SULFONYLUREA RECEPTORS AND KATP CHANNELS

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The long-term objective of this proposal is to understand the structure and function of ion channels that link cellular metabolism with membrane electrical activity. We are characterizing a pancreatic alpha/Beta-cell K+ channel whose electrical activity is modulated by ATP and sulfonylureas. This channel, I/ATP, is thought either to be the high affinity sulfonylurea receptor found in alpha/Beta-cells or to be tightly associated with this receptor. K/ATP sets the resting membrane potential in Beta-cells and is a key ion channel in the glucose-induced depolarization that leads to insulin release. We have developed and characterized an iodinated sulfonylurea, termed iodoglyburide, which specifically photolabels a high affinity, 140 kDa sulfonylurea receptor in isolated membranes from various cell types including neurons, pituitary cell lines, pancreatic alpha-and Beta-cells and several alpha- and Beta-cell models, i.e., a hamster insulin secreting tumor (HIT cells), a rat insulinoma (RIN cells) and a mouse glucagon secreting cell line (alphaTC-6 cells). Iodoglyburide is fully biologically active, has a K/D for binding equivalent to the ED50 for induction of insulin secretion in HIT cells and inhibits K/ATP. The pharmacological properties of the photolabeling reaction are those expected for the high affinity receptor defined in isolated HIT cell membranes. We have isolated the 140 kDa photolabeled receptor and obtained peptide sequence. Antibodies produced against this peptide sequence crossreact with the photolabeled protein and are competed by the appropriate synthetic peptides. A degenerate oligonucleotide/PCR strategy has been used to clone a cDNA fragment coding the N-terminus of the receptor. The major goals of the revised application are: a. To clone and sequence a cDNA for the Beta-cell receptor. b. To express this cDNA in Xenopus oocytes and COS cells in order to ascertain whether the receptor has channel activity and, if so, characterize this activity. c. To use the receptor clone to characterize the receptor, and, in the event the receptor does not have K+ channel activity, to isolate channel subunits. d. To use the receptor clone to isolate other members of this family of proteins.