Ca2+/Calmodulin-Dependent Protein Kinase II-Dependent Regulation of TREK-1
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Date
2014-05
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The Ohio State University
Abstract
Ion channels serve as core constituents of macromolecular complexes that regulate cell membrane excitability and cell signaling pathways. Thus, proper ion channel function is essential for heart function. Importantly, ion channel activity is heavily regulated by posttranslational modifications. The mechano-sensitive two pore K+ channel TREK-1 has been identified as an important channel for proper repolarization in the heart and has been shown to induce actin reorganization resulting in filopodia-like protrusions in COS M6 cells. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an important multifunctional serine/threonine kinase that has been linked to cardiac diseases such as hypertrophy, arrhythmias, and cardiac failure, has been identified as a potential regulator of TREK-1 activity. However, little is known about the mechanism and/or role of CaMKII-dependent regulation of TREK-1. We created a library of TREK-1 mutants using site-directed mutagenesis to ablate putative CaMKII phosphorylation sites located in the intracellular domains of the channel. Wildtype (WT) and mutant TREK-1 constructs were engineered into a GFP-expressing pIRES vector and expressed in COS M6 cells. Cells were immunostained with TREK-1 antibody and the cell shape, size, and structure were analyzed using confocal microscopy. In parallel, TREK-1 fragments corresponding to the major intracellular domains (N-terminal, linker, and C-terminal) were generated as GST fusion proteins and used for in vitro CaMKII phosphorylation assays. Our studies indicate that CaMKII phosphorylates TREK-1 at a site in the N-terminal intracellular domain. Cells expressing the TREK-1 N-terminal mutant (Ser46Ala) showed little difference in cytoskeletal organization compared to WT-expressing cells. However, these cells were significantly smaller than both untransfected and WT cells. These studies suggest that CaMKII phosphorylates TREK-1 at a site in the N-terminus and that although this site may not play a significant role in the formation of membrane protrusions, this site may be part of cell signaling pathways that affect cell size. Future studies will determine whether CaMKII targets additional sites on the channel and whether CaMKII-dependent phosphorylation of TREK-1 is important for regulating other cell functions.
Description
2nd Place at Denman Undergraduate Research Forum in Health Sciences: Cellular/Laboratory
3rd Place Undergraduate Research Forum for the College of Engineering and Architechture
3rd Place Undergraduate Research Forum for the College of Engineering and Architechture
Keywords
TREK-1, CaMKII, phosphorylation, mutant, actin