Both Ca2+ and cyclic nucleotides play pivotal roles in the regulation of cardiac function. Our goal in the Program Project will be to provide structural information aimed at understanding the mechanisms of regulation by Ca2+ and cyclic nucleotides in the heart. There are three specific structural targets from among the variety of proteins that are currently under study by member of this Project that are amenable to structural analysis by solution-state NMR spectroscopy: the L-type calcium channel and two forms of phosphodiesterase. We will concentrate our efforts on characterizing the regions of each protein, and its protein binding partners, that are responsible for the regulation of the protein's activity. The L-type calcium channel is regulated by a negative feedback mechanism in which increased concentration of Ca2+ that enters through the channel inactivates the channel. Thus, the channel both regulates, and is regulated by, Ca2+, A approximately residue portion of the cytoplasmic tail of the channel and the interactions and relevant structures of the channel inactivation region and calmodulin using NMR and other spectroscopies. Cyclic nucleotides also play key roles in the regulation of cardiac function. Their concentrations are therefore carefully regulated, both the level of synthesis and degradation. The phosphodiesterases (PDEs) carry out the hydrolysis of cyclic nucleotides: We will study two of the predominant forms of the enzyme found in heart: the Ca2+/calmodulin-dependent form, PDE1, and the cGMP- stimulated form, PDE2A. As is clear from their names, each of these PDEs are regulated via a different mechanism. The regulatory domains of the PDEs will be studied by NMR in the presence and absence of their respective ligands to provide structural insights into the mechanisms by which the enzyme are regulated.
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