Activation of the KATP channel by Mg-nucleotide interaction with SUR1

The mechanism of adenosine triphosphate (ATP)-sensitive potassium (KATP) channel activation by Mg-nucleotides was studied using a mutation (G334D) in the Kir6.2 subunit of the channel that renders KATP channels insensitive to nucleotide inhibition and has no apparent effect on their gating. KATP channels carrying this mutation (Kir6.2-G334D/SUR1 channels) were activated by MgATP and MgADP with an EC50 of 112 and 8 µM, respectively. This activation was largely suppressed by mutation of the Walker A lysines in the nucleotide-binding domains of SUR1: the remaining small (∼10%), slowly developing component of MgATP activation was fully inhibited by the lipid kinase inhibitor LY294002. The EC50 for activation of Kir6.2-G334D/SUR1 currents by MgADP was lower than that for MgATP, and the time course of activation was faster. The poorly hydrolyzable analogue MgATPγS also activated Kir6.2-G334D/SUR1. AMPPCP both failed to activate Kir6.2-G334D/SUR1 and to prevent its activation by MgATP. Maximal stimulatory concentrations of MgATP (10 mM) and MgADP (1 mM) exerted identical effects on the single-channel kinetics: they dramatically elevated the open probability (PO > 0.8), increased the mean open time and the mean burst duration, reduced the frequency and number of interburst closed states, and eliminated the short burst states. By comparing our results with those obtained for wild-type KATP channels, we conclude that the MgADP sensitivity of the wild-type KATP channel can be described quantitatively by a combination of inhibition at Kir6.2 (measured for wild-type channels in the absence of Mg2+) and activation via SUR1 (determined for Kir6.2-G334D/SUR1 channels). However, this is not the case for the effects of MgATP

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

Molecular Analysis of ATP-sensitive K Channel Gating and Implications for Channel Inhibition by ATP

The β cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2ΔC26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2ΔC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2ΔC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 μM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state

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

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

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

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

Lipophilicity predicts the ability of nonsulphonylurea drugs to block pancreatic beta-cell KATP channels and stimulate insulin secretion: statins as a test case

Aims KATP ion channels play a key role in glucose‐stimulated insulin secretion. However, many drugs block KATP as “off targets” leading to hyperinsulinaemia and hypoglycaemia. As such drugs are often lipophilic, the aim was to examine the relationship between drug lipophilicity (P) and IC50 for KATP block and explore if the IC50's of statins could be predicted from their lipophilicity and whether this would allow one to forecast their acute action on insulin secretion. Materials and methods A meta‐analysis of 26 lipophilic, nonsulphonylurea, blockers of KATP was performed. From this, the IC50's for pravastatin and simvastatin were predicted and then tested experimentally by exploring their effects on KATP channel activity via patch‐clamp measurement, calcium imaging and insulin secretion in murine beta cells and islets. Results Nonsulphonylurea drugs inhibited KATP channels with a Log IC50 linearly related to their logP. Simvastatin blocked KATP with an IC50 of 25 nmol/L, a value independent of cytosolic factors, and within the range predicted by its lipophilicity (21‐690 nmol/L). 10 μmol/L pravastatin, predicted IC50 0.2‐12 mmol/L, was without effect on the KATP channel. At 10‐fold therapeutic levels, 100 nmol/L simvastatin depolarized the beta‐cell membrane potential and stimulated Ca2+ influx but did not affect insulin secretion; the latter could be explained by serum binding. Conclusions The logP of a drug can aid prediction for its ability to block beta‐cell KATP ion channels. However, although the IC50 for the block of KATP by simvastatin was predicted, the difference between this and therapeutic levels, as well as serum sequestration, explains why hypoglycaemia is unlikely to be observed with acute use of this statin