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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK044311-04
Application #
2518310
Study Section
Physiology Study Section (PHY)
Program Officer
Laughlin, Maren R
Project Start
1994-09-20
Project End
1999-08-31
Budget Start
1997-09-30
Budget End
1998-08-31
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
Slucca, Michela; Harmon, Jamie S; Oseid, Elizabeth A et al. (2010) ATP-sensitive K+ channel mediates the zinc switch-off signal for glucagon response during glucose deprivation. Diabetes 59:128-34
Russ, U; Kuhner, P; Prager, R et al. (2009) Incomplete dissociation of glibenclamide from wild-type and mutant pancreatic K ATP channels limits their recovery from inhibition. Br J Pharmacol 156:354-61
Winkler, Marcus; Lutz, Rebekka; Russ, Ulrich et al. (2009) Analysis of two KCNJ11 neonatal diabetes mutations, V59G and V59A, and the analogous KCNJ8 I60G substitution: differences between the channel subtypes formed with SUR1. J Biol Chem 284:6752-62
Düfer, M; Haspel, D; Krippeit-Drews, P et al. (2009) Activation of the Na+/K+-ATPase by insulin and glucose as a putative negative feedback mechanism in pancreatic beta-cells. Pflugers Arch 457:1351-60
Aguilar-Bryan, Lydia; Bryan, Joseph (2008) Neonatal diabetes mellitus. Endocr Rev 29:265-91
Dufer, Martina; Haspel, Dirk; Krippeit-Drews, Peter et al. (2007) The KATP channel is critical for calcium sequestration into non-ER compartments in mouse pancreatic beta cells. Cell Physiol Biochem 20:65-74
Bryan, Joseph; Munoz, Alvaro; Zhang, Xinna et al. (2007) ABCC8 and ABCC9: ABC transporters that regulate K+ channels. Pflugers Arch 453:703-18
Vila-Carriles, Wanda H; Zhao, Guiling; Bryan, Joseph (2007) Defining a binding pocket for sulfonylureas in ATP-sensitive potassium channels. FASEB J 21:18-25
Babenko, Andrey P; Polak, Michel; Cave, Helene et al. (2006) Activating mutations in the ABCC8 gene in neonatal diabetes mellitus. N Engl J Med 355:456-66
Babenko, Andrey P (2005) K(ATP) channels ""vingt ans apres"": ATG to PDB to Mechanism. J Mol Cell Cardiol 39:79-98

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