Inward rectifier K (Kir) channels are essential for the normal function of both excitable and nonexcitable cells.
The specific aims of this proposal are two-fold: to better understand the mechanisms of inward strong rectification in Kir2.1 (IRK1), the major component of the high resting K conductance in many cell types, and to investigate the basis of mechanosensitivity of the G-protein regulated K channel Kir3.4 (GIRK4), a major component of the resting K conductance in atrial muscle and brain. We will apply electrophysiological (patch-clamp), molecular biological and biochemical techniques to various cloned Kir channels expressed in Xenopus oocytes or mammalian cell lines. In the first specific aim, we will determine how an intrinsic gating mechanism, which we have recently identified and postulate to be a tethered gating particle, interacts with polyamines and Mg to contribute to strong inward reactivation in Kir channels. We will test the novel hypothesis that the tethered gating particle contains binding sites for polyamines and Mg which enhances its ability to cause inward rectification, providing further insight into the molecular basis of strong inward rectification. In the second specific aim, we will characterize the molecular mechanisms underlying stretch-induced inactivation of Kir3.x channels, a property which we have recently identified in Kir3.4 and native cardiac KACh channels. We will determine: whether mechanosensitivity is also a property of other members of the Kir3.x family, the regions of the Kir3.4 channel required for mechanosensitivity, using chimeric constructs and site-directed mutagenesis; the role of G proteins; and the cytoskeletal and/or extracellular matrix elements responsible for transducing mechanosensitivity. The mechanosensitivity of Kir3.x channels may contribute to a variety of stretch-induced responses, including stretch- induced arrhythmias, atrial natriuretic peptide (ANP) release, and/or hypertrophic gene programming. Together, these studies in Kir channels will provide important insights into the regulation of excitability in ventricular and atrial cardiac muscle, as well as in other excitable tissues.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HL060025-04
Application #
6389875
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Spooner, Peter
Project Start
1998-06-01
Project End
2003-05-31
Budget Start
2001-06-01
Budget End
2002-05-31
Support Year
4
Fiscal Year
2001
Total Cost
$308,861
Indirect Cost
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
John, Scott; Weiss, James N; Ribalet, Bernard (2011) Subcellular localization of hexokinases I and II directs the metabolic fate of glucose. PLoS One 6:e17674
John, Scott A; Ottolia, Michela; Weiss, James N et al. (2008) Dynamic modulation of intracellular glucose imaged in single cells using a FRET-based glucose nanosensor. Pflugers Arch 456:307-22
Xie, Lai-Hua; John, Scott A; Ribalet, Bernard et al. (2008) Phosphatidylinositol-4,5-bisphosphate (PIP2) regulation of strong inward rectifier Kir2.1 channels: multilevel positive cooperativity. J Physiol 586:1833-48
Xie, Lai-Hua; John, Scott A; Ribalet, Bernard et al. (2007) Activation of inwardly rectifying potassium (Kir) channels by phosphatidylinosital-4,5-bisphosphate (PIP2): interaction with other regulatory ligands. Prog Biophys Mol Biol 94:320-35
Ribalet, Bernard; John, Scott A; Xie, Lai-Hua et al. (2006) ATP-sensitive K+ channels: regulation of bursting by the sulphonylurea receptor, PIP2 and regions of Kir6.2. J Physiol 571:303-17
Ribalet, Bernard; John, Scott A; Xie, Lai-Hua et al. (2005) Regulation of the ATP-sensitive K channel Kir6.2 by ATP and PIP(2). J Mol Cell Cardiol 39:71-7
Xie, Lai-Hua; John, Scott A; Ribalet, Bernard et al. (2005) Long polyamines act as cofactors in PIP2 activation of inward rectifier potassium (Kir2.1) channels. J Gen Physiol 126:541-9
John, Scott A; Weiss, James N; Ribalet, Bernard (2005) ATP sensitivity of ATP-sensitive K+ channels: role of the gamma phosphate group of ATP and the R50 residue of mouse Kir6.2. J Physiol 568:931-40
Xie, Lai-Hua; John, Scott A; Ribalet, Bernard et al. (2004) Regulation of gating by negative charges in the cytoplasmic pore in the Kir2.1 channel. J Physiol 561:159-68
Xie, Lai-Hua; John, Scott A; Weiss, James N (2003) Inward rectification by polyamines in mouse Kir2.1 channels: synergy between blocking components. J Physiol 550:67-82

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