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Degree Name

Doctor of Philosophy (PhD)




One mechanism by which calcium is mobilized from intracellular stores is via the activation of inositol 1,4,5-trisphosphate receptors (InsP3Rs) by the second messenger, inositol 1,4,5-trisphosphate (InsP3). InsP3 is produced when certain factors bind to G protein-coupled receptors on the plasma membrane, causing activation of phospholipase C which cleaves InsP3 from its membrane tether, allowing InsP3 to diffuse through the cytoplasm to bind to its receptor. Activation of InsP3Rs leads to release of calcium into the cytoplasm of the cell, where it can activate a multitude of signal transduction cascades and a variety of local and global cellular events. One such enzyme activated by a rise in intracellular [Ca2+] is Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII is a multifunctional Serine/Threonine protein kinase involved in many signaling pathways in various cell types including regulating cardiac gene expression. Recent results from our lab have shown that the predominant InsP3R isoform in the heart, the InsP3R2, is primarily targeted to the nuclear envelope in ventricular myocytes. Here it forms a macromolecular complex with the nuclear-localized cardiac isoform of CaMKII, CaMKIIB. Upon stimulation of InsP3 production, Ca2+ released through the InsP3R2 activates CaMKIIB, allowing it to act on downstream targets. The results of this study and others suggest that the activity of InsP3Rs can be inhibited by CaMKII-mediated feedback phosphorylation. In this study, I use exogenously expressed fragments of the InsP3R2 and site-directed mutagenesis to show that CaMKII can phosphorylate the InsP3R2 at Serine-150 and determine that phosphorylation of this residue is responsible for modulation of channel activity. Non-phosphorylatable (S150A) and phospho-mimetic (S150E) mutations were constructed in the full-length InsP3R2, expressed in COS cells and studied in planar lipid bilayers. Upon treatment with CaMKII, the non-phosphorylatable channel showed no decrease in activity. Conversely, the phosphomimetic channel displayed a very low Po under normal recording conditions in the absence of CaMKII, thus mimicking a channel that has been phosphorylated by CaMKII. The results of this study show for the first time that Serine-150 of the InsP3R2 is phosphorylated by CaMKII and results in a decrease in the channel's open probability.

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