Phospholamban thiols play a central role in activation of the cardiac muscle sarcoplasmic reticulum calcium pump by nitroxyl

Nitroxyl (HNO) donated by Angeli's salt activates uptake of Ca 2+ by the cardiac SR Ca 2+ pump (SERCA2a). To determine whether HNO achieves this by a direct interaction with SERCA2a or its regulatory protein, phospholamban (PLN), we measured its effects on SERCA2a activation (as reflected in dephosphoryla- tion) using insect cell microsomes expressing SERCA2a with or without PLN (wild-type and Cys f Ala mutant). The results show that activation of SERCA2a dephos- phorylation by HNO is PLN-dependent and that PLN thiols are targets for HNO. We conclude that HNO produces a disulfide bond that alters the conformation of PLN, relieving inhibition of the Ca 2+ pump. Stimulation of the cardiac SR Ca 2+ pump (SERCA2a) 1 in isolated SR vesicles by cAMP/protein kinase A-dependent phosphorylation of phospholamban (PLN) was originally demonstrated by Tada et al. (1). Subsequent studies showed that phosphorylation of PLN at Ser 16 increases the apparent Ca 2+ affinity of SERCA2a (2), the rates of phosphorylation and dephosphorylation (2, 3), and the Vmax of Ca 2+ transport (3, 4). Phosphorylation of Ser 16 induces a change in the conformation of PLN (5, 6), which relieves its inhibition and activates the pump. Recent work with ex- pressed Ca 2+ pump proteins suggests that activation of the cardiac SR Ca 2+ pump following the relief of PLN inhibition involves changes in the kinetic behavior of the Ca 2+ -ATPase consistent with SERCA2a oligomerization (7). These changes include stabilization of the ADP-sensitive phosphoenzyme, E1P, and allosteric activation of dephosphorylation by ATP, resulting in an increased rate of turnover of the ADP- insensitive phosphoenzyme, E2P. We have recently shown that nitroxyl (HNO) donated by Angeli's salt (AS, Na2N2O3) activates the SR Ca 2+ pump in isolated murine cardiac myocytes (8). This effect is inde- pendent of � -adrenergic activation (9) and results from a direct effect of HNO on the SR Ca 2+ pump as evidenced by stimulation of Ca 2+ uptake by HNO in isolated murine heart SR vesicles (8). We hypothesize that HNO stimulates Ca 2+ uptake by covalently modifying critical thiol residues in the SERCA2a pump and/or its regulatory protein, PLN, altering the conformation of these proteins, and relieving the inhibi- tion of the pump. Chemical modification of protein thiols by HNO can proceed by two pathways leading to either (a) the formation of a sulfinamide (RS(O)NH2) when one thiol group is involved or (b) the formation of a disulfide (RSSR) plus hydroxylamine (H2NOH) when two thiols are in the proximity of each other (10, 11). The fact that HNO-induced activation of RyR2 receptors reconstituted in lipid planar bilayers can be reversed by DTT (8) suggests that disulfide bond formation may be important in activating Ca 2+ release. In this study, we investigated the mechanism of activation of the SR Ca 2+ pump by HNO using SERCA2a expressed in the absence or presence of PLN in High Five (HF) insect cell microsomes. To study Ca 2+ pump activation by HNO, we measured the kinetics of dephosphorylation of SERCA2a, which is accelerated by HNO. To test whether thiol residues in PLN are critical for activation, we carried out these experiments in microsomes expressing SERCA2a and null-Cys PLN (PLN(-C)) in which the three transmembrane (TM) domain cysteine residues were replaced with alanine. Immunoblotting with an anti-PLN antibody was used to test whether HNO-induced PLN oligomer formation contributes to the relief of inhibition of SERCA2a by PLN. The results show that PLN is necessary for activation of SERCA2a by HNO and that cysteine residues in the TM domain of PLN play a critical role in PLN-dependent HNO activation of the Ca 2+ pump.