investigator's application): ATP-sensitive K+ channels, KATP, can be reconstituted from SUR1, a member of the ATP-binding cassette superfamily and KIR6.2, a member of the inward rectifier K+ channel family. The reconstituted channel is a moderate inward rectifier whose conductance is appropriately modulated by ATP/ADP, by sulfonylurea channel blockers and potassium channel openers. Preliminary data suggest SUR1 and KIR6.2 assemble with a 1:1 stoichiometry into large complexes, presumably KATP channels. The overall objective of the application is to test the hypothesis that KATP channels have a tetrameric architecture, (SUR1/KIR6.2)4. The specific objectives are: 1) To establish that SUR1 and KIR6.2 associate to form a heteromultimeric complex. Association is being monitored using his-tagged SUR1 and KIR6.2 subunits, by utilizing specific lectins that bind SUR1, by cophotolabeling with 125I-azidoglibenclamide and by the appearance of a complex glycosylation pattern of SUR1. 2) To determine the stoichiometry of the complex. This is being done using sedimentation velocity measurements to estimate the molecular size(s) of the complexes and by engineering and expressing active channel forming fusion proteins with defined stoichiornetries, e.g., SUR1-KIR6.2 and SUR1-(KIR6.2)2. 3) To determine the stoichiometry of active KATP channels. This is being done using a mutant of KIR6.2, N 160D, that confers strong rectification on reconstituted KATP channels. Co-expression of wildtype KIR6.2 with the N 160D mutation gives heteromeric channels whose properties, along with SUR1-N160D fusion channels, will be used to determine if active channels are tetrameric. 4) To start to map the domains of interaction between SUR1 and KIR6.2. This is being done by engineering KIR6.2/KIR3.4 chimeras to determine which parts of KIR6.2 are needed for association and which for formation of active channels. KIR3.4 is similar to 6.2, but does not associate or form active channels with SUR1. This work will provide a model for ion channel regulation by members of the ATP-binding cassette superfamily.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK052771-03
Application #
2906036
Study Section
Physiology Study Section (PHY)
Program Officer
Laughlin, Maren R
Project Start
1997-09-01
Project End
2001-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
3
Fiscal Year
1999
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
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Bryan, Joseph; Munoz, Alvaro; Zhang, Xinna et al. (2007) ABCC8 and ABCC9: ABC transporters that regulate K+ channels. Pflugers Arch 453:703-18
Szollosi, Andras; Nenquin, Myriam; Aguilar-Bryan, Lydia et al. (2007) Glucose stimulates Ca2+ influx and insulin secretion in 2-week-old beta-cells lacking ATP-sensitive K+ channels. J Biol Chem 282:1747-56
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Hussain, Khalid; Bryan, Joseph; Christesen, Henrick T et al. (2005) Serum glucagon counterregulatory hormonal response to hypoglycemia is blunted in congenital hyperinsulinism. Diabetes 54:2946-51
Lam, Tony K T; Pocai, Alessandro; Gutierrez-Juarez, Roger et al. (2005) Hypothalamic sensing of circulating fatty acids is required for glucose homeostasis. Nat Med 11:320-7
Kikuta, Toshiteru; Ohara-Imaizumi, Mica; Nakazaki, Mitsuhiro et al. (2005) Docking and fusion of insulin secretory granules in SUR1 knock out mouse beta-cells observed by total internal reflection fluorescence microscopy. FEBS Lett 579:1602-6
Munoz, Alvaro; Hu, Min; Hussain, Khalid et al. (2005) Regulation of glucagon secretion at low glucose concentrations: evidence for adenosine triphosphate-sensitive potassium channel involvement. Endocrinology 146:5514-21
Sieg, Andrea; Su, Jiping; Munoz, Alvaro et al. (2004) Epinephrine-induced hyperpolarization of islet cells without KATP channels. Am J Physiol Endocrinol Metab 286:E463-71

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