E23K, a common single nucleotide polymorphism in K IR 6.2, the pore-forming subunit of pancreatic -cell ATP-sensitive K ؉ channels, significantly enhanced open probability of these channels, thus reducing their sensitivity toward inhibitory ATP 4؊ and increasing the threshold concentration for insulin release. Previous association studies and high allelic frequency suggest this effect to critically inhibit secretion and play a major role in pathogenesis of common type 2 diabetes. Based on evidence for functional relevance of E23K in both the heterozygous (E/K; with E in position 23 of K IR 6.2 in one allele and K in the other) and homozygous (K/K; with K in position 23 of K IR 6.2 in both alleles) genotype, we propose a model in which enhanced susceptibility to type 2 diabetes is associated with evolutionary advantage of the E/K state. Diabetes 51:875-879, 2002 T ype 2 diabetes is generally perceived as a polygenic disorder, with disease development being influenced by both hereditary and environmental factors (1). Genes encoding for key components of insulin secretion and glucose metabolism pathways have been widely considered as targets for defects in type 2 diabetes. However, despite intensive investigations, little progress has been made in identifying the genes that impart susceptibility to the common late-onset forms of the disease (2).In pancreatic -cells, ATP-sensitive K ϩ (K ATP ) channels critically control insulin secretion by coupling metabolism to electrical activity (3). Recent advances resulted in cloning of these channels and elucidation of their subunit composition (4). The -cell channels are assembled, with tetradimeric stoichiometry, from two structurally distinct subunits: an inwardly rectifying K ϩ channel subunit (K IR 6.2), forming the pore, and the regulatory sulfonylurea receptor subunit-1 (SUR1). While hypoglycemic sulfonylureas (e.g., glibenclamide) exert their effects on channel activity by interaction with SUR1, there is strong evidence that the receptor site for inhibitory ATP 4Ϫ is formed by K IR 6.2.Three common missense single nucleotide polymorphisms (SNPs) have been observed in K IR 6.2 (E23K, L270V, and I337V) (5-9), and their potential impact in type 2 diabetes led us to analyze their functional relevance. Whereas L270V and I337V were without effect on the properties of reconstituted human SUR1/K IR 6.2 channels (including expression rate, single channel conductance, spontaneous open probability [P O ], and nucleotide and drug sensitivities [results not shown]), E23K markedly affected channel gating, significantly reducing the time spent in long interburst closed states (17 Ϯ 3% for SUR1/ the mutant isoform of K IR 6.2 with K instead of E in position 23 [K IR 6.2 E23K ] vs. 54 Ϯ 6% for wild-type channels, n ϭ 10 each, P Ͻ 0.001) (Fig. 1A and B), thus producing a 1.6-fold increase of P O (P O ϭ 0.66 Ϯ 0.05 for SUR1/ K IR 6.2 E23K vs. 0.41 Ϯ 0.04 for wild-type channels, n ϭ 10 each, P Ͻ 0.001). The increase of P O was confirmed by noise analysis (patches with 100 -500 chann...
Diversity of sulfonylurea receptor (SUR
Hypoglycemic sulfonylureas (e.g., glibenclamide, glipizide, and tolbutamide) exert their stimulatory effect on excitatory cells by closure of ATP-sensitive potassium (KATP) channels. These channels are heteromultimers composed with a 4:4 stoichiometry of an inwardly rectifying K+ channel (KIR) subunit 6.x plus a sulfonylurea receptor (SUR). SUR1/KIR6.2 reconstitutes the neuronal/pancreatic beta-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. SUR2A and SUR2B are splice variants of a single gene differing only in their C-terminal 42 amino acids. Affinities of sulfonylureas for rat SUR2A, rat or human SUR2B, and a SUR2 chimera containing the C-terminal 42 amino acids of SUR1 did not differ significantly, implying that the C terminus does not form part of the binding pocket. Consistent with these findings, reconstituted SUR2A/KIR6.2 and SUR2B/KIR6.2 channels revealed similar sensitivities for glibenclamide and tolbutamide. Dissociation constants of sulfonylureas for SUR2A and SUR2B were 10- to 400-fold higher than for SUR1, however, amazingly the benzoic acid derivative meglitinide did not show lower affinity for SUR2 isoforms. Potencies of glibenclamide, glipizide, tolbutamide, and meglitinide to inhibit activity of SUR1/KIR6.2 and SUR2B/KIR6.2 channels were 3- to 6-fold higher than binding affinities of these drugs with concentration-inhibition relations being significantly steeper (Hill coefficients 1.23-1.32) than binding curves (Hill coefficients 0.93-1.06). The data establish that the C terminus of SURs does not affect sulfonylurea affinity and sensitivity. We conclude that occupation of one of the four SUR sites per channel complex is sufficient to induce KATP channel closure.
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 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 P-cell line) was similar to the KD value for tolbutamide and fourfold higher than the KD value for compound III. 4 In mouse pancreatic P-cells, drug concentrations inhibiting adenosine 5'-triphosphate-sensitive K+ channels (KATP-channels) half-maximally (EC5() 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. 5In mouse pancreatic P-cells, measurement of KATp-channel activity in cell-attached patches and recording of insulin release displayed much higher EC.% values for compound IV than inside-out patch experiments. A corresponding, but less pronounced difference in EC.V values was observed for compound III, whereas the ECv values for tolbutamide did not differ significantly.6 It is concluded that the sulphonylurea receptor is located at the cytoplasmic face of the P-cell plasma membrane. Receptor activation is induced by the anionic forms of sulphonylureas and their analogues.
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