The goal of this research is to elucidate the molecular structural basis of calcium regulation in muscle. Muscle contraction and relaxation are regulated by dynamic protein-protein complexes that cycle calcium through cell membranes. The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) transports Ca2+ from the cytosol into the SR lumen. SERCA's function is regulated by two membrane inhibitors: phospholamban (PLN) and sarcolipin (PLN), expressed primarily in cardiac and skeletal muscle, respectively. The intramembrane interactions of these two proteins with SERCA regulate calcium flux and thus muscle function. ?-adrenergic phosphorylation of PLN reverses SERCA inhibition, while the reversal of SLN inhibition is due to the variation in expression levels or T5 phosphorylation. Since several myopathies are associated with these calcium handling proteins, understanding how they regulate SERCA is central to muscle physiology and pathophysiology. We will pursue the following aims:
AIM 1. Defining allosteric regulation of SERCA/PLN and SERCA/SLN complexes.
AIM 2. Ascertaining the role of single (T17) and double (S16/T17) phosphorylation of PLN.
AIM 3. Elucidating the allosteric transitions of SERCA/PLN and SERCA/SLN in lipid bilayers.
AIM 4. Determining the oligomeric structure of PLN in lipid bilayers. In the first five years of this project, we focused on understanding the structural dynamics of PLN and SLN free and in interaction with SERCA, laying the foundation for a deeper knowledge of calcium transport regulation at the molecular level. In the next five years, building on our original proposed mechanisms, we seek to gain control of these mechanisms. Our long-term goal is to harness the concepts and models we have developed for the rational design of PLN and SLN mutants for therapeutic purposes.
This research program focuses on the elucidation of the molecular mechanisms that regulate calcium homeostasis in muscle. SERCA/PLN and SERCA/SLN are two membrane protein complexes embedded in the sarcoplasmic reticulum that are involved in the excitation-contraction-relaxation cycle in both cardiac and skeletal muscle. SERCA mutations are linked to Brody disease, a clinical condition characterized by exercise-induced muscle cramps, stiffness, and relaxation impairments. Also, PLN mutations have been directly linked with specific heart conditions. For instance, R9C mutation, R14 deletion, and L39 truncation of PLN have been detected in patients affected by dilated cardiomyopathy. Also, down regulation of SLN mRNA expression has been correlated with atrial fibrillation-induced atrial remodeling. There is now direct evidence that manipulation of calcium cycling, either via the over-expression of SERCA or by tuning SERCA inhibition can reverse many important clinical endpoints of late stage failing heart. Therefore, understanding SERCA regulation by both SLN and PLN is central to the development of non-conventional drug therapy such as gene therapy.
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