The overall thrust is to deploy new molecular constructs---many inspired from channel mechanistic studies-for the discovery of fundamental, newly accessible arenas of CaM/Ca channel physiology in heart. This thrust drives three aims, addressing successively more general realms of cardiac physiology, each with fundamental and therapeutic implications. (1) To clarify facilitation of cardiac L-type Ca channels by Ca2+/CaM. By contrast to CDI, a distinct process of facilitated channel opening by Ca 2+ (CDF) remains mysterious, despite its probable role in strengthening the heartbeat at faster heart rates. Still unclear is the actual strength of CDF in heart, and whether CDF shares rich CaM signaling features found in model experimental systems. Those systems permit study of engineered recombinant L-type channels that lack CDI and thereby permit maximal resolution of CDF. By contrast, incomplete separation of CDF from CDI seriously complicates study in heart. We will thus express engineered L-type channels (lacking CDI and dihydropyridine block) in myocytes. During dihydropyridine block of native channels, selective resolution of recombinant channels will permit unambiguous assessment and mechanistic dissection of CDF in the native setting. (2) To define the capabilities of cardiac L-type Ca channels to activate nuclear CREB. Such Ca 2+ signaling appears crucial to the dynamic regulation of cardiac genes. In neurons, CaM not only regulates the channel to which it is bound, such CaM may also bridge preferential signaling of L-type channels to CREB. Here, we will define basic aspects of CREB signaling in myocytes, using distinctive methodologies such as CaM/L-type channel fusions to test whether the very CaM that modulates a channel is essential for triggering CREB. Optical FRET-based sensors of CREB activation also promise rapid temporal correlation of Ca 2+ entry patterns and CREB activation. (3) To estimate the concentration of local endogenous CaM near L-type channels in heart cells. As CaMs responsive to local Ca 2+ influx through L-type channels may be the initiatory Ca 2+ sensors that ultimately trigger CREB and other nuclear factors, the number of CaMs privy to the local Ca 2+ signal from channels is key to downstream signaling strength. Here, we will utilize CaM/L-type channel fusions, with polymer chain theory, to estimate the local concentration of endogenous CaM near channels. Preliminary results hint at mM concentrations, suggesting that a 'school'of local CaMs resides near channels. Overall, this proposal will answer fundamental unknowns of CaM/Ca channel physiology in the heart.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Lathrop, David A
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Johns Hopkins University
Biomedical Engineering
Schools of Medicine
United States
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Limpitikul, Worawan B; Viswanathan, Meera C; O'Rourke, Brian et al. (2018) Conservation of cardiac L-type Ca2+ channels and their regulation in Drosophila: A novel genetically-pliable channelopathic model. J Mol Cell Cardiol 119:64-74
Dick, Ivy E; Joshi-Mukherjee, Rosy; Yang, Wanjun et al. (2016) Arrhythmogenesis in Timothy Syndrome is associated with defects in Ca(2+)-dependent inactivation. Nat Commun 7:10370
Limpitikul, Worawan B; Dick, Ivy E; Ben-Johny, Manu et al. (2016) An autism-associated mutation in CaV1.3 channels has opposing effects on voltage- and Ca(2+)-dependent regulation. Sci Rep 6:27235
Dick, Ivy E; Limpitikul, Worawan B; Niu, Jacqueline et al. (2016) A rendezvous with the queen of ion channels: Three decades of ion channel research by David T Yue and his Calcium Signals Laboratory. Channels (Austin) 10:20-32
Bazzazi, Hojjat; Sang, Lingjie; Dick, Ivy E et al. (2015) Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes. J Physiol 593:3865-84
Ben-Johny, Manu; Dick, Ivy E; Sang, Lingjie et al. (2015) Towards a Unified Theory of Calmodulin Regulation (Calmodulation) of Voltage-Gated Calcium and Sodium Channels. Curr Mol Pharmacol 8:188-205
Adams, Paul J; Ben-Johny, Manu; Dick, Ivy E et al. (2014) Apocalmodulin itself promotes ion channel opening and Ca(2+) regulation. Cell 159:608-22
Park, Sarah A; Lee, Shin-Rong; Tung, Leslie et al. (2014) Optical mapping of optogenetically shaped cardiac action potentials. Sci Rep 4:6125
Limpitikul, Worawan B; Dick, Ivy E; Joshi-Mukherjee, Rosy et al. (2014) Calmodulin mutations associated with long QT syndrome prevent inactivation of cardiac L-type Ca(2+) currents and promote proarrhythmic behavior in ventricular myocytes. J Mol Cell Cardiol 74:115-24
Yang, Philemon S; Johny, Manu Ben; Yue, David T (2014) Allostery in Ca²? channel modulation by calcium-binding proteins. Nat Chem Biol 10:231-8

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