma proteins – a KATP channel perspective

Sulphonylurea drugs stimulate insulin secretion from pancreatic β-cells primarily by inhibiting ATP sensitive potassium (KATP) channels in the β-cell membrane. The effective sulphonylurea concentration at its site of action is significantly attenuated by binding to serum albumin, which makes it difficult to compare in vitro and in vivo data. We therefore measured the ability of gliclazide and glibenclamide to inhibit KATP channels and stimulate insulin secretion in the presence of serum albumin. We used this data, together with estimates of free drug concentrations from binding studies, to predict the extent of sulphonylurea inhibition of KATP channels at therapeutic concentrations in vivo. KATP currents from mouse pancreatic β-cells and Xenopus oocytes were measured using the patch-clamp technique. Gliclazide and glibenclamide binding to human plasma were determined in spiked plasma samples using an ultrafiltration-mass spectrometry approach. Bovine serum albumin (60g/l) produced a mild, non-significant reduction of gliclazide block of KATP currents in pancreatic β-cells and Xenopus oocytes. In contrast, glibenclamide inhibition of recombinant KATP channels was dramatically suppressed by albumin (predicted free drug concentration <0.1%). Insulin secretion was also reduced. Free concentrations of gliclazide and glibenclamide in the presence of human plasma measured in binding experiments were 15% and 0.05%, respectively. Our data suggest the free concentration of glibenclamide in plasma is too low to account for the drug’s therapeutic effect. In contrast, the free gliclazide concentration in plasma is high enough to close KATP channels and stimulate insulin secretion

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

Vertebral Formulae and Congenital Vertebral Anomalies in Guinea Pigs: A Retrospective Radiographic Study

The objectives of this retrospective study of 240 guinea pigs (148 females and 92 males) were to determine the prevalence of different vertebral formulae and the type and anatomical localization of congenital vertebral anomalies (CVA). Radiographs of the cervical (C), thoracic (Th), lumbar (L), sacral (S), and caudal (Cd) part of the vertebral column were reviewed. Morphology and number of vertebrae in each segment of the vertebral column and type and localization of CVA were recorded. In 210/240 guinea pigs (87.50%) with normal vertebral morphology, nine vertebral formulae were found with constant number of C but variable number of Th, L, and S vertebrae: C7/Th13/L6/S4/Cd5-7 (75%), C7/Th13/L6/S3/Cd6-7 (4.17%), C7/Th13/L5/S4/Cd6-7 (2.50%), C7/Th13/L6/S5/Cd5-6 (1.67%), C7/Th12/L6/S4/Cd6 (1.25%), C7/Th13/L7/S4/Cd6 (1.25%), C7/Th13/L7/S3/Cd6-7 (0.83%), C7/Th12/L7/S4/Cd5 (0.42%), C7/Th13/L5/S5/Cd7 (0.42%). CVA were found in 30/240 (12.5%) of guinea pigs, mostly as a transitional vertebra (28/30), which represents 100% of single CVA localised in cervicothoracic (n = 1), thoracolumbar (n = 22) and lumbosacral segments (n = 5). Five morphological variants of thoracolumbar transitional vertebrae (TTV) were identified. Two (2/30) guinea pigs had a combination of CVA: cervical block vertebra and TTV (n = 1) and TTV and lumbosacral transitional vertebra (LTV) (n = 1). These findings suggest that guinea pigs’ vertebral column displays more morphological variants with occasional CVA predominantly transitional vertebrae

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

Diabetes mellitus and renal failure: Prevention and management.

Nowadays, diabetes mellitus (DM) and hypertension are considered as the most common causes of end-stage renal disease (ESRD). In this paper, other than presenting the role of DM in ESRD, glucose metabolism and the management of hyperglycemia in these patients are reviewed. Although in several large studies there was no significant relationship found between tight glycemic control and the survival of ESRD patients, it is recommended that glycemic control be considered as the main therapeutic goal in the treatment of these patients to prevent damage to other organs. Glycemic control is perfect when fasting blood sugar is less than 140 mg/dL, 1-h postprandial blood glucose is less than 200 mg/dL, and glycosylated hemoglobin (HbA1c) is 6-7 in patients with type 1 diabetes and 7-8 in patients with type 2 diabetes. Administration of metformin should be avoided in chronic renal failure (CRF) because of lactic acidosis, the potentially fatal complication of metformin, but glipizide and repaglinide seem to be good choices

The use of dopamine-hyaluronate associate-coated maghemite nanoparticles to label cells

Sodium hyaluronate (HA) was associated with dopamine (DPA) and introduced as a coating for maghemite (γ-Fe2O3) nanoparticles obtained by the coprecipitation of iron(II) and iron(III) chlorides and oxidation with sodium hypochlorite. The effects of the DPA anchorage of HA on the γ-Fe2O3 surface on the physicochemical properties of the resulting colloids were investigated. Nanoparticles coated at three different DPA-HA/γ-Fe2O3 and DPA/HA ratios were chosen for experiments with rat bone marrow mesenchymal stem cells and human chondrocytes. The nanoparticles were internalized into rat bone marrow mesenchymal stem cells via endocytosis as confirmed by Prussian Blue staining. The efficiency of mesenchymal stem cell labeling was analyzed. From among the investigated samples, efficient cell labeling was achieved by using DPA-HA-γ-Fe2O3 nanoparticles with DPA-HA/γ-Fe2O3 = 0.45 (weight/ weight) and DPA/HA = 0.038 (weight/weight) ratios. The particles were used as a contrast agent in magnetic resonance imaging for the labeling and visualization of cells