Protein phosphorylation is a common cellular mechanism used to regulate the function of most proteins. Cardiac inwardly rectifying potassium (Kir) channels are also regulated by protein phosphorylation that changes their activity and modulates cardiac excitability. Over the past ten years it has been appreciated that the activity of all Kir channels depends critically on interactions with the membrane phospholipid phosphatidylinositol-bis-phosphate (PIP2). Moreover great advances over the past five years have been made in solving the three-dimensional structures of representative Kir family members. The long term goal of our laboratory in general is to understand ion channel function and regulation in terms of molecular structure and in particular to gain mechanistic insight for the dependence of Kir activity on PIP2. We have found that many different types of Kir channel modulation, including phosphorylation, depend on channel-PIP2 interactions and we aim to understand the molecular basis of such dependence. Evidence from the literature and from our own preliminary studies suggest that phosphorylation changes the sensitivity of the channel to activation by PIP2. Examination in the three-dimensional structures of the position of putative sites that have been implicated to be involved in phosphorylation effects reveal a striking clustering around amino acid residues that affect sensitivity to PIP2. We have thus formulated the following hypothesis that we propose to test in this application: """"""""Phosphorylation can exert its functional effects on the cardiac Kir channels by modulating channel-PIP2 interactions"""""""". Although the problem of protein phosphorylation and its mechanism of action has attracted great effort from many outstanding investigators, the experimental tools we have had to unequivocally identify single phosphorylation sites have been limiting. Thus, in the ion channel field we do not yet have mechanistic structural understanding of how phosphorylation affects channel activity. Here, we propose to use Mass Spectrometry to identify phosphorylation sites in Kir3 channels in order to test our hypothesis in a three- dimensional context. Our preliminary results have identified a protein kinase A-targeted phosphorylation site (Kir3.1-S385), using a combination of Mass Spectrometry methods (MALDI-TOF and tandem Mass Spectrometry). This result has demonstrated to us the feasibility of this approach in identifying phosphorylation sites. We propose to test electrophysiologically whether specific phosphorylation sites affect sensitivity to PIP2. A comprehensive account of sites used by different protein kinases, the assessment of which sites exert their effects through PIP2, and development of experimentally testable computational models ought to give us good mechanistic insights as to how phosphorylation regulates channel activity. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 1 Continuation Format Page.

Public Health Relevance

Phosphorylation processes regulate cardiac performance, such as heart rate and strength of contraction, under many conditions, including exercise. This project aims to identify amino acid residues of cardiac potassium channel proteins that are phosphorylated. The hypothesis to be tested in the three-dimensional context of the proteins is that phosphorylated residues can exert their functional effects by altering directly or allosterically interactions of these channels with the key membrane phospholipid PIP2. If true, this hypothesis will provide a framework on which phosphorylation effects on channel activity could be explained mechanistically.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
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Wang, Lan-Hsiang
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Virginia Commonwealth University
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