The proposed research has two long-range goals: 1) to establish the relationship between the Beta-cell sulfonylurea receptor and the ATP-sensitive K+ channel, and 2) to understand the role of sulfonylurea receptors on non-Beta-cells in the therapy of Type II diabetes. To achieve the first goal, we will make use of a new, potent sulfonylurea, iodo'glyburide', which can be conveniently synthesized in an unlabeled, or 125I-labeled form. We will compare the Kd of receptor binding of this drug to the K0.5 for half- inhibition of K+ efflux, the ED50 for insulin secretion, and the lC50 for inhibition of ATP-sensitive K+ channel activity using hamster insulinoma tumor (HIT) cells. We will determine whether the number of sulfonylurea receptors per cell is equivalent to the number of ATP-sensitive K+ channels and will examine how drugs that are known to alter ATP-sensitive K+ channel activity affect the binding of iodo'glyburide' to its receptor. The molecular weight of the receptor will be established, the protein partially purified, reconstituted into a planar bilayer, and assayed for ATP- sensitive K+ channel activity. We expect these experiments to strengthen the proposal that the ATP-sensitive K+ channel protein contains the sulfonylurea binding site. To prove this, the HIT cell sulfonylurea receptor cDNA will be cloned and expressed, and the expressed protein tested for ATP-sensitive K+ channel activity. Cloning of the receptor cDNA will be by standard protocols. A partial receptor protein sequence will be obtained, oligonucleotides designed based on this sequence, and HIT cell cDNA libraries screened with the oligonucleotides. The putative sulfonylurea receptor encoded by the isolated cDNA will be expressed in Xenopus oocytes, transfected mammalian cells, and E. coli and the protein assayed for 125I-labeled iodo'glyburide' binding and ATP-sensitive K+ channel activity. To begin to understand the role of the sulfonylurea receptor in the therapy of Type II diabetes, we will examine whether non-Beta-cell tissues and cell types also contain this protein, or a similar protein, using as probes the 125I-labeled iodo'glyburide, and the HIT cell receptor cDNA. If the sulfonylurea receptor is also present on peripheral tissue membranes, this would indicate a second role for this protein, possibly related to glucose uptake into these tissues.
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