scholarly journals Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: A mechanistic study

2014 ◽  
Vol 144 (5) ◽  
pp. 469-486 ◽  
Author(s):  
Peter Proks ◽  
Heidi de Wet ◽  
Frances M. Ashcroft
Keyword(s):  
Katp Channel ◽  
Neonatal Diabetes ◽  
Katp Channels ◽  
Nucleotide Binding ◽  
Mechanistic Study ◽  
Xenopus Laevis Oocytes ◽  
Sulfonylurea Receptor ◽  
Β Cell ◽  
Stimulatory Action ◽  
Channel Inhibition

Sulfonylureas, which stimulate insulin secretion from pancreatic β-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K+ (KATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on β-cell and cardiac types of KATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the KATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on β-cell KATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas.

KATP channels and insulin secretion: a key role in health and disease

2006 ◽  
Vol 34 (2) ◽  
pp. 243-246 ◽  
Author(s):  
F.M. Ashcroft
Keyword(s):  
Glucose Intolerance ◽  
Katp Channel ◽  
Cell Function ◽  
Neonatal Diabetes ◽  
Katp Channels ◽  
Β Cell ◽  
Channel Inhibition ◽  
Β Cell Function ◽  
Health And Disease ◽  
And Function

This review summarizes advances in our understanding of the structure and function of the ATP-sensitive potassium (KATP) channel of the pancreatic β-cell that have been made over the last 5 years. It discusses recent structural studies of the octameric KATP channel complex and studies of the regulation of KATP channel activity by nucleotides. It then considers the molecular mechanism by which gain-of-function mutations in the Kir6.2 subunit of the KATP channel reduce channel inhibition by ATP and thereby lead to neonatal diabetes, and how identification of these mutations has led to changes in therapy. Finally, it illustrates how mouse models of glucose intolerance or diabetes can provide fresh insight into β-cell function, using the C57BL/6J mouse, whose glucose intolerance arises from mutations in nicotinamide nucleotide transhydrogenase, as an example.

scholarly journals Regulation of KATP Channel Activity by Diazoxide and MgADP

1997 ◽  
Vol 110 (6) ◽  
pp. 643-654 ◽  
Author(s):  
S.-L. Shyng ◽  
T. Ferrigni ◽  
C.G. Nichols
Keyword(s):  
Katp Channel ◽  
Katp Channels ◽  
Nucleotide Binding ◽  
Sulfonylurea Receptor ◽  
Channel Activity ◽  
Nucleotide Hydrolysis ◽  
Inward Rectifier Potassium Channel ◽  
Hydrolysis Rates ◽  
Atp Inhibition

KATP channels were reconstituted in COSm6 cells by coexpression of the sulfonylurea receptor SUR1 and the inward rectifier potassium channel Kir6.2. The role of the two nucleotide binding folds of SUR1 in regulation of KATP channel activity by nucleotides and diazoxide was investigated. Mutations in the linker region and the Walker B motif (Walker, J.E., M.J. Saraste, M.J. Runswick, and N.J. Gay. 1982. EMBO [Eur. Mol. Biol. Organ.] J. 1:945–951) of the second nucleotide binding fold, including G1479D, G1479R, G1485D, G1485R, Q1486H, and D1506A, all abolished stimulation by MgADP and diazoxide, with the exception of G1479R, which showed a small stimulatory response to diazoxide. Analogous mutations in the first nucleotide binding fold, including G827D, G827R, and Q834H, were still stimulated by diazoxide and MgADP, but with altered kinetics compared with the wild-type channel. None of the mutations altered the sensitivity of the channel to inhibition by ATP4−. We propose a model in which SUR1 sensitizes the KATP channel to ATP inhibition, and nucleotide hydrolysis at the nucleotide binding folds blocks this effect. MgADP and diazoxide are proposed to stabilize this desensitized state of the channel, and mutations at the nucleotide binding folds alter the response of channels to MgADP and diazoxide by altering nucleotide hydrolysis rates or the coupling of hydrolysis to channel activation.

scholarly journals Neonatal Diabetes Caused by Mutations in Sulfonylurea Receptor 1: Interplay between Expression and Mg-Nucleotide Gating Defects of ATP-Sensitive Potassium Channels

2010 ◽  
Vol 24 (10) ◽  
pp. 2070-2070
Author(s):  
Qing Zhou ◽  
Intza Garin ◽  
Luis Castaño ◽  
Jesús Argente ◽  
Ma. Teresa Muñoz-Calvo ◽  
...  
Keyword(s):  
Neonatal Diabetes ◽  
Katp Channels ◽  
Nucleotide Binding ◽  
Sulfonylurea Receptor ◽  
Channel Response ◽  
Potential Gain ◽  
Channel Expression ◽  
Transient Neonatal Diabetes ◽  
Sulfonylurea Receptor 1 ◽  
Diabetes Severity

Abstract Context: ATP-sensitive potassium (KATP) channels regulate insulin secretion by coupling glucose metabolism to β-cell membrane potential. Gain-of-function mutations in the sulfonylurea receptor 1 (SUR1) or Kir6.2 channel subunit underlie neonatal diabetes. Objective: The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect KATP channel function. Design: E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gating properties of the resulting channels were assessed biochemically and electrophysiologically. Results: Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP4− or sulfonylureas. Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on β-cells. When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone. Conclusion: The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Furthermore, they suggest that diabetes severity is determined by interplay between effects of a mutation on channel expression and channel gating.

scholarly journals Regulation of Cloned Atp-Sensitive K Channels by Adenine Nucleotides and Sulfonylureas

2001 ◽  
Vol 118 (4) ◽  
pp. 391-406 ◽  
Author(s):  
Scott A. John ◽  
James N. Weiss ◽  
Bernard Ribalet
Keyword(s):  
Katp Channel ◽  
Adenine Nucleotides ◽  
Katp Channels ◽  
Functional Coupling ◽  
Sulfonylurea Receptor ◽  
K Channels ◽  
Channel Regulation ◽  
Channel Opening ◽  
Insight Into ◽  
Positively Charged

KATP channels, comprised of the pore-forming protein Kir6.x and the sulfonylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interactions, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP2, on channel regulation by adenine nucleotides and the sulfonylurea glyburide. Our data show that the Kir6.2 “PIP2-insensitive” mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburide. These results suggest that R176 and R177 are required for functional coupling to SUR1, which confers MgADP and sulfonylurea sensitivity to the KATP channel. In contrast, the R301C and R314C mutants, which are also “PIP2-insensitive,” remained sensitive to stimulation by MgADP in the absence of ATP and were inhibited by glyburide. Based on these findings, as well as previous data, we propose a model of the KATP channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that interacts with NBF1 bound to ATP on SUR1, promoting channel opening by counteracting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the absence of ATP, since KATP channels are not blocked by ATP, they do not require the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence of ATP. The region delimited by R301 and R314 is not involved in the interaction with NBF1 or NBF2, but confers additional PIP2 sensitivity.

scholarly journals Diabetes induced by gain-of-function mutations in the Kir6.1 subunit of the KATP channel

2016 ◽  
Vol 149 (1) ◽  
pp. 75-84 ◽  
Author(s):  
Maria S. Remedi ◽  
Jonathan B. Friedman ◽  
Colin G. Nichols
Keyword(s):  
Insulin Secretion ◽  
Katp Channel ◽  
Vascular System ◽  
Wild Type Mouse ◽  
Neonatal Diabetes ◽  
Katp Channels ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Gain Of Function ◽  
Insulin Promoter

Gain-of-function (GOF) mutations in the pore-forming (Kir6.2) and regulatory (SUR1) subunits of KATP channels have been identified as the most common cause of human neonatal diabetes mellitus. The critical effect of these mutations is confirmed in mice expressing Kir6.2-GOF mutations in pancreatic β cells. A second KATP channel pore-forming subunit, Kir6.1, was originally cloned from the pancreas. Although the prominence of this subunit in the vascular system is well documented, a potential role in pancreatic β cells has not been considered. Here, we show that mice expressing Kir6.1-GOF mutations (Kir6.1[G343D] or Kir6.1[G343D,Q53R]) in pancreatic β cells (under rat-insulin-promoter [Rip] control) develop glucose intolerance and diabetes caused by reduced insulin secretion. We also generated transgenic mice in which a bacterial artificial chromosome (BAC) containing Kir6.1[G343D] is incorporated such that the transgene is only expressed in tissues where Kir6.1 is normally present. Strikingly, BAC-Kir6.1[G343D] mice also show impaired glucose tolerance, as well as reduced glucose- and sulfonylurea-dependent insulin secretion. However, the response to K+ depolarization is intact in Kir6.1-GOF mice compared with control islets. The presence of native Kir6.1 transcripts was demonstrated in both human and wild-type mouse islets using quantitative real-time PCR. Together, these results implicate the incorporation of native Kir6.1 subunits into pancreatic KATP channels and a contributory role for these subunits in the control of insulin secretion.

scholarly journals Mechanism of ATP-sensitive K Channel Inhibition by Sulfhydryl Modification

1998 ◽  
Vol 112 (3) ◽  
pp. 325-332 ◽  
Author(s):  
Stefan Trapp ◽  
Stephen J. Tucker ◽  
Frances M. Ashcroft
Keyword(s):  
Katp Channel ◽  
Membrane Surface ◽  
Katp Channels ◽  
Cysteine Residue ◽  
K Channel ◽  
Atp Binding Site ◽  
Channel Inhibition ◽  
Cysteine Scanning ◽  
Sulfhydryl Modification ◽  
Cysteine Scanning Mutagenesis

ATP-sensitive potassium (KATP) channels are reversibly inhibited by intracellular ATP. Agents that interact with sulfhydryl moieties produce an irreversible inhibition of KATP channel activity when applied to the intracellular membrane surface. ATP appears to protect against this effect, suggesting that the cysteine residue with which thiol reagents interact may either lie within the ATP-binding site or be inaccessible when the channel is closed. We have examined the interaction of the membrane-impermeant thiol-reactive agent p-chloromercuriphenylsulphonate (pCMPS) with the cloned β cell KATP channel. This channel comprises the pore-forming Kir6.2 and regulatory SUR1 subunits. We show that the cysteine residue involved in channel inhibition by pCMPS resides on the Kir6.2 subunit and is located at position 42, which lies within the NH2 terminus of the protein. Although ATP protects against the effects of pCMPS, the ATP sensitivity of the KATP channel was unchanged by mutation of C42 to either valine (V) or alanine (A), suggesting that ATP does not interact directly with this residue. These results are consistent with the idea that C42 is inaccessible to the intracellular solution, and thereby protected from interaction with pCMPS when the channel is closed by ATP. We also observed that the C42A mutation does not affect the ability of SUR1 to endow Kir6.2 with diazoxide sensitivity, and reduces, but does not prevent, the effects of MgADP and tolbutamide, which are mediated via SUR1. The Kir6.2-C42A (or V) mutant channel may provide a suitable background for cysteine-scanning mutagenesis studies.

A disrupter of actin microfilaments impairs sulfonylurea-inhibitory gating of cardiac KATP channels

1996 ◽  
Vol 271 (6) ◽  
pp. H2710-H2716 ◽  
Author(s):  
P. A. Brady ◽  
A. E. Alekseev ◽  
L. A. Aleksandrova ◽  
L. A. Gomez ◽  
A. Terzic
Keyword(s):  
Katp Channel ◽  
Katp Channels ◽  
Hill Coefficient ◽  
Kinetic Properties ◽  
Actin Microfilaments ◽  
Actin Microfilament ◽  
Channel Inhibition ◽  
Dnase Treatment ◽  
Single Channel Currents ◽  
Sulfonylurea Drugs

The efficacy with which sulfonylurea drugs inhibit cardiac ATP-sensitive K+ (KATP) channels is reduced during metabolic compromise and cellular contracture. Disruption of the actin microfilament network, which occurs under similar conditions, reduces the sensitivity of the channel toward intracellular ATP. To investigate whether a disrupter of actin microfilaments could also affect the responsiveness of the KATP channel to sulfonylurea drugs, single-channel currents were measured in the inside-out configuration of excised patches from guinea pig ventricular myocytes. Treatment of the internal side of patches with deoxyribonuclease (DNase) I (100 micrograms/ml), which forms complexes with G actin and prevents actin filament formation, antagonized sulfonylurea-induced inhibition of KATP channels that was coupled with a loss of sensitivity to ATP. The apparent dissociation constant and Hill coefficient for the inhibitory effect of glyburide, a prototype sulfonylurea, on KATP-channel opening were, respectively, 0.13 microM and 0.95 before and 2.7 microM and 0.98 after DNase treatment. DNase did not alter intraburst kinetic properties of the channel. When DNase was denatured or coincubated with purified actin (200 micrograms/ml), it no longer decreased glyburide-induced channel inhibition. This suggests that sulfonylurea-inhibitory gating of cardiac KATP channels may also be regulated through a mechanism involving subsarcolemmal actin microfilament networks.

Molecular Mechanisms of the Inhibitory Effects of Propofol and Thiamylal on Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channels

2004 ◽  
Vol 100 (2) ◽  
pp. 338-346 ◽  
Author(s):  
Takashi Kawano ◽  
Shuzo Oshita ◽  
Akira Takahashi ◽  
Yasuo Tsutsumi ◽  
Yoshinobu Tomiyama ◽  
...  
Keyword(s):  
Adenosine Triphosphate ◽  
Katp Channel ◽  
Molecular Mechanisms ◽  
Katp Channels ◽  
Site Directed Mutagenesis ◽  
Sulfonylurea Receptor ◽  
Inhibitory Effects ◽  
Inwardly Rectifying Potassium Channel ◽  
Mutagenesis Study ◽  
Inwardly Rectifying

Background Both propofol and thiamylal inhibit adenosine triphosphate-sensitive potassium (KATP) channels. In the current study, the authors investigated the effects of these anesthetics on the activity of recombinant sarcolemmal KATP channels encoded by inwardly rectifying potassium channel (Kir6.1 or Kir6.2) genes and sulfonylurea receptor (SUR1, SUR2A, or SUR2B) genes. Methods The authors used inside-out patch clamp configurations to investigate the effects of propofol and thiamylal on the activity of recombinant KATP channels using COS-7 cells transfected with various types of KATP channel subunits. Results Propofol inhibited the activities of the SUR1/Kir6.2 (EC50 = 77 microm), SUR2A/Kir6.2 (EC50 = 72 microm), and SUR2B/Kir6.2 (EC50 = 71 microm) channels but had no significant effects on the SUR2B/Kir6.1 channels. Propofol inhibited the truncated isoform of Kir6.2 (Kir6.2DeltaC36) channels (EC50 = 78 microm) that can form functional KATP channels in the absence of SUR molecules. Furthermore, the authors identified two distinct mutations R31E (arginine residue at position 31 to glutamic acid) and K185Q (lysine residue at position 185 to glutamine) of the Kir6.2DeltaC36 channel that significantly reduce the inhibition of propofol. In contrast, thiamylal inhibited the SUR1/Kir6.2 (EC50 = 541 microm), SUR2A/Kir6.2 (EC50 = 248 microm), SUR2B/Kir6.2 (EC50 = 183 microm), SUR2B/Kir6.1 (EC50 = 170 microm), and Kir6.2DeltaC36 channels (EC50 = 719 microm). None of the mutants significantly affects the sensitivity of thiamylal. Conclusions These results suggest that the major effects of both propofol and thiamylal on KATP channel activity are mediated via the Kir6.2 subunit. Site-directed mutagenesis study suggests that propofol and thiamylal may influence Kir6.2 activity by different molecular mechanisms; in thiamylal, the SUR subunit seems to modulate anesthetic sensitivity.

scholarly journals Protein kinase Cε activation induces EHD-dependent degradation and downregulation of KATP channels: Implications for glucose stimulated insulin secretion

2020 ◽  
Author(s):  
Christopher J Cockcroft ◽  
Paul Manna ◽  
Rucha Karnik ◽  
Tarvinder K Taneja ◽  
David Wrighton ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Protein Kinase ◽  
Katp Channels ◽  
Published Data ◽  
Lysosomal Degradation ◽  
Β Cell ◽  
Channel Inhibition ◽  
Channel Degradation ◽  
Glucose Stimulated Insulin Secretion ◽  
Pkc Activation

AbstractPancreatic β-cells have the unique ability to couple glucose metabolism to insulin secretion. This capacity is generally attributed to the ability of ATP to inhibit KATP channels, and the consequent β-cell membrane depolarization and excitation. This notion has recently been challenged by a study which demonstrated that high glucose (HG) downregulates the cell surface KATP channels, and thereby leads to β-cell depolarisation and excitation. The authors attributed the downregulation to HG-induced protein kinase C (PKC) activation and the consequent increase in channel endocytosis. This interpretation, however, is inconsistent with our previous findings that PKC activation does not affect endocytosis. To address this controversy, we revisited the problem: we have used cell biological and electrophysiological approaches combined with the pharmacological activator of PKC, PMA (phorbol 12-myristate 13-acetate). We first confirm that PKC does not play a role in KATP channel endocytosis; instead, it downregulates the channel by promoting lysosomal degradation coupled with reduced recycling. We then show that (i) mutation of the dileucine motif (355LL356) in the C-terminal domain of the Kir6.2 subunit of the KATP channel complex prevents lysosomal degradation; (ii) lysosomal targeting is mediated by the EHD (Eps15 homology domain– containing) proteins; and (iii) the PKC isoform responsible for channel degradation is PKCε. Taken together with the published data, we suggest that HG promotes β-cell excitability via two mechanisms: ATP-dependent channel inhibition and ATP-independent, PKCε-dependent channel degradation. The results likely have implications for glucose induced biphasic insulin secretion.

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