The long-term objectives are to understand the function and structure of inward rectifier K channels (IRKs). IRKs are important in excitable tissues such as nerve and muscle and our particular emphasis is on heart muscle. Here IRKs regulate resting membrane potential and the terminal phase of cardiac repolarization. Consequently their function is of great importance in evaluation and discovery of cardiac antiarrhythmic drugs, especially Class III antiarrhythmics. Three major hypotheses are examined: first whether specific naturally occurring cytoplasmic polyamines (PAs) regulate IRK function in cardiomyocytes; second whether the present topological model of IRKs is correct; and third whether a human cardiac IRK, hIRK is a major component of I-Kr, the rapid part of I-K the delayed rectifier K+ current of cardiomyocytes.
The specific aims are to: 1) localize C-terminus binding sites for both Mg2+ and PAs which together confer the unique property of inward rectification; 2) compare the effective valence and time-dependence of PA block with so-called """"""""intrinsic"""""""" gating of IRKs; 3) test the relationship between PA levels and IRK activity in cardiomyocytes; 4) est specific topological models of IRKs using glycosylation site insertion mutagenesis; and 5) identify the binding site for the Class Ill antiarrhythmic dofetilide, on hIRK and clone accessory subunit modifiers of this channel.
For Aims 1 and 2 and the first part of Aim 5, the research design uses methods of mutagenesis, heterologous expression and electrophysiology in an iterative manner.
For Aim 2 chemical interruption of PA metabolism is used to manipulate PA levels of cardiomyocytes while testing IRK function electrophysiologically.
For Aim 4 a genetically engineered IRK protein is overexpressed in a baculovirus-Sf9 system, immunopurified and tested for N-glycosylation at sites predicted to be extracellular by present topological models. Functional tests using patch clamp are performed simultaneously. For the second part of Aim 5, a variety of candidate modifier subunits that we have cloned will be tested for their ability to make hIRK more closely mimic I-Kr.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL055404-02
Application #
2332552
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Project Start
1996-03-08
Project End
2001-01-31
Budget Start
1997-02-01
Budget End
1998-01-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Physiology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Kuryshev, Yuri A; Ficker, Eckhard; Wang, Lu et al. (2005) Pentamidine-induced long QT syndrome and block of hERG trafficking. J Pharmacol Exp Ther 312:316-23
Ficker, Eckhard; Kuryshev, Yuri A; Dennis, Adrienne T et al. (2004) Mechanisms of arsenic-induced prolongation of cardiac repolarization. Mol Pharmacol 66:33-44
Ficker, Eckhard; Dennis, Adrienne T; Wang, Lu et al. (2003) Role of the cytosolic chaperones Hsp70 and Hsp90 in maturation of the cardiac potassium channel HERG. Circ Res 92:e87-100
Obejero-Paz, Carlos A; Yang, Tianen; Dong, Wei-Qiang et al. (2003) Deferoxamine promotes survival and prevents electrocardiographic abnormalities in the gerbil model of iron-overload cardiomyopathy. J Lab Clin Med 141:121-30
Laurita, Kenneth R; Chuck, Emil Thomas; Yang, Tianen et al. (2003) Optical mapping reveals conduction slowing and impulse block in iron-overload cardiomyopathy. J Lab Clin Med 142:83-9
Schwalbe, Ruth A; Rudin, Alicia; Xia, Shen-Ling et al. (2002) Site-directed glycosylation tagging of functional Kir2.1 reveals that the putative pore-forming segment is extracellular. J Biol Chem 277:24382-9
Wible, Barbara A; Wang, Liming; Kuryshev, Yuri A et al. (2002) Increased K+ efflux and apoptosis induced by the potassium channel modulatory protein KChAP/PIAS3beta in prostate cancer cells. J Biol Chem 277:17852-62
Peri, R; Wible, B A; Brown, A M (2001) Mutations in the Kv beta 2 binding site for NADPH and their effects on Kv1.4. J Biol Chem 276:738-41
Kuryshev, Y A; Wible, B A; Gudz, T I et al. (2001) KChAP/Kvbeta1.2 interactions and their effects on cardiac Kv channel expression. Am J Physiol Cell Physiol 281:C290-9
Thomas, D; Wendt-Nordahl, G; Rockl, K et al. (2001) High-affinity blockade of human ether-a-go-go-related gene human cardiac potassium channels by the novel antiarrhythmic drug BRL-32872. J Pharmacol Exp Ther 297:753-61

Showing the most recent 10 out of 20 publications