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Diabetes, Vol 47, Issue 8 1274-1280, Copyright © 1998 by American Diabetes Association

ARTICLES

Cloning of the promoters for the beta-cell ATP-sensitive K-channel subunits Kir6.2 and SUR1

R Ashfield and SJ Ashcroft
Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, UK. rebecca.ashfield@ndcb.ox.ac.uk

The beta-cell ATP-sensitive potassium channel (K-ATP channel), which regulates insulin secretion, is composed of two types of subunits: 1) a sulfonylurea receptor (SUR1) and 2) an inwardly rectifying potassium channel (Kir6.2). We have isolated clones containing 5'-flanking DNA for both genes by hybridization screening of a human genomic library. Sequencing of over one kilobase of each upstream region has revealed that the putative promoters are G + C rich, with no TATA box. Several E-boxes and potential Sp1 sites are present in both promoters, and the Kir6.2 upstream region contains an Alu repeat. Using a luciferase reporter gene in transient transfection assays, we demonstrate that the upstream DNA contains promoters that are active in the beta-cell lines HIT T15 and MIN6. The promoters are completely inactive in the fibroblast cell line COS7 but show some activity in HepG2 (liver) and HEK293 (epithelial) cell lines. Deletion analysis suggests that a short (173-base pair [bp]) fragment of SUR1 5'-flanking sequence is sufficient for maximal promoter activity. In contrast, over 900 bp of Kir6.2 5' sequence are required for similar high level expression, and deletion of the Alu repeat results in an increase in promoter activity.
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