scholarly journals Differential mechanisms of Cantú syndrome–associated gain of function mutations in the ABCC9 (SUR2) subunit of the KATP channel

2015 ◽  
Vol 146 (6) ◽  
pp. 527-540 ◽  
Author(s):  
Paige E. Cooper ◽  
Monica Sala-Rabanal ◽  
Sun Joo Lee ◽  
Colin G. Nichols
Keyword(s):  
Cell Metabolism ◽  
Katp Channels ◽  
Sulfonylurea Receptor ◽  
Membrane Excitability ◽  
Gain Of Function ◽  
Equivalent Position ◽  
Functional Consequences ◽  
Atp Inhibition ◽  
Patch Clamp Electrophysiology ◽  
Inwardly Rectifying

Cantú syndrome (CS) is a rare disease characterized by congenital hypertrichosis, distinct facial features, osteochondrodysplasia, and cardiac defects. Recent genetic analysis has revealed that the majority of CS patients carry a missense mutation in ABCC9, which codes for the sulfonylurea receptor SUR2. SUR2 subunits couple with Kir6.x, inwardly rectifying potassium pore-forming subunits, to form adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, which link cell metabolism to membrane excitability in a variety of tissues including vascular smooth muscle, skeletal muscle, and the heart. The functional consequences of multiple uncharacterized CS mutations remain unclear. Here, we have focused on determining the functional consequences of three documented human CS-associated ABCC9 mutations: human P432L, A478V, and C1043Y. The mutations were engineered in the equivalent position in rat SUR2A (P429L, A475V, and C1039Y), and each was coexpressed with mouse Kir6.2. Using macroscopic rubidium (86Rb+) efflux assays, we show that KATP channels formed with P429L, A475V, or C1039Y mutants enhance KATP activity compared with wild-type (WT) channels. We used inside-out patch-clamp electrophysiology to measure channel sensitivity to ATP inhibition and to MgADP activation. For P429L and A475V mutants, sensitivity to ATP inhibition was comparable to WT channels, but activation by MgADP was significantly greater. C1039Y-dependent channels were significantly less sensitive to inhibition by ATP or by glibenclamide, but MgADP activation was comparable to WT. The results indicate that these three CS mutations all lead to overactive KATP channels, but at least two mechanisms underlie the observed gain of function: decreased ATP inhibition and enhanced MgADP activation.

scholarly journals Engineered interaction between SUR1 and Kir6.2 that enhances ATP sensitivity in KATP channels

2012 ◽  
Vol 140 (2) ◽  
pp. 175-187 ◽  
Author(s):  
Emily B. Pratt ◽  
Qing Zhou ◽  
Joel W. Gay ◽  
Show-Ling Shyng
Keyword(s):  
Cell Metabolism ◽  
Katp Channels ◽  
Dynamic Parameter ◽  
Sulfonylurea Receptor ◽  
Wild Type ◽  
Membrane Excitability ◽  
Close Proximity ◽  
Secretion Inhibition ◽  
Sulfonylurea Receptor 1 ◽  
Hallmark Feature

The ATP-sensitive potassium (KATP) channel consisting of the inward rectifier Kir6.2 and SUR1 (sulfonylurea receptor 1) couples cell metabolism to membrane excitability and regulates insulin secretion. Inhibition by intracellular ATP is a hallmark feature of the channel. ATP sensitivity is conferred by Kir6.2 but enhanced by SUR1. The mechanism by which SUR1 increases channel ATP sensitivity is not understood. In this study, we report molecular interactions between SUR1 and Kir6.2 that markedly alter channel ATP sensitivity. Channels bearing an E203K mutation in SUR1 and a Q52E in Kir6.2 exhibit ATP sensitivity ∼100-fold higher than wild-type channels. Cross-linking of E203C in SUR1 and Q52C in Kir6.2 locks the channel in a closed state and is reversible by reducing agents, demonstrating close proximity of the two residues. Our results reveal that ATP sensitivity in KATP channels is a dynamic parameter dictated by interactions between SUR1 and Kir6.2.

scholarly journals Cantu syndrome-associated SUR2 (ABCC9) mutations in distinct structural domains result in KATP channel gain-of-function by differential mechanisms

2017 ◽  
Author(s):  
Conor McClenaghan ◽  
Alex Hanson ◽  
Monica Sala-Rabanal ◽  
Helen I. Roessler ◽  
Dragana Josifova ◽  
...  
Keyword(s):  
Katp Channel ◽  
Molecular Mechanisms ◽  
Cardiovascular Disorder ◽  
Major Effect ◽  
Channel Activity ◽  
Gain Of Function ◽  
Structural Domains ◽  
Equivalent Position ◽  
Genes Encoding ◽  
Patch Clamp Electrophysiology

AbstractThe complex cardiovascular disorder Cantu Syndrome arises from gain-of-function mutations in either KCNJ8 or ABCC9, the genes encoding the Kir6.1 and SUR2 subunits of ATP-sensitive potassium (KATP) channels. Recent reports indicate that such mutations can increase channel activity by multiple molecular mechanisms. In this study, we determine the mechanism by which KATP function is altered by several mutations in distinct structural domains of SUR2: D207E in the intracellular L0-linker and Y985S, G989E, M1060I, and R1154Q/W in TMD2. Mutations were engineered at their equivalent position in rat SUR2A (D207E, Y981S, G985E, M1056I and R1150Q/W) and functional effects were investigated using macroscopic rubidium (86Rb+) efflux assays and patch clamp electrophysiology. The results show that D207E increases KATP activity by increasing intrinsic stability of the open state, whilst the cluster of Y981S/G985E/M1056I mutations, as well as R1150Q/W, augment Mg-nucleotide activation. The response of mutant channels to inhibition by the sulfonylurea drug glibenclamide, a potential pharmacotherapy for CS, was also tested. There was no major effect on glibenclamide sensitivity for the D207E, Y981S, G985E or M1056I mutations, but glutamine and tryptophan substitution at R1150 resulted in significant decreases in potency.

ATP-sensitive and inwardly rectifying potassium channels in smooth muscle

1997 ◽  
Vol 77 (4) ◽  
pp. 1165-1232 ◽  
Author(s):  
J. M. Quayle ◽  
M. T. Nelson ◽  
N. B. Standen
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Protein Kinase ◽  
Potassium Channels ◽  
Membrane Conductance ◽  
Katp Channels ◽  
Muscle Membrane ◽  
Sulfonylurea Receptor ◽  
Kir Channels ◽  
Inwardly Rectifying

The properties and roles of ATP-sensitive (KATP) and inwardly rectifying (KIR) potassium channels are reviewed. Potassium channels regulate the membrane potential of smooth muscle, which controls calcium entry through voltage-dependent calcium channels, and thereby contractility through changes in intracellular calcium. The KATP channel is likely to be composed of members of the inward rectifier channel gene family (Kir6) and sulfonylurea receptor proteins. The KIR channels do not appear to be as widely distributed as KATP channels in smooth muscle and may provide a mechanism by which changes in extracellular K+ can alter smooth muscle membrane potential, and thereby arterial diameter. The KATP channels contribute to the resting membrane conductance of some types of smooth muscle and can open under situations of metabolic compromise. The KATP channels are targets of a wide variety of vasodilators and constrictors, which act, respectively, through adenosine 3',5'-cyclic monophosphate/protein kinase A and protein kinase C. The KATP channels are also activated by a number of synthetic vasodilators (e.g., diazoxide and pinacidil) and are inhibited by the oral hypoglycemic sulfonylurea drugs (e.g., glibenclamide). Together, KATP and KIR channels are important regulators of smooth muscle function and represent important therapeutic targets.

Sulfonylurea-insensitive permanent neonatal diabetes caused by a severe gain-of-function Tyr330His substitution in Kir6.2

2022 ◽  
Author(s):  
Conor McClenaghan ◽  
Novella Rapini ◽  
Domenico Umberto De Rose ◽  
Jian Gao ◽  
Jacob Roeglin ◽  
...  
Keyword(s):  
Developmental Delay ◽  
Molecular Basis ◽  
Glucose Monitoring ◽  
Neonatal Diabetes ◽  
Katp Channels ◽  
Gain Of Function ◽  
Gene Encoding ◽  
Permanent Neonatal Diabetes ◽  
Mild Developmental Delay ◽  
Atp Inhibition

Background/Aims: Mutations in KCNJ11, the gene encoding the Kir6.2 subunit of pancreatic and neuronal KATP channels, are associated with a spectrum of neonatal diabetes diseases. Methods: Variant screening was used to identify cause of neonatal diabetes, and continuous glucose monitoring used to assess effectiveness of sulfonylurea treatment. Electrophysiological analysis of variant KATP channel function was used to determine molecular basis. Results: We identified a previously uncharacterized KCNJ11 mutation, c.988T>C [pTyr330His], in an Italian child diagnosed with sulfonylurea-resistant permanent neonatal diabetes and developmental delay (iDEND). Functional analysis of recombinant KATP channels reveals that this mutation causes a drastic gain-of-function, due to a reduction in ATP-inhibition. Further, we demonstrate that the Tyr330His substitution causes a significant decrease in sensitivity to the sulfonylurea, glibenclamide. Conclusions: In this subject, the KCNJ11(c.988T>C) mutation provoked neonatal diabetes, with mild developmental delay, which was insensitive to correction by sulfonylurea therapy. This is explained by the molecular loss of sulfonylurea sensitivity conferred by the Tyr330His substitution, and highlights the need for molecular analysis of such mutations.

scholarly journals Sulfonylurea Receptors Type 1 and 2A Randomly Assemble to Form Heteromeric KATP Channels of Mixed Subunit Composition

2007 ◽  
Vol 131 (1) ◽  
pp. 43-58 ◽  
Author(s):  
Kim W. Chan ◽  
Adam Wheeler ◽  
László Csanády
Keyword(s):  
Katp Channels ◽  
Sulfonylurea Receptor ◽  
Pharmacological Characterization ◽  
Mixing Ratios ◽  
Metabolic Stresses ◽  
Inwardly Rectifying ◽  
Global Fitting ◽  
Tandem Constructs ◽  
Heteromeric Channels

ATP-sensitive potassium (KATP) channels play important roles in regulating insulin secretion, controlling vascular tone, and protecting cells against metabolic stresses. KATP channels are heterooctamers of four pore-forming inwardly rectifying (Kir6.2) subunits and four sulfonylurea receptor (SUR) subunits. KATP channels containing SUR1 (e.g. pancreatic) and SUR2A (e.g. cardiac) display distinct metabolic sensitivities and pharmacological profiles. The reported expression of both SUR1 and SUR2 together with Kir6.2 in some cells raises the possibility that heteromeric channels containing both SUR subtypes might exist. To test whether SUR1 can coassemble with SUR2A to form functional KATP channels, we made tandem constructs by fusing SUR to either a wild-type (WT) or a mutant N160D Kir6.2 subunit. The latter mutation greatly increases the sensitivity of KATP channels to block by intracellular spermine. We expressed, individually and in combinations, tandem constructs SUR1-Kir6.2 (S1-WT), SUR1-Kir6.2[N160D] (S1-ND), and SUR2A-Kir6.2[N160D] (S2-ND) in Xenopus oocytes, and studied the voltage dependence of spermine block in inside-out macropatches over a range of spermine concentrations and RNA mixing ratios. Each tandem construct expressed alone supported macroscopic K+ currents with pharmacological properties indistinguishable from those of the respective native channel types. Spermine sensitivity was low for S1-WT but high for S1-ND and S2-ND. Coexpression of S1-WT and S1-ND generated current components with intermediate spermine sensitivities indicating the presence of channel populations containing both types of Kir subunits at all possible stoichiometries. The relative abundances of these populations, determined by global fitting over a range of conditions, followed binomial statistics, suggesting that WT and N160D Kir6.2 subunits coassemble indiscriminately. Coexpression of S1-WT with S2-ND also yielded current components with intermediate spermine sensitivities, suggesting that SUR1 and SUR2A randomly coassemble into functional KATP channels. Further pharmacological characterization confirmed coassembly of not only S1-WT and S2-ND, but also of coexpressed free SUR1, SUR2A, and Kir6.2 into functional heteromeric channels.

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 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 Muscle KATP Channels: Recent Insights to Energy Sensing and Myoprotection

2010 ◽  
Vol 90 (3) ◽  
pp. 799-829 ◽  
Author(s):  
Thomas P. Flagg ◽  
Decha Enkvetchakul ◽  
Joseph C. Koster ◽  
Colin G. Nichols
Keyword(s):  
Cell Metabolism ◽  
Katp Channels ◽  
Sulfonylurea Receptor ◽  
Channel Activity ◽  
Tissue Specific ◽  
Fiber Damage ◽  
Cellular Energetics ◽  
Muscle Types ◽  
Energy Sensing ◽  
The Cost

ATP-sensitive potassium (KATP) channels are present in the surface and internal membranes of cardiac, skeletal, and smooth muscle cells and provide a unique feedback between muscle cell metabolism and electrical activity. In so doing, they can play an important role in the control of contractility, particularly when cellular energetics are compromised, protecting the tissue against calcium overload and fiber damage, but the cost of this protection may be enhanced arrhythmic activity. Generated as complexes of Kir6.1 or Kir6.2 pore-forming subunits with regulatory sulfonylurea receptor subunits, SUR1 or SUR2, the differential assembly of KATP channels in different tissues gives rise to tissue-specific physiological and pharmacological regulation, and hence to the tissue-specific pharmacological control of contractility. The last 10 years have provided insights into the regulation and role of muscle KATP channels, in large part driven by studies of mice in which the protein determinants of channel activity have been deleted or modified. As yet, few human diseases have been correlated with altered muscle KATP activity, but genetically modified animals give important insights to likely pathological roles of aberrant channel activity in different muscle types.

scholarly journals Cryo-electron microscopy structures and progress toward a dynamic understanding of KATP channels

2018 ◽  
Vol 150 (5) ◽  
pp. 653-669 ◽  
Author(s):  
Michael C. Puljung
Keyword(s):  
Electron Microscopy ◽  
Ligand Binding ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Cell Metabolism ◽  
Katp Channels ◽  
K Channel ◽  
Sulfonylurea Receptor ◽  
Binding Domains ◽  
Cryo Electron Microscopy

Adenosine triphosphate (ATP)–sensitive K+ (KATP) channels are molecular sensors of cell metabolism. These hetero-octameric channels, comprising four inward rectifier K+ channel subunits (Kir6.1 or Kir6.2) and four sulfonylurea receptor (SUR1 or SUR2A/B) subunits, detect metabolic changes via three classes of intracellular adenine nucleotide (ATP/ADP) binding site. One site, located on the Kir subunit, causes inhibition of the channel when ATP or ADP is bound. The other two sites, located on the SUR subunit, excite the channel when bound to Mg nucleotides. In pancreatic β cells, an increase in extracellular glucose causes a change in oxidative metabolism and thus turnover of adenine nucleotides in the cytoplasm. This leads to the closure of KATP channels, which depolarizes the plasma membrane and permits Ca2+ influx and insulin secretion. Many of the molecular details regarding the assembly of the KATP complex, and how changes in nucleotide concentrations affect gating, have recently been uncovered by several single-particle cryo-electron microscopy structures of the pancreatic KATP channel (Kir6.2/SUR1) at near-atomic resolution. Here, the author discusses the detailed picture of excitatory and inhibitory ligand binding to KATP that these structures present and suggests a possible mechanism by which channel activation may proceed from the ligand-binding domains of SUR to the channel pore.

scholarly journals Sulfonylurea and K+-Channel Opener Sensitivity of KATP Channels

1999 ◽  
Vol 114 (2) ◽  
pp. 203-213 ◽  
Author(s):  
J.C. Koster ◽  
Q. Sha ◽  
C.G. Nichols
Keyword(s):  
Katp Channels ◽  
K Channel ◽  
Sulfonylurea Receptor ◽  
Common Pathway ◽  
Stimulatory Action ◽  
High Affinity ◽  
Apparent Loss ◽  
Regulatory Input ◽  
Atp Inhibition ◽  
K Channel Opener

The sensitivity of KATP channels to high-affinity block by sulfonylureas and to stimulation by K+ channel openers and MgADP (PCOs) is conferred by the regulatory sulfonylurea receptor (SUR) subunit, whereas ATP inhibits the channel through interaction with the inward rectifier (Kir6.2) subunit. Phosphatidylinositol 4,5-bisphosphate (PIP2) profoundly antagonized ATP inhibition of KATP channels expressed from cloned Kir6.2+SUR1 subunits, but also abolished high affinity tolbutamide sensitivity. By stabilizing the open state of the channel, PIP2 drives the channel away from closed state(s) that are preferentially affected by high affinity tolbutamide binding, thereby producing an apparent loss of high affinity tolbutamide inhibition. Mutant KATP channels (Kir6.2[ΔN30] or Kir6.2[L164A], coexpressed with SUR1) also displayed an “uncoupled” phenotype with no high affinity tolbutamide block and with intrinsically higher open state stability. Conversely, Kir6.2[R176A]+SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block. In addition to antagonizing high-affinity block by tolbutamide, PIP2 also altered the stimulatory action of the PCOs, diazoxide and MgADP. With time after PIP2 application, PCO stimulation first increased, and then subsequently decreased, probably reflecting a common pathway for activation of the channel by stimulatory PCOs and PIP2. The net effect of increasing open state stability, either by PIP2 or mutagenesis, is an apparent “uncoupling” of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with poly-l-lysine.

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