The Na/K-ATPase (NKA) is the main Na extrusion pathway and is essential in [Na]i regulation. In heart, [Na]i critically modulates [Ca]i, contractiity and triggered arrhythmias via Na/Ca exchange. Thus, NKA regulation is functionally important, and our prior work on this project has made several seminal observations. First, phospholemman (PLM) regulates NKA similar to the way phospholamban (PLB) regulates SR Ca-ATPase (SERCA). That is, PLM binds to and inhibits NKA by reducing the [Na]i-affinity, but PLM phosphorylation relieves this inhibition to increase NKA function. Second, PLM phosphorylation and the consequent NKA stimulation is integral to the sympathetic fight or flight response (tempering the rise in [Na]i and cellular Ca load, and limiting Ca overload-induced arrhythmias). Third, PLM physically associates with NKA, but in bimolecular FRET measurements, PLM phosphorylation alters the PLM-NKA interaction. Fourth, PLM also forms homo-oligomers, which are favored by PLM phosphorylation. Fifth, we identified sites on both NKA (E960) and PLM (F28) that are essential for the regulatory interaction. Sixth, that block of NKA-?2 (vs. ?1) has a preferential inotropic effect. Here we will further decipher NKA-PLM regulation in heart, using FRET, [Na]i and [Ca]i measurements, patch-clamp and molecular biology in myocytes and HEK 293 cells and structural modeling.
Aim 1 will identify PLM (& NKA) sites that are critical for PLM-PLM interaction, and also elucidate the molecular structure of PLM multimers (which we suspect are tetramers). We will also test whether PLM-PLM disruption promotes PLM-NKA interaction and pump inhibition.
Aim 2 will use novel NKA mutants to test the molecular basis of and functional effects of NKA-NKA dimerization on pump activity, PLM interaction and signaling. We will also examine how ouabain disrupts these natural dimers.
Aim 3 will test whether membrane domains from PLB or FXYD4 (which can stimulate NKA), can alter PLM effects on NKA in PLM/PLB or PLM/FXYD4 chimeras. We will also test if novel selective NKA-?2 inhibitors might be an improved modern-day digitalis therapy. These studies will greatly enrich our mechanistic understanding of NKA regulation in heart and explore novel therapeutic strategies.

Public Health Relevance

Altered cardiac myocyte sodium concentration [Na] is known to be a critical factor in modulating both mechanical action of the heart and induction of arrhythmias. Here we extend our novel work on the interaction and modulation of the Na/K-ATPase (responsible for regulating [Na]) by the small protein phospholemman. The proposed work will add important fundamental insight into the interaction, and how this regulatory complex regulates [Na]i in the heart (using state-of-the-art molecular, biophysical and cell biology approaches).

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL081562-13
Application #
9246558
Study Section
Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
Program Officer
Lathrop, David A
Project Start
2005-07-01
Project End
2019-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
13
Fiscal Year
2017
Total Cost
$409,732
Indirect Cost
$142,771
Name
University of California Davis
Department
Pharmacology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Morotti, Stefano; McCulloch, Andrew D; Bers, Donald M et al. (2016) Atrial-selective targeting of arrhythmogenic phase-3 early afterdepolarizations in human myocytes. J Mol Cell Cardiol 96:63-71
Wagner, Stefan; Maier, Lars S; Bers, Donald M (2015) Role of sodium and calcium dysregulation in tachyarrhythmias in sudden cardiac death. Circ Res 116:1956-70
Clancy, Colleen E; Chen-Izu, Ye; Bers, Donald M et al. (2015) Deranged sodium to sudden death. J Physiol 593:1331-45
Bers, Donald M; Chen-Izu, Ye (2015) Sodium and calcium regulation in cardiac myocytes: from molecules to heart failure and arrhythmia. J Physiol 593:1327-9
Chen-Izu, Ye; Shaw, Robin M; Pitt, Geoffrey S et al. (2015) Na+ channel function, regulation, structure, trafficking and sequestration. J Physiol 593:1347-60
Kwong, Jennifer Q; Lu, Xiyuan; Correll, Robert N et al. (2015) The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart. Cell Rep 12:15-22
Shattock, Michael J; Ottolia, Michela; Bers, Donald M et al. (2015) Na+/Ca2+ exchange and Na+/K+-ATPase in the heart. J Physiol 593:1361-82
Xie, Yuanfang; Liao, Zhandi; Grandi, Eleonora et al. (2015) Slow [Na]i Changes and Positive Feedback Between Membrane Potential and [Ca]i Underlie Intermittent Early Afterdepolarizations and Arrhythmias. Circ Arrhythm Electrophysiol 8:1472-80
Ping, Peipei; Gustafsson, Ã…sa B; Bers, Don M et al. (2015) Harnessing the Power of Integrated Mitochondrial Biology and Physiology: A Special Report on the NHLBI Mitochondria in Heart Diseases Initiative. Circ Res 117:234-8
Negroni, Jorge A; Morotti, Stefano; Lascano, Elena C et al. (2015) β-adrenergic effects on cardiac myofilaments and contraction in an integrated rabbit ventricular myocyte model. J Mol Cell Cardiol 81:162-75

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