HNO Uncouples PLN from SERCA2a Enhancing Pump Activity

Abstract

The sarco(endo)plasmic Ca-ATPase (SERCA2a) sequesters Ca2+ ions from the cytosol into the cardiac sarcoplasmic reticulum (CSR) to promote cardiac relaxation. Phospholamban (PLN) is an integral membrane protein that regulates SERCA2a in CSR. Phosphorylation of PLN in response to β-adrenergic stimulation enhances cardiac inotropy by increasing CSR Ca2+ uptake. Nitroxyl (HNO), a new candidate drug therapy for congestive heart failure (CHF), improves overall cardiovascular function by increasing Ca2+ release and Ca2+ re-uptake in CSR through direct effects on RyR2 and SERCA2a, respectively. We propose that HNO, a thiol oxidant, modifies one or more PLN sulfhydryls and thereby decreases the regulatory interaction of PLN with SERCA2a, contributing to SERCA2a activation. To test this model, we have used fluorescence and EPR spectroscopy to determine the effects of HNO on the physical regulation of SERCA2a by PLN. We used expressed SERCA2a ± coexpressed PLN in High Five insect cells microsomes and Angeli's Salt (AS) as our HNO donor. Steady-state fluorescence intensity studies of FITC-labeled SERCA2a coexpressed with PLN showed that AS/HNO increased the amplitude of the Ca2+-dependent E2 to E1Ca2 conformational change by nearly two fold, similar to the effect observed when PLN is uncoupled from SERCA2a by anti-PLN antibody treatment. This matches our previous finding that AS/HNO stimulates SERCA2a activity nearly two fold. Saturation-transfer EPR studies of maleimide spin-labeled SERCA2a coexpressed with PLN showed that AS/HNO decreased the rotational mobility of SERCA2a, but not significantly more than that of control SERCA2a without PLN. Taken together, our results suggest HNO physically uncouples PLN from SERCA2a increasing SERCA2a conformational flexibility and SERCA2a activity. We are investigating the role of individual PLN cysteines in this mechanism by repeating these experiments using SERCA2a coexpressed with PLN cysteine mutants, and direct effects of HNO on SERCA2a are also being investigated.