Intermolecular interactions in the mechanism of skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1): evidence for a triprotomer.

Native membrane sarcoplasmic reticulum (SR) Ca(2+)-ATPase isolated from skeletal muscle (SERCA1) exhibits oligomeric kinetic behavior [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry 43, 4400-4416]. In the present study we used quenched-flow mixing, electron paramagnetic resonance (EPR), and chemical cross-linking to probe for intermolecular interactions at physiological (0.1 M) and high (0.4 M) KCl. Exposure of SR membranes to water- and lipid-soluble cross-linking reagents revealed a mixture of SERCA1 oligomeric species consisting mainly of dimers and trimers. Titration of iodoacetamide spin-labeled SERCA1 with AMPPCP in the presence of 10 microM Ca(2+) and 0.1 M KCl revealed high- (K(D) = 45 microM) and low-affinity (K(D) = 315 microM) nucleotide binding sites in a 2:1 ratio, respectively. Raising the [KCl] to 0.4 M increased the fraction of weak binding sites and lowered the K(D) of the high-affinity component (20 microM). Phosphorylation by 10 microM ATP at 21 degrees C and 0.1 M KCl produced an early burst of P(i) production without a corresponding decline in the steady-state phosphoenzyme (EP) level. The steady-state EP level was twice as large as the P(i) burst and received equal contributions from E1P and E2P. Chasing the phosphoenzyme at 0.4 M KCl and 2 degrees C with ADP revealed a biphasic time course of E1P formation with a slow phase that matched the kinetics of the transient EPR signal from the spin-labeled Ca(2+)-ATPase. The absence of a fast component in the EPR signal excludes E1P as its source. Instead, it arises from a slow, KCl-dependent transformation at the start of the cycle which controls the formation of downstream intermediates with an increased mole fraction of rotationally restricted probes. We modeled this behavior with a SERCA1 trimer in which the formation of E1P/E2/E2P from E1ATP/E2P/E1P results from concerted transformations in the subunits coupling phosphorylation (E1ATP --> E1P + ADP) to dephosphorylation (E2P --> E2 + P(i)) and the conversion of E1P to E2P.

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 dephosphorylation) using insect cell microsomes expressing SERCA2a with or without PLN (wild-type and Cys --> Ala mutant). The results show that activation of SERCA2a dephosphorylation 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.

Phospholamban inhibits Ca-ATPase conformational changes involving the E2 intermediate.

We have used steady-state fluorescence spectroscopy in combination with enzyme kinetic assays to test the hypothesis that phospholamban (PLB) stabilizes the Ca-ATPase in the E2 intermediate state. The cardiac muscle Ca-ATPase (SERCA2a) isoform was expressed either alone or coexpressed with PLB in High-Five insect cells and was isolated as insect cell microsomes. Fluorescence studies of the Ca-ATPase covalently labeled with the probe 5-(2-((iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid showed that PLB decreased the amplitude of the Ca-ATPase E2 --> E1 conformational transition by 45 +/- 3% and shifted the [Ca2+] dependence of the transition to higher Ca2+ levels (DeltaKCa = 230 nM), similar to the effect of PLB on Ca-ATPase activity. Similarly, PLB decreased the amplitude of Ca-ATPase phosphorylation by inorganic phosphate (Pi) by 55 +/- 2% and decreased slightly the affinity for Pi (DeltaK0.5 = 70 microM). However, PLB did not affect the Ca2+-dependent inhibition of Ca-ATPase phosphorylation by Pi. Finally, PLB decreased Ca-ATPase sensitivity to vanadate, increasing the IC50 value by 300 nM. The results suggest that PLB binding to Ca-ATPase stabilizes the enzyme in a conformation distinct from E2, decreasing the number of enzymes in the E2 state capable of undergoing ligand-dependent conformational changes involving the Ca-free E2 intermediate. The inability of conformation-specific ligands to fully convert this E2-like state into E1 or E2 implies that these states are not in a simple equilibrium relationship.

Nitroxyl improves cellular heart function by directly enhancing cardiac sarcoplasmic reticulum Ca2+ cycling.

Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pump function is common, development of effective therapies to stimulate contraction has proven difficult. This is thought to be attributable to their frequent reliance on cAMP stimulation to increase activator Ca(2+). A potential alternative is nitroxyl (HNO), the 1-electron reduction product of nitric oxide (NO) that improves contraction and relaxation in normal and failing hearts in vivo. The mechanism for myocyte effects remains unknown. Here, we show that this activity results from a direct interaction of HNO with the sarcoplasmic reticulum Ca(2+) pump and the ryanodine receptor 2, leading to increased Ca(2+) uptake and release from the sarcoplasmic reticulum. HNO increases the open probability of isolated ryanodine-sensitive Ca(2+)-release channels and accelerates Ca(2+) reuptake into isolated sarcoplasmic reticulum by stimulating ATP-dependent Ca(2+) transport. Contraction improves with no net rise in diastolic calcium. These changes are not induced by NO, are fully reversible by addition of reducing agents (redox sensitive), and independent of both cAMP/protein kinase A and cGMP/protein kinase G signaling. Rather, the data support HNO/thiolate interactions that enhance the activity of intracellular Ca(2+) cycling proteins. These findings suggest HNO donors are attractive candidates for the pharmacological treatment of heart failure.

Intermolecular conformational coupling and free energy exchange enhance the catalytic efficiency of cardiac muscle SERCA2a following the relief of phospholamban inhibition.

Activation of cardiac muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) by beta1-agonists involves cAMP- and PKA-dependent phosphorylation of phospholamban (PLB), which relieves the inhibitory effects of PLB on SERCA2a. To investigate the mechanism of SERCA2a activation, we compared the kinetic properties of SERCA2a expressed with (+) and without (-) PLB in High Five insect cell microsomes to those of SERCA1 and SERCA2a in native skeletal and cardiac muscle SR. Both native SERCA1 and expressed SERCA2a without PLB exhibited high-affinity (10-50 microM) activation of pre-steady-state catalytic site dephosphorylation by ATP, steady-state accumulation of the ADP-sensitive phosphoenzyme (E1P), and a rapid phase of EGTA-induced phosphoenzyme (E2P) hydrolysis. In contrast, SERCA2a in native cardiac SR vesicles and expressed SERCA2a with PLB lacked the high-affinity activation by ATP and the rapid phase of E2P hydrolysis, and exhibited low steady-state levels of E1P. The results indicate that the kinetic differences in Ca2+ transport between skeletal and cardiac SR are due to the presence of phospholamban in cardiac SR, and not due to isoform-dependent differences between SERCA1 and SERCA2a. Therefore, the results are discussed in terms of a model in which PLB interferes with SERCA2a oligomeric interactions, which are important for the mechanism of Ca2+ transport in skeletal muscle SERCA1 [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry 43, 4400-4416]. We propose that intermolecular coupling of SERCA2a molecules during catalytic cycling is obligatory for the changes in Ca2+ transport activity that accompany the relief of PLB inhibition of the cardiac SR Ca2+-ATPase.

The time-dependent distribution of phosphorylated intermediates in native sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle is not compatible with a linear kinetic model.

Quenched-flow mixing was used to characterize the kinetic behavior of the intermediate reactions of the skeletal muscle sarcoplasmic reticulum (SR) Ca-ATPase (SERCA1) at 2 and 21 degrees C. At 2 degrees C, phosphorylation of SR Ca-ATPase with 100 microM ATP labeled one-half of the catalytic sites with a biphasic time dependence [Mahaney, J. E., Froehlich, J. P., and Thomas, D. D. (1995) Biochemistry 34, 4864-4879]. Chasing the phosphoenzyme (EP) with 1.66 mM ADP 10 ms after the start of phosphorylation revealed mostly ADP-insensitive E2P (95% of EP(total)), consistent with its rapid formation from ADP-sensitive E1P. The consecutive relationship of the phosphorylated intermediates predicts a decrease in the proportion of E1P ([E1P]/[EP(total)]) with increasing phosphorylation time. Instead, after 10 ms the proportion of E1P increased and that of E2P decreased until they reached a constant 1:1 stoichiometry ([E1P]:[E2P] approximately 1). At 21 degrees C, phosphorylation displayed a transient overshoot associated with an inorganic phosphate (P(i)) burst, reflecting increased turnover of E2P at the higher temperature. The P(i) burst exceeded the decay of the EP overshoot, suggesting that rephosphorylation of the enzyme occurs before the recycling step (E2 --> E1). This behavior and the reversed order of accumulation of phosphorylated intermediates at 2 degrees C are not compatible with the conventional linear consecutive reaction mechanism: E1 + ATP --> E1.ATP --> E1P + ADP --> E2P --> E2.P(i) --> E1 + P(i). Solubilization of the Ca-ATPase into monomers using the nonionic detergent C(12)E(8) gave a pattern of phosphorylation in which E1P and E2P behave like consecutive intermediates. Kinetic modeling of the C(12)E(8)-solubilized SR Ca-ATPase showed that it behaves according to the conventional Ca-ATPase reaction mechanism, consistent with monomeric catalytic function. We conclude that the nonconforming features of native SERCA1 arise from oligomeric protein conformational interactions that constrain the subunits to a staggered or out-of-phase mode of operation.