New mechanisms of SERCA2a regulation: role of luminal calcium

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PROJECT SUMMARY/ABSTRACT SERCA2a Ca pump plays a central role in heart function. The speed at which SERCA2a removes Ca from the cytosol is the main determinant of the rate of cardiac muscle relaxation. SERCA2a also sets the total amount of Ca in the sarcoplasmic reticulum (SR), which determines the strength of cardiac contraction. It is not surprising, that impaired SERCA2a function has been reported in a number of pathological conditions, including heart failure (HF). Thus, understanding mechanisms of SERCA regulation is of great clinical importance. Besides activation of muscle contraction, SR luminal Ca ([Ca]SR) plays an important role in regulation of SR protein function. While SERCA2a activity controls [Ca]SR, less is known about how changes in [Ca]SR affect SERCA2a Ca transport. Our preliminary results suggest that SR luminal Ca plays an important role in regulation of SERCA2a. In this project we will use advanced structural analyses, innovative molecular biological techniques, new organelle-specific sensors, state-of-the-art optical methods and in vivo gene delivery to explore this new mechanism of SERCA2a regulation. In Aim 1, we will test the hypothesis that luminal Ca regulates SERCA2a by increasing the pump’s catalytic efficacy and by relieving the phospholamban (PLB) inhibitory effect. Molecular dynamic simulations will be used to select specific domains on the SERCA2a luminal side that are involved in Ca regulation. Site-directed mutagenesis will be used to identify the specific amino acids that form the luminal Ca-binding sites and to develop the luminal Ca-insensitive SERCA2a mutant. We will assess effects of the luminal Ca regulation on Ca transport, the ATPase activity and the PLB interaction. Then, myocytes expressing the luminal Ca-insensitive SERCA2a mutant will be studied to define the role of this novel mechanism in cardiac Ca cycling. We expect that the outcome of this work will greatly advance our understanding of SERCA2a function. We expect that the outcome of these studies will provide a detailed view of this new mechanism of SERCA2a regulation, advancing our understanding of the Ca pump’s function. In Aim 2, we will test the hypothesis that luminal redox potential regulates SERCA2a by stabilizing its luminal Ca binding sites, thus, improving the pump’s regulation by [Ca]SR. Molecular dynamic simulations will be used to forecast the role of the SERCA2a luminal disulfide bond in the pump catalytic cycle. We will assess whether mutation of luminal cysteines leads to a loss-of-function phenotype by abolishing SERCA2a regulation by luminal Ca. Newly developed approaches to measure luminal redox potential and [Ca]SR will be used to define the cross-talk between luminal redox potential and SERCA2a activity. We will investigate the contribution of this new mechanism to SERCA2a dysfunction and Ca mishandling in HF. The likely outcome of these studies is a new concept that can explain how alterations in SERCA2a structure/function cause defects in Ca regulation in HF.