This proposal focuses on the mechanisms and electrophysiological consequences of the molecular interactions between the inward rectifier potassium channel protein Kir2.1 and the ?ubunit of the major cardiac sodium channel NaV1.5. Our preliminary results strongly suggest that NaV1.5 and Kir2.1 modulate each other's surface expression and function through their respective PDZ binding domains within a macromolecular complex to control cardiac excitability. Such a dynamic reciprocity is post-translational, involving, at least in part, mutual regulation of trafficking and targeting of both channel proteins at common membrane compartments, as well as internalization. We focus on inheritable mutations that are known to disrupt trafficking of NaV1.5 (Brugada Syndrome, BS) or Kir2.1 (Andersen-Tawil Syndrome, ATS). We surmise that a mutation that disrupts the expression of one channel protein type (e.g., NaV1.5) will also affect the other type (e.g., Kir2.1 by disturbing the common macromolecular complex through which they interact, thus contributing to both the electrophysiological phenotype and arrhythmogenic potential. We will test the following three major hypotheses: 1) NaV1.5 and Kir2.1 protein channels undergo PDZ-domain mediated interactions with common partners in a macromolecular complex that controls their membrane stability; 2) macromolecular complex formation affects anterograde and/or retrograde trafficking of Kir2.1 and NaV1.5; and 3) human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CMs) expressing either ATS or BS mutations that affect protein trafficking will show reduced excitability reflecting altered expression of both channel proteins, which should contribute strongly to the inherited arrhythmia phenotype. We propose to combine proteomics (e.g., protein purification, yeast two-hybrid assay and interaction domain mapping) and genetic (e.g., mutagenesis and silencing) tools, confocal microscopy, live cell imaging, fluorescence recovery after photobleaching, patch clamping, optical mapping, gene transfer and silencing in heterologous systems, and in single, highly mature ventricular-like hiPSC-CMs and hiPSC-CM monolayers. We will also conduct computer simulations to enable the virtual dissection and interpretation of the electrophysiological and arrhythmogenic changes resulting from NaV1.5-Kir2.1 interactions and their mutants in a macromolecular complex.

Public Health Relevance

The work proposed has strong implications for understanding the regulation of cardiac ion channels in health, as well as in heart failure and acquired and inheritable ion channel diseases. Posttranslational modifications and protein-protein interactions in macromolecular complexes may play critical roles in ion channel dysfunction and life-threatening arrhythmias, and may great potential to improved medical care.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL122352-01A1
Application #
8816386
Study Section
Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
Program Officer
Krull, Holly
Project Start
2014-11-14
Project End
2018-10-31
Budget Start
2014-11-14
Budget End
2015-10-31
Support Year
1
Fiscal Year
2015
Total Cost
$489,932
Indirect Cost
$143,368
Name
University of Michigan Ann Arbor
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Goldstein, Daniel R; Jalife, José (2017) Synergistic Research Between the Center of Arrhythmia Research and the Michigan Biology of Cardiovascular Aging at the University of Michigan. Circ Res 121:1221-1223
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Jalife, José (2016) Dynamics and Molecular Mechanisms of Ventricular Fibrillation in Structurally Normal Hearts. Card Electrophysiol Clin 8:601-12

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