Molecular Insights Into the SERCA – Phospholamban Regulatory Axis
Creators: Armanious, Gareth
Publisher: name: University of Alberta Library
Year: 2023

<p>When calcium enters the cytosol of a muscle cell from the SR, it initiates muscle contraction, while its removal by the sarco(endo)plasmic reticulum calcium ATPase (SERCA) induces muscle relaxation. SERCA&#39;s activity is regulated by Phospholamban (PLN), which can reversibly inhibit its apparent calcium affinity. This inhibition is reversed by phosphorylation of PLN through the action of protein kinase A (PKA). The significance of SERCA in heart disease has been highlighted through the discovery of hereditary mutations in the cytoplasmic and transmembrane domains of PLN. These variants, such as Arg9-Cys, Arg9-Leu, Arg9-His, deletion of Arg14 (R14-del), and truncation at Leu39 (Leu39-X) have been associated with heart disease. This thesis aims to offer valuable insights into the mechanisms of PLN variants and their ability to influence SERCA activity, in addition to probing the fundamental interactions and regulatory action of PLN on SERCA.<br>
    I examined how mutations in distinct regions of PLN important for phosphorylation, interaction with SERCA, and membrane anchoring impact the SERCA-PLN regulatory axis. In addition, I explored the fundamental ability of PLN to influence SERCA activity and how this inhibitory effect is achieved. While there are well- known disease-associated and disease-causing variants of PLN, there are many variants of uncertain significance (VUS) which have not been characterized. I sought to proactively assess PLN residue significance in the regulation of SERCA and characterize newly identified and uncharacterized PLN variants. Additionally, despite multiple crystal structures of SERCA that represent various conformations throughout the calcium transport cycle, the structure and function of PLN throughout the entirety of the SERCA transport cycle has gaps in our understanding of the mode of interaction and how PLN exerts control on SERCA. Using sequential alanine, truncation, and selective mutagenesis of PLN, and steady-state vs. pre-steady-state SERCA activity measurements, I was able to reveal mechanistic insight into how hereditary mutations alter regulation of SERCA in addition to uncovering key residues of WT PLN of critical importance for SERCA regulation. <br>
    Using sequential truncation and alanine mutations, I discovered that PLN C-terminal residues are critical determinants of the regulatory control of SERCA, with truncation of only 1 C-terminal residue resulting in a 50% loss of regulatory control over SERCA, compared to a complete loss of function in the case of Leu39-truncation. Truncation of two or more C-terminal residues resulted in a complete loss of function akin to the PLN-null effect observed with Leu39-truncation PLN. I conclude that PLN C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLN C terminus is an important determinant of the quaternary structure of the SERCA regulatory complex. <br>
With respect to investigation of how PLN regulates SERCA, I evaluated the effects of PLN on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. I observed that PLN altered ATP-dependent calcium translocation by SERCA within the first transport cycle. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under different substrate conditions (various calcium and/or ATP concentrations) promoting distinct conformational states of SERCA, we found that the effect of PLN on SERCA depends on substrate preincubation conditions. The results indicated that PLN can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA&#39;s kinetic properties. Moreover, I noted multiple modes of interaction between SERCA and PLN and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events, providing insights into the physiological role of PLN and its regulatory effect on SERCA transport activity.<br>
Lastly, I focused on novel missense variants in PLN, an Ala15-Thr variant found in a 4-year-old female and a Pro21-Thr variant found in a 60 -year-old female, both with a family history and clinical diagnosis of dilated cardiomyopathy. Both patients also harboured a Val896-Met variant in cardiac myosin binding protein. The PLN variants caused defects in the function, phosphorylation, and dephosphorylation of PLN, and we classified these variants as potentially pathogenic. While the cardiac myosin binding protein variant Val896-Met has been previously classified as benign, it has the potential to be a low-risk susceptibility variant. My studies provide new evidence for missense variants previously classified as benign or VUS.</p>