Mathias Schwanstecher

Potassium channel openers require ATP to bind to and act through sulfonylurea receptors

KATP channels are composed of a small inwardly rectifying K+ channel subunit, either KIR6.1 or KIR6.2, plus a sulfonylurea receptor, SUR1 or SUR2 (A or B), which belong to the ATP‐binding cassette superfamily. SUR1/KIR6.2 reconstitute the neuronal/pancreatic β‐cell channel, whereas SUR2A/KIR6.2 and SUR2B/KIR6.1 (or KIR6.2) are proposed to reconstitute the cardiac and the vascular‐smooth‐muscle‐type KATP channels, respectively. We report that potassium channel openers (KCOs) bind to and act through SURs and that binding to SUR1, SUR2A and SUR2B requires ATP. Non‐hydrolysable ATP‐analogues do not support binding, and Mg2+ or Mn2+ are required. Point mutations in the Walker A motifs or linker regions of both nucleotide‐binding folds (NBFs) abolish or weaken [3H]P1075 binding to SUR2B, rendering reconstituted SUR2B/KIR6.2 channels insensitive towards KCOs. The C‐terminus of SUR affects KCO affinity with SUR2B ∼ SUR1 > SUR2A. KCOs belonging to different structural classes inhibited specific [3H]P1075 binding to SUR2B in a monophasic manner, with the exception of minoxidil sulfate, which induced a biphasic displacement. The affinities of KCO binding to SUR2B were 3.5–8‐fold higher than their potencies for activation of SUR2B/KIR6.2 channels. The results establish that SURs are the KCO receptors of KATP channels and suggest that KCO binding requires a conformational change induced by ATP hydrolysis in both NBFs.

Control of insulin secretion by sulfonylureas, meglitinide and diazoxide in relation to their binding to the sulfonylurea receptor in pancreatic islets

Sulfonylureas inhibit an ATP-dependent K+ channel in the B-cell plasma membrane and thereby initiate insulin release. Diazoxide opens this channel and inhibits insulin release. In mouse pancreatic islets, we have explored whether other targets for these drugs must be postulated to explain their hypo- or hyperglycaemic properties. At non-saturating drug concentrations the rates of increase in insulin secretion declined in the order tolbutamide = meglitinide greater than glipizide greater than glibenclamide. The same rank order was observed when comparing the rates of disappearance of insulin-releasing and K+ channel-blocking effects. The different kinetics of response depend on the lipid solubility of the drugs, which controls their penetration into the intracellular space. Allowing for the different kinetics, the same maximum secretory rates were caused by saturating concentrations of tolbutamide, meglitinide, glipizide and glibenclamide. A close correlation between insulin-releasing and K+ channel-blocking potencies of these drugs was observed. The relative potencies of tolbutamide, meglitinide, glipizide and glibenclamide corresponded well to their relative affinities for binding to islet-cell membranes, suggesting that the binding site represents the sulfonylurea receptor. The biphasic time-course of dissociation of glibenclamide binding indicates a complex receptor-drug interaction. For diazoxide there was no correlation between affinity of binding to the sulfonylurea receptor and potency of inhibition of insulin secretion. Thus, opening or closing of the ATP-dependent K+ channel by diazoxide or sulfonylureas, respectively, appears to be due to interaction with different binding sites in the B-cell plasma membrane. The free concentrations of tolbutamide, glipizide, glibenclamide and diazoxide which are effective on B-cells are in the range of therapeutic plasma concentrations of the free drugs. It is concluded that the hypo- and hyperglycaemic effects of these drugs result from changing the permeability of the ATP-dependent K+ channel in the B-cell plasma membrane.

IDENTIFICATION OF THE POTASSIUM CHANNEL OPENER SITE ON SULFONYLUREA RECEPTORS

Diversity of sulfonylurea receptor (SUR) subunits underlies tissue specific pharmacology of KATPchannels, which represent critical regulators of electrical activity in numerous cells. Notably, the neuronal/pancreatic β-cell receptor, SUR1, imparts high sensitivity to hypoglycemic sulfonylureas (SUs;e.g. glibenclamide) and low to potassium channel openers (KCOs; e.g. P1075), whereas the opposite drug sensitivities are conferred by cardiovascular receptors, SUR2A and SUR2B. By exchanging domains between SUR1 and SUR2B, we identify two regions (KCO I: Thr1059–Leu1087 and KCO II: Arg1218–Asn1320; rat SUR2 numbering) within the second set of transmembrane domains (TMDII) as critical for KCO binding. Swapping both regions reconstitutes KCO affinities and sensitivities of the donor SUR isoform. High glibenclamide affinity of SUR1 is not reduced by transfer of KCO I plus II from SUR2B, demonstrating that high SU and KCO affinity can coexist in the same SUR molecule. Consistently, high SU affinity was imparted on SUR2B by substituting the region separating KCO I and II (Ile1088–Val1217) with the corresponding domain of SUR1. We infer the receptor sites for KCOs and SUs to be closely associated within a regulatory domain (Thr1059–Asn1320) in TMDII of SURs.

Effect of MgATP on pinacidil‐induced displacement of glibenclamide from the sulphonylurea receptor in a pancreatic β‐cell line and rat cerebral cortex

1 The effects of blockers and openers of K+ channels on binding of [3H]‐glibenclamide to microsomes obtained from a pancreatic β‐cell line (HIT‐T15) or rat cerebral cortex were examined. 2 The blockers quinine, chlorpromazine and thiopentone and the openers cromakalim [(±) 6‐cyano‐3,4‐dihydro‐2,2‐dimethyl‐trans‐4‐(2‐oxo‐1‐pyrrolidyl)‐2H‐benzo[b]pyran‐3‐ol] and minoxidil sulphate did not significantly interact with the sulphonylurea receptor of HIT‐cells both at phosphorylating (presence of MgATP) and dephosphorylating (absence of MgATP) conditions. 3 In the absence of MgATP, pinacidil (200‐ 500 μm) did not significantly displace [3H]‐glibenclamide binding to microsomes from HIT‐cells. The displacement of [3H]‐glibenclamide binding was strongly enhanced by MgATP and was due to a decrease in the number of high affinity binding sites for glibenclamide. 4 MgATP enhanced pinacidil‐induced inhibition of [3H]‐glibenclamide binding to microsomes from rat cerebral cortex. 5 The effect of MgATP on pinacidil‐induced inhibition of [3H]‐glibenclamide binding was maintained after solubilization of the membranes from HIT‐cells or rat cerebral cortex. 6 It is concluded that the sulphonylurea receptor is regulated not only by sulphonylureas but also by the K+ channel openers, diazoxide and pinacidil, and by protein phosphorylation. The binding sites for sulphonylureas and these K+ channel openers are not identical, but appear to be located at a single protein or at tightly associated proteins.

Phosphate and thiophosphate group donating adenine and guanine nucleotides inhibit glibenclamide binding to membranes from pancreatic islets.

SummaryIn microsomes obtained from mouse pancreatic islets, the Mg complex of adenosine 5′-triphosphate (MgATP) increased the dissociation constant (KD) for binding of [3H]glibenclamide by sixfold. In the presence of Mg2+, not only ATP but also adenosine 5′-0-(3-thiotriphosphate) (ATPγS), adenosine 5′-diphosphate (ADP), guanosine 5′-triphosphate (GTP), guanosine 5′-diphosphate (GDP), guanosine 5′-0-(3-thiotriphosphate) (GTPγTS) and guanosine 5′-0-(2-thiodiphosphate) (GDPβ S) inhibited binding of [3H]glibenclamide. These effects were not observed in the absence of Mg2+. Half maximally effective concentrations of the Mg complexes of ATP, ADP, ATPγS and GDP were 11.6, 19.0, 62.3 and 90.1 μmol/l, respectively. The non-hydrolyzable analogues adenosine 5′-(β,γ-imidotriphosphate) (AMP-PNP) and guanosine 5′-(β,γ-imidotriphosphate) (GMP-PNP) did not alter [3H]glibenclamide binding in the presence of Mg2+. MgADP acted much more slowly than MgATP and both MgADP and MgADP did not inhibit [3H]glibenclamide binding when the concentrations of MgATP and MgATP were kept low by the hexokinase reaction. Development of MgATP-induced inhibition of [3H]glibenclamide binding and dissociation of [3H]-glibenclamide binding occurred at similar rates. However, the reversal of MgATP-induced inhibition of [3H]glibenclamide binding was slower than the association of [3H]glibenclamide with its binding site. Exogenous alkaline phosphatase accelerated the reversal of MgATP-induced inhibition of [3H]glibenclamide binding. MgATP enhanced displacement of [3H]glibenclamide binding by diazoxide. The data suggest that sulfonylureas and diazoxide exert their effects by interaction with the same binding site at the sulfonylurea receptor and that protein phosphorylation modulates the affinity of the receptor.

Glucose both inhibits and stimulates insulin secretion from isolated pancreatic islets exposed to maximally effective concentrations of sulfonylureas

SummaryIsolated pancreatic islets from mice were perifused with media containing maximally effective concentrations of glibenclamide (0.1–10 μmol/l) or glipizide (1 μmol/l). In these islets an increase of the glucose concentration from 10 mmol/l to 40 mmol/l or addition of d-glyc-eraldehyde (20 mmol/1) caused a temporary decrease in insulin release which was followed by a sustained enhancement of release. α-Ketoisocaproate (3 or 20 mmol/1) did not inhibit insulin release; at high concentration it was an even stronger secretagogue than d-glucose or d-glyceraldehyde. It is concluded that high energy phosphates couple B-cell fuel metabolism and insulin release by acting both on the ATP-dependent K+ channel and on other targets not yet identified.

Location of the sulphonylurea receptor at the cytoplasmic face of the β‐cell membrane

1 In insulin‐secreting cells the location of the sulphonylurea receptor was examined by use of a sulphonylurea derivative representing the glibenclamide molecule devoid of its cyclohexyl moiety (compound III) and a benzenesulphonic acid derivative representing the glibenclamide molecule devoid of its cyclohexylurea moiety (compound IV). At pH7.4 compound IV is only present in charged form. 2 Lipid solubility declined in the order tolbutamide > compound III > compound IV. 3 The dissociation constant (KD) for binding of compound IV to the sulphonylurea receptor in HIT‐cells (pancreatic β‐cell line) was similar to the KD value for tolbutamide and fourfold higher than the KD value for compound III. 4 In mouse pancreatic β‐cells, drug concentrations inhibiting adenosine 5′‐triphosphate‐sensitive K+ channels (KATP‐channels) half‐maximally (EC50) were determined by use of the patch‐clamp technique. When the drugs were applied to the extracellular side of outside‐out or the intracellular side of inside‐out membrane patches, the ratio of extracellular to intracellular EC50 values was 281 for compound IV, 25.5 for compound III and 1.2 for tolbutamide. 5 In mouse pancreatic β‐cells, measurement of KATP‐channel activity in cell‐attached patches and recording of insulin release displayed much higher EC50 values for compound IV than inside‐out patch experiments. A corresponding, but less pronounced difference in EC50 values was observed for compound III, whereas the EC50 values for tolbutamide did not differ significantly. 6 It is concluded that the sulphonylurea receptor is located at the cytoplasmic face of the β‐cell plasma membrane. Receptor activation is induced by the anionic forms of sulphonylureas and their analogues.

Photoaffinity Labeling of the Cerebral Sulfonylurea Receptor Using a Novel Radioiodinated Azidoglibenclamide Analogue

Abstract: In previous studies evidence has been presented by photoaffinity labeling that a polypeptide of 145–150 kDa represents the cerebral sulfonylurea receptor. However, covalent incorporation of [3H]glibenclamide or a 125I‐labeled glibenclamide analogue into the sulfonylurea receptor required high amounts of photoenergy and took place with low yield of photoinsertion. To provide a probe with increased photoreactivity a 4‐azido‐5‐iodosalicyloyl analogue of glibenclamide was synthesized. Binding experiments revealed specific and reversible high‐affinity binding of this novel probe to the particulate (KD = 0.13 nM) and solubilized (KD = 0.56 nM) sulfonylurea receptor from cerebral cortex. The novel probe showed >100‐fold higher sensitivity to irradiation at 356 nm than glibenclamide. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed specific photoincorporation into a cerebral protein of 175 kDa and indicated an efficiency of photoincorporation of 9%. From dissociation binding curves following irradiation photoincorporation was estimated as 28% of specifically bound ligand. Photoincorporation into the 175‐kDa protein following saturation binding of the novel probe to particulate sites from cerebral cortex indicated a KD value of 0.38 nM. Inhibition of photoincorporation into this protein by glibenclamide, glipizide, and tolbutamide revealed KD values for these sulfonylureas of 0.06 nM, 1.6 nM, and 1.2 µM, respectively. These results show that the novel photoaffinity ligand can be used as a probe for detection and characterization of the sulfonylurea receptor and suggest that a 175‐kDa protein represents the cerebral sulfonylurea receptor.

The Binding Properties of the Particulate and Solubilized Sulfonylurea Receptor from Cerebral Cortex Are Modulated by the Mg2+ Complex of ATP

Abstract: Glibenclamide closes an ATP‐sensitive K+ channel (K‐ATP channel) by interaction with the sulfonylurea receptor in the plasma membrane of pancreatic B cells and thereby initiates insulin release. Previous studies demonstrated that the Mg2+ complex of ATP decreases glibenclamide binding to the sulfonylurea receptor from pancreatic islets. The aim of the present study was to examine the effect of adenine and guanine nucleotides on binding of sulfonylureas to the cerebral sulfonylurea receptor. For this purpose, binding properties of the particulate and solubilized site from rat or pig cerebral cortex were analyzed. Maximum recovery of receptors in detergent extracts amounted to 40–50%. Specific binding of [3H]glibenclamide to the solubilized receptors corresponded well to specific binding to microsomes. In microsomes and detergent extracts, the Mg2+ complexes of ATP, ADP, GTP, and GDP inhibited binding of [3H]glibenclamide. These effects were not observed in the absence of Mg2+. In detergent extracts, Mg‐ATP (300 μM) reduced the number of high‐affinity sites for [3H]‐glibenclamide by 52% and increased the dissociation constant for [3H]glibenclamide by eightfold; Mg‐ATP was half‐maximally effective at 41 μM. Alkaline phosphatase accelerated the reversal of Mg‐ATP‐induced inhibition of [3H]glibenclamide binding. The data suggest similar control of the sulfonylurea receptor from brain and pancreatic islets by protein phosphorylation.

Interaction of Fluorescein Derivatives with Sulfonylurea Binding in Insulin-Secreting Cells

Recently evidence was presented that fluorescein derivatives (e.g. phloxine B) inhibit glibenclamide binding by occupation of a nucleotide-binding site at the ATP-sensitive potassium channel (KATP channel). However, this conclusion was inconsistent with the results of previous studies testing the effects of nucleotides on glibenclamide binding. To elucidate the interaction mode of fluorescein derivatives with sulfonylurea binding, the effect of phloxine B on binding of [3H]glibenclamide to microsomes obtained from a pancreatic beta-cell line (HIT-T15) was examined. Phloxine B inhibited specific binding of glibenclamide half-maximally at 3.2 mumol/l. The slope parameter for the displacement curve was close to one, suggesting a competitive interaction between both drugs. In accordance with this assumption 4 mumol/l phloxine B did not show an effect on the number of high-affinity binding sites but increased the apparent dissociation constant for glibenclamide by 3.1-fold and 30 mumol/l phloxine B did not alter the rate of dissociation of [3H]glibenclamide. Moreover, MgATP (300 mumol/l) significantly reduced the apparent affinity for binding of phloxine B to the sulfonylurea receptor. This finding resembled the action of MgATP on binding of sulfonylureas to their receptor site. It is concluded that fluorescein derivatives inhibit glibenclamide binding due to competition for the same site at the sulfonylurea receptor.