ATP-sensitive potassium channels.
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ATP-Binding Cassette Transporters↗
Biomedical subjects
F Chudziak
Publications and source records attributed to F Chudziak.
Structural requirements of sulphonylureas and analogues for interaction with sulphonylurea receptor subtypes.
1. The structure-activity relationship for hypoglycaemic sulphonylureas and analogues was examined. Binding affinities were compared using membranes from HIT-T15 cells (beta-cell line) and from COS-7 cells transiently expressing sulphonylurea receptor subtypes (SUR1, SUR2A and SUR2B). Inhibition of adenosine-triphosphate-sensitive K+ channels (KATP-channels) was measured in mouse pancreatic beta-cells. 2. The tested compounds displayed similar binding affinities for SUR2A and SUR2B. 3. Meglitinide (benzoic acid derivative) bound to SUR1 and the SUR2 isoforms with similar affinities. Replacement of the carboxyl group of meglitinide by a methyl group significantly decreased the binding affinities for SUR1 and the SUR2 isoforms (>4 fold) and the potency to inhibit KATP-channel activity of beta-cells (24 fold). Replacement of the carboxyl group of meglitinide by a sulphonylurea group significantly increased the affinities for SUR1 (5 fold) and the SUR2 isoforms (13 - 16 fold). 4. Glibenclamide bound to the SUR2 isoforms with 300 - 500 fold lower affinity than to SUR1. Exchanging the cyclohexyl ring of glibenclamide by a methyl group or removal of the lipophilic side chain of glibenclamide (5-chloro-2-methoxy-benzamidoethyl chain) markedly reduced but did not abolish the selectivity for SUR1. 5. In conclusion, interaction of sulphonylureas and acidic analogues with SUR1, SUR2A and SUR2B is favoured by the anionic group of these drugs. Hypoglycaemic sulphonylureas (e.g. glibenclamide) owe selectivity for SUR1 to lipophilic substitution on their urea group. Sulphonylureas without lipophilic substitution on the urea group could represent lead compounds for the development of SUR2-selective drugs.
ATP-Binding Cassette Transporters↗
Identification of a 38-kDa high affinity sulfonylurea-binding peptide in insulin-secreting cells and cerebral cortex.
Previous studies have described specific photoincorporation of radiolabeled sulfonylureas into a peptide with high molecular mass (140-175 kDa), which thus has been suggested to represent the sulfonylurea receptor. In the present study, a 125I-labeled 4-azidosalicyloyl analog of glibenclamide, 125I-N3-GA (N-[4-(2-(4-azido-2-hydroxy-5-125I- iodobenzamido)ethyl)benzenesulfonyl]-N'-cyclohexylurea), was used for photoaffinity labeling. This novel probe was specifically photoincorporated into a peptide with an apparent molecular mass of 160-175 kDa when samples from insulin-secreting HIT cells or cerebral cortex were boiled in a SDS-buffer prior to separation with SDS-polyacrylamide gel electrophoresis. However, omitting the heating step revealed specific labeling of an additional peptide with an apparent molecular mass of 38 kDa. The amount of radioactivity specifically photoincorporated into this peptide was 3-4-fold higher than that incorporated into the 160-175-kDa peptide. Both peptides displayed similar dissociation constants for binding of the sulfonylureas IN3-GA (N-[4-(2-(4-azido-2-hydroxy- 5-iodobenzamido)ethyl)benzenesulfonyl]-N'-cyclohexylurea), glibenclamide, glipizide, and tolbutamide. Analysis of photoaffinity labeling of solubilized fractions indicated an almost exclusive specific linkage to the 38-kDa peptide. The data support the view that the sulfonylurea receptor in insulin-secreting cells and cerebral cortex consists of a peptide with an apparent molecular mass of 38 kDa, which seems to be tightly coupled to a 160-175-kDa peptide.
ATP-Binding Cassette Transporters↗
Photoaffinity labeling of the cerebral sulfonylurea receptor using a novel radioiodinated azidoglibenclamide analogue.
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 microM, 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.
ATP-Binding Cassette Transporters↗
Location of the sulphonylurea receptor at the cytoplasmic face of the beta-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 cyclohexy moiety (compound III) and a benzenesulphonic acid derivative representing the glibenclamide molecule devoid of its cyclohexylurea moiety (compound IV). At pH 7.4 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 beta-cell line) was similar to the KD value for tolbutamide and fourfold higher than the KD value for compound III. 4. In mouse pancreatic beta-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 beta-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 beta-cell plasma membrane. Receptor activation is induced by the anionic forms of sulphonylureas and their analogues.
ATP-Binding Cassette Transporters↗