Cytoplasmic Domain Of Phospholamban Research Articles

Effect of Phosphorylation on Interactions between Transmembrane Domains of SERCA and Phospholamban

We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to map interactions between the transmembrane (TM) domains of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLB) as affected by PLB phosphorylation. In the cardiac sarcoplasmic reticulum, PLB binding to SERCA results in Ca-dependent enzyme inhibition, which is reversed by PLB phosphorylation at Ser16. Previous spectroscopic studies on SERCA-PLB have largely focused on the cytoplasmic domain of PLB, showing that phosphorylation induces a structural shift in this domain relative to SERCA. However, SERCA inhibition is due entirely to TM domain interactions. Therefore, we focus here on PLB’s TM domain, attaching Cys-reactive spin labels at five different positions. In each case, continuous-wave EPR indicated moderate spin-label mobility, with the addition of SERCA revealing two populations, one indistinguishable from PLB alone and another with more restricted rotational mobility, presumably due to SERCA-binding. Phosphorylation had no effect on the rotational mobility of either component but significantly decreased the mole fraction of the restricted component. Solvent-accessibility experiments using power-saturation EPR and saturation-recovery EPR confirmed that these two spectral components were SERCA-bound and unbound PLB and showed that phosphorylation increased the overall lipid accessibility of the TM domain by increasing the fraction of unbound PLB. However—based on these results—at physiological levels of SERCA and PLB, most SERCA would have bound PLB even after phosphorylation. Additionally, no structural shift in the TM domain of SERCA-bound PLB was detected, as there were no significant changes in membrane insertion depth or its accessibility. Therefore, we conclude that under physiological conditions, the phosphorylation of PLB induces little or no change in the interaction of the TM domain with SERCA, so relief of inhibition is predominantly due to the previously observed structural shift in the cytoplasmic domain.

Intrinsically Disordered HAX-1 Regulates SERCA in a Calcium-Dependent Manner

Hematopoietic-subtrate-1 associated protein X-1 (HAX-1) is a 279 aa protein expressed ubiquitously. In the cardiac muscle, HAX-1 was found to inhibit the sarco-endoplasmic reticulum calcium ATPase (SERCA) by shifting the pCa to higher Ca2+ concentrations. Co-immunoprecipitation assays suggest that HAX-1 binds to phospholamban (PLN), enhancing its inhibitory action. HAX-1 function is reversed upon PLN phosphorylation by cAMP-dependent protein kinase A. Our activity assays, show that HAX-1 shifts SERCA's pCa to lower values, inhibiting the ATPase activity even in the absence of PLN. Using a combination of NMR and CD spectroscopy, we found that HAX-1 is intrinsically disordered in the absence of a binding partner. Chemical denaturation experiments show only a few portions of the protein might adopt a defined secondary structure conformation, while the remainder is essentially unfolded. In agreement with previous reports, we found that HAX-1 binds Ca2+ ions and PLN's cytoplasmic domain. We propose that HAX-1 regulates the SERCA/PLN complex in a Ca2+-dependent manner. This would add another layer of control in Ca2+ homeostatic balance in the heart muscle.

Combining Solution and Solid State NMR Techniques in the Analysis of Arg to Cys Mutations in Phospholamban Associated with Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is characterized by the enlargement and weakening of the left ventricle. This dilation of the ventricle prevents the heart from contracting normally and can spread to the right ventricle and atria as the disease progresses. Inherited forms of DCM have been identified in families that carry missense, deletion, or nonsense mutations in the regulatory protein, phospholamban (PLN). Two of the identified missense mutations, R9C and R25C, result in cysteine substitutions for arginine residues in PLN's cytoplasmic domain. In vitro studies with these mutants indicate that they have an altered ability to regulate SERCA, however it is unclear how these mutations lead to dysregulation of SERCA.Here we have applied both solution and solid-state NMR techniques to characterize the structure, dynamics and topology of the monomeric constructs for the R9C and R25C mutants to better understand how these mutations affect PLN's regulation of SERCA in the development of DCM. These studies were pursued under both reducing and oxidizing conditions to better reflect the physiological conditions of DCM progression and for comparison to coupled enzyme activity assays used to quantify the effect of these mutants on SERCA activity. We have utilized chemical shift perturbations as sensitive probes toward the local environment of the cytoplasmic and juxta-membrane domains of PLN which harbor the aforementioned mutations. Dynamics in lipid bilayers have been probed through cross-polarization or J-coupling enabled experiments which are exclusively sensitive toward either the rigid or mobile regions of the protein. Heteronuclear NMR was employed to probe the fast timescale dynamics of these mutants. Finally, separated local field experiments in magnetically aligned bicelles were employed to probe the topology.

Role of conformational sampling of Ser16 and Thr17-phosphorylated phospholamban in interactions with SERCA

Phosphorylation of phospholamban (PLB) at Ser16 and/ or Thr17 is believed to release its inhibitory effect on sarcoplasmic reticulum calcium ATPase. Ser16 phosphorylation of PLB has been suggested to cause a conformational change that alters the interaction between the enzyme and protein. Using computer simulations, the conformational sampling of Ser16 phosphorylated PLB in implicit membrane environment is compared here with the unphosphorylated PLB system to investigate these conformational changes. The results suggest that conformational changes in the cytoplasmic domain of PLB upon phosphorylation at Ser16 increase the likelihood of unfavorable interactions with SERCA in the E2 state prompting a conformational switch of SERCA from E2 to E1. Phosphorylation of PLB at Thr17 on the other hand does not appear to affect interactions with SERCA significantly suggesting that the mechanism of releasing the inhibitory effect is different between Thr17 phosphorylated and Ser16 phosphorylated PLB.

Lethal, Hereditary Mutants of Phospholamban Elude Phosphorylation by Protein Kinase A

The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, phospholamban, are essential components of cardiac contractility. Phospholamban modulates contractility by inhibiting SERCA, and this process is dynamically regulated by β-adrenergic stimulation and phosphorylation of phospholamban. Herein we reveal mechanistic insight into how four hereditary mutants of phospholamban, Arg(9) to Cys, Arg(9) to Leu, Arg(9) to His, and Arg(14) deletion, alter regulation of SERCA. Deletion of Arg(14) disrupts the protein kinase A recognition motif, which abrogates phospholamban phosphorylation and results in constitutive SERCA inhibition. Mutation of Arg(9) causes more complex changes in function, where hydrophobic substitutions such as cysteine and leucine eliminate both SERCA inhibition and phospholamban phosphorylation, whereas an aromatic substitution such as histidine selectively disrupts phosphorylation. We demonstrate that the role of Arg(9) in phospholamban function is multifaceted: it is important for inhibition of SERCA, it increases the efficiency of phosphorylation, and it is critical for protein kinase A recognition in the context of the phospholamban pentamer. Given the synergistic consequences on contractility, it is not surprising that the mutants cause lethal, hereditary dilated cardiomyopathy.

Hydrophobic Imbalance in the Cytoplasmic Domain of Phospholamban Is a Determinant for Lethal Dilated Cardiomyopathy

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) and its regulatory partner phospholamban (PLN) are essential for myocardial contractility. Arg(9) → Cys (R9C) and Arg(14) deletion (R14del) mutations in PLN are associated with lethal dilated cardiomyopathy in humans. To better understand these mutations, we made a series of amino acid substitutions in the cytoplasmic domain of PLN and tested their ability to inhibit SERCA. R9C is a complete loss-of-function mutant of PLN, whereas R14del is a mild loss-of-function mutant. When combined with wild-type PLN to simulate heterozygous conditions, the mutants had a dominant negative effect on SERCA function. A series of targeted mutations in this region of the PLN cytoplasmic domain ((8)TRSAIRR(14)) demonstrated the importance of hydrophobic balance in proper PLN regulation of SERCA. We found that Arg(9) → Leu and Thr(8) → Cys substitutions mimicked the behavior of the R9C mutant, and an Arg(14) → Ala substitution mimicked the behavior of the R14del mutant. The results reveal that the change in hydrophobicity resulting from the R9C and R14del mutations is sufficient to explain the loss of function and persistent interaction with SERCA. Hydrophobic imbalance in the cytoplasmic domain of PLN appears to be a predictor for the development and progression of dilated cardiomyopathy.

Structural and Functional Dynamics of an Integral Membrane Protein Complex Modulated by Lipid Headgroup Charge

We have used membrane surface charge to modulate the structural dynamics of an integral membrane protein, phospholamban (PLB), and thereby its functional inhibition of the sarcoplasmic reticulum Ca-ATPase (SERCA). It was previously shown by electron paramagnetic resonance, in vesicles of neutral lipids, that the PLB cytoplasmic domain is in equilibrium between an ordered T state and a dynamically disordered R state and that phosphorylation of PLB increases the R state and relieves SERCA inhibition, suggesting that R is less inhibitory. Here, we sought to control the T/R equilibrium by an alternative means—varying the lipid headgroup charge, thus perturbing the electrostatic interaction of PLB's cationic cytoplasmic domain with the membrane surface. We resolved the T and R states not only by electron paramagnetic resonance in the absence of SERCA but also by time-resolved fluorescence resonance energy transfer from SERCA to PLB, thus probing directly the SERCA–PLB complex. Compared to neutral lipids, anionic lipids increased both the T population and SERCA inhibition, while cationic lipids had the opposite effects. In contrast to conventional models, decreased inhibition was not accompanied by decreased binding. We conclude that PLB binds to SERCA in two distinct structural states of the cytoplasmic domain: an inhibitory T state that interacts strongly with the membrane surface and a less inhibitory R state that interacts more strongly with the anionic SERCA cytoplasmic domain. Modulating membrane surface charge provides an effective way of investigating the correlation between structural dynamics and function of integral membrane proteins.

Phosphorylation and Mutation Induce an Order-To-Disorder Transition in the Cytoplasmic Domain of Phospholamban

Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We have studied this interaction using electron microscopy of two-dimensional crystals of SERCA in complex with phospholamban. In previous studies, phospholamban oligomers were found interspersed between SERCA dimer ribbons and a three-dimensional model was constructed to show interactions with SERCA. In the present study, we have examined the effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the two-dimensional crystals. Based on projection maps from negatively-stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild-type phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27-to-Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of phospholamban (Arg14-to-Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34-to-Ala) was disordered. The functional state of phospholamban correlated with an order-to-disorder transition of phospholamban's cytoplasmic domain in the two-dimensional co-crystals. Furthermore, the residues studied (Ser16, Lys27 and Asn34) may implicate domain Ib of phospholamban in the order-to-disorder transition. In summary, the two-dimensional co-crystals with SERCA require a functional pentameric form of phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the phospholamban monomer for the inhibitory association. Phosphorylation or mutation of phospholamban alters the SERCA-pentamer interaction in a manner normally associated with inhibition by the monomer.

Lethal Arg9Cys phospholamban mutation hinders Ca 2+ -ATPase regulation and phosphorylation by protein kinase A

The regulatory interaction of phospholamban (PLN) with Ca(2+)-ATPase controls the uptake of calcium into the sarcoplasmic reticulum, modulating heart muscle contractility. A missense mutation in PLN cytoplasmic domain (R9C) triggers dilated cardiomyopathy in humans, leading to premature death. Using a combination of biochemical and biophysical techniques both in vitro and in live cells, we show that the R9C mutation increases the stability of the PLN pentameric assembly via disulfide bridge formation, preventing its binding to Ca(2+)-ATPase as well as phosphorylation by protein kinase A. These effects are enhanced under oxidizing conditions, suggesting that oxidative stress may exacerbate the cardiotoxic effects of the PLN(R9C) mutant. These results reveal a regulatory role of the PLN pentamer in calcium homeostasis, going beyond the previously hypothesized role of passive storage for active monomers.

Heparin-derived oligosaccharides interact with the phospholamban cytoplasmic domain and stimulate SERCA function

The association between the cardiac transmembrane proteins phospholamban and sarcoplasmic reticulum Ca 2+ ATPase (SERCA2a) regulates the active transport of Ca 2+ into the sarcoplasmic reticulum (SR) lumen and controls the contraction and relaxation of the heart. Heart failure (HF) and cardiac hypertrophy have been linked to defects in Ca 2+ uptake by the cardiac SR and stimulation of calcium transport by modulation of the PLB-SERCA interaction is a potential therapy. This work is part of an effort to identify compounds that destabilise the PLB-SERCA interaction in well-defined membrane environments. It is shown that heparin-derived oligosaccharides (HDOs) interact with the cytoplasmic domain of PLB and consequently stimulate SERCA activity. These results indicate that the cytoplasmic domain of PLB is functionally important and could be a valid target for compounds with drug-like properties.

Structural Dynamics and Topology of Phospholamban in Oriented Lipid Bilayers Using Multidimensional Solid-State NMR

Phospholamban (PLN), a single-pass membrane protein, regulates heart muscle contraction and relaxation by reversible inhibition of the sarco(endo)plasmic reticulum Ca-ATPase (SERCA). Studies in detergent micelles and oriented lipid bilayers have shown that in its monomeric form PLN adopts a dynamic L shape (bent or T state) that is in conformational equilibrium with a more dynamic R state. In this paper, we use solid-state NMR on both uniformly and selectively labeled PLN to refine our initial studies, describing the topology and dynamics of PLN in oriented lipid bilayers. Two-dimensional PISEMA (polarization inversion spin exchange at the magic angle) experiments carried out in DOPC/DOPE mixed lipid bilayers reveal a tilt angle of the transmembrane domain with respect to the static magnetic field, of 21 +/- 2 degrees and, at the same time, map the rotation angle of the transmembrane domain with respect to the bilayer. PISEMA spectra obtained with selectively labeled samples show that the cytoplasmic domain of PLN is helical and makes an angle of 93 +/- 6 degrees with respect to the bilayer normal. In addition, using samples tilted by 90 degrees , we find that the transmembrane domain of PLN undergoes fast long-axial rotational diffusion about the bilayer normal with the cytoplasmic domain undergoing this motion and other complex dynamics, scaling the values of chemical shift anisotropy. While this dynamic was anticipated by previous solution NMR relaxation studies in micelles, these measurements in the anisotropic lipid environment reveal new dynamic and conformational features encoded in the free protein that might be crucial for SERCA recognition and subsequent inhibition.

Structural Changes in the Cytoplasmic Domain of Phospholamban by Phosphorylation at Ser16: A Molecular Dynamics Study

Phospholamban is a 52-residue integral membrane protein that regulates the activity of the sarcoplasmic reticulum calcium pump in cardiac muscle. Its inhibitory action is relieved when phospholamban is phosphorylated at Ser16 by cAMP-dependent protein kinase. To computationally explore all possible conformations of the phosphorylated form, and thereby to understand the structural effects of phosphorylation, replica-exchange molecular dynamics (REMD) was applied to the cytoplasmic domain that includes Ser16. The simulations showed that (i) without phosphorylation, the region from Lys3 to Ser16 takes all alpha-helical conformations; (ii) when phosphorylated, the alpha-helix is partially unwound in the C-terminal part (from Ser10 to Ala15) resulting in less extended conformations; (iii) the phosphate at Ser16 forms salt bridges with Arg9, Arg13, and/or Arg14; and (iv) the salt bridges with Arg13 and Arg14 distort the alpha-helix and induce unwinding of the C-terminal part. We then applied conventional all-atom molecular dynamics simulations to the full-length phospholamban in the phospholipid bilayer. The results were consistent with those obtained with REMD simulations, suggesting that the transmembrane part of phospholamban and the lipid bilayer itself have only minor effects on the conformational changes in the cytoplasmic domain. The distortions caused by the salt bridges involving the phosphate at Ser16 readily explain the relief of the inhibitory effect of phospholamban by phosphorylation, as they will substantially reduce the population of all helical conformations, which are presumably required for the binding to the calcium pump. This will also be the mechanism for releasing the phosphorylated phospholamban from kinase.

A116. The cytoplasmic domain of phospholamban inhibits serca activity by direct interaction alone

A116. The cytoplasmic domain of phospholamban inhibits serca activity by direct interaction alone

Phosphorylation-dependent Conformational Switch in Spin-labeled Phospholamban Bound to SERCA

We have used chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) to probe the functional dynamics of phospholamban (PLB), which regulates the Ca-ATPase (SERCA) in cardiac sarcoplasmic reticulum. The transmembrane domain of PLB inhibits SERCA at low [Ca(2+)], but the cytoplasmic domain relieves this inhibition upon Ser16 phosphorylation. Monomeric PLB was synthesized with Ala11 replaced by the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) spin label, which reports peptide backbone dynamics directly. PLB was reconstituted into membranes in the presence or absence of SERCA. TOAC-PLB showed normal inhibitory function, which was reversed by phosphorylation at Ser16 or by micromolar [Ca(2+)]. EPR showed that the PLB cytoplasmic domain exhibits two resolved conformations, a tense T state that is ordered and a relaxed R state that is dynamically disordered and extended. PLB phosphorylation shifts this equilibrium toward the R state and makes it more dynamic (hyperextended). Phosphorylation strongly perturbs the dynamics of SERCA-bound PLB without dissociating the complex, while micromolar [Ca(2+)] has no effect on PLB dynamics. A lipid anchor synthetically attached to the N terminus of PLB permits Ca-dependent SERCA inhibition but prevents the phosphorylation-induced disordering and reversal of inhibition. We conclude that the relief of SERCA inhibition by PLB phosphorylation is due to an order-to-disorder transition in the cytoplasmic domain of PLB, which allows this domain to extend above the membrane surface and induce a structural change in the cytoplasmic domain of SERCA. This mechanism is distinct from the one that relieves PLB-dependent SERCA inhibition upon the addition of micromolar [Ca(2+)].

Spectroscopic studies of phospholamban variants in phospholipid bilayers

Phospholamban (PLB) is a 52 amino acid transmembrane protein found in the sarcoplasmic reticulum of cardiac myocytes, where it regulates the transport of calcium ions by SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase). This work has shown that the cytoplasmic domain of PLB associates with phospholipid vesicles, possibly with the lipid polar head groups, and, in doing so, undergoes a transition from a random coil to a more helical conformation. These findings support a recent hypothesis that the cytoplasmic domain of PLB acts as a conformational switch, alternating between an orientation that lies across the membrane surface and an upright orientation that associates with the regulatory site of SERCA.

Phospholamban structural dynamics in lipid bilayers probed by a spin label rigidly coupled to the peptide backbone.

We have used chemical synthesis and electron paramagnetic resonance to probe the structural dynamics of phospholamban (PLB) in lipid bilayers. Derivatives of monomeric PLB were synthesized, each of which contained a single spin-labeled 2,2,6,6,-Tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid amino acid, with the nitroxide-containing ring covalently and rigidly attached to the alpha-carbon, providing direct insight into the conformational dynamics of the peptide backbone. 2,2,6,6,-tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid was attached at positions 0, 11, and 24 in the cytoplasmic domain or at position 46 in the transmembrane domain. The electron paramagnetic resonance spectrum of the transmembrane domain site (position 46) indicates a single spectral component corresponding to strong immobilization of the probe, consistent with the presence of a stable and highly ordered transmembrane helix. In contrast, each of the three cytoplasmic domain probes has two clearly resolved spectral components (conformational states), one of which indicates nearly isotropic nanosecond dynamic disorder. For the probe at position 11, an N-terminal lipid anchor shifts the equilibrium toward the restricted component, whereas Mg(2+) shifts it in the opposite direction. Relaxation enhancement, due to Ni(2+) ions chelated to lipid head-groups, provides further information about the membrane topology of PLB, allowing us to confirm and refine a structural model based on previous NMR data. We conclude that the cytoplasmic domain of PLB is in a dynamic equilibrium between an ordered conformation, which is in direct contact with the membrane surface, and a dynamically disordered form, which is detached from the membrane and poised to interact with its regulatory target.

Electron paramagnetic resonance reveals a large-scale conformational change in the cytoplasmic domain of phospholamban upon binding to the sarcoplasmic reticulum Ca-ATPase.

We used EPR spectroscopy to probe directly the interaction between phospholamban (PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at residue 11, which was then spin labeled. PLB was reconstituted into membranes in the presence or absence of SERCA, and spin label mobility and accessibility were measured. The spin label was quite rotationally mobile in the absence of SERCA, but became more restricted in the presence of SERCA. SERCA also decreased the dependence of spin label mobility on PLB concentration in the membrane, indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the absence of SERCA. In the presence of SERCA, the spin label was released and a covalent bond was formed between PLB and SERCA, indicating direct interaction of one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the PLB-bound spin label IPSL to paramagnetic agents, localized in different phases of the membrane, indicates that SERCA greatly reduces the level of interaction of the spin label with the membrane surface. We propose that the cytoplasmic domain of PLB associates with the lipid surface, and that association with SERCA induces a major conformational change in PLB in which the cytoplasmic domain is drawn away from the lipid surface by SERCA.

Global phosphorylation at Ser16 of the 32-residue cytoplasmic domain of phospholamban: Comparison of di-t-butyl- and dibenzyl-N,N-diisopropylphosphoramidites

Phospholamban (PLB), a 52-residue integral membrane protein of the cardiac sarcoplasmic reticulum, is known as the regulator of the Ca2+-ATPase pump via its phosphorylation-dephosphorylation at Ser16. In order to investigate the structural effects of the phosphorylation on the cytoplasmic domain of PLB, we synthesized PLB 2–33 in its nonphosphorylated and phosphorylated forms using Fmoc chemistry. PLB 2–33 was phosphorylated using the global phosphorylation method. Phosphorylation with di-t-butyl-N,N-diisopropylphosphoramidite led to an incomplete reaction (nonphosphorylated peptide was recovered) and to the formation of an H-phosphonate. In contrast, the use of dibenzyl-N,N-diisopropylphosphoramidite yielded the desired phosphorylated peptide quantitatively and did not give rise to the H-phosphonate. Our results show the effectiveness of the dibenzyl-protected phosphoramidite when the site to be phosphorylated is particularly hindered.

Amino acids Glu2 to Ile18 in the cytoplasmic domain of phospholamban are essential for functional association with the Ca(2+)-ATPase of sarcoplasmic reticulum.

Phospholamban inhibits the Ca(2+)-ATPase of cardiac sarcoplasmic reticulum by lowering its affinity for Ca2+. In earlier studies (Toyofuku, T., Kazimierz, K., Tada, M., and MacLennan, D. H. (1993) J. Biol. Chem. 268, 2809-2815), parts of the phosphorylation and nucleotide binding/hinge domains of the Ca(2+)-ATPase were shown to be essential for phospholamban interaction. In order to identify the sites in phospholamban which interact with the Ca(2+)-ATPase, a series of mutants containing amino acid replacements in the cytoplasmic and transmembrane regions of phospholamban were co-expressed with the cardiac/slow-twitch muscle Ca(2+)-ATPase isozyme, SERCA2a, in HEK-293 cells. Mutation of residues in the cytoplasmic 1A domain of phospholamban, including positively charged residues, Lys3, Arg9, Arg13, and Arg14, negatively charged residue, Glu2, hydrophobic residues, Val4, Leu7, Ala11, Ile12, Ala15, and Ile18, and phosphorylation site residues, Ser16 and Thr17, resulted in loss of the inhibitory effect of phospholamban on Ca2+ transport by the Ca(2+)-ATPase. By contrast, mutation of Met1, Gln5, Tyr6, Thr8, Ser10, Glu19, or Met20 or of residues in the cytoplasmic 1B domain (Pro21 to Asn30) and of Cys41 in the transmembrane domain (Leu31 to Leu52) did not diminish the inhibitory effects of phospholamban on Ca2+ transport. These results suggest that a region essential for functional association of phospholamban with the Ca(2+)-ATPase lies in the cytoplasmic 1A domain of phospholamban.

Residues 2-25 of phospholamban are insufficient to inhibit Ca2+ transport ATPase of cardiac sarcoplasmic reticulum

We have used sarcoplasmic reticulum (SR) vesicles isolated from AT-1 cardiomyocytes to examine the mechanism of phospholamban (PLB) inhibition of the Ca2+ pump of cardiac SR. By immunoblotting with monoclonal antibodies, we observed that these SR vesicles contained a normal amount of the cardiac isoform of the Ca2+ pump (SERCA 2) but only a trace level of PLB. A monoclonal antibody that recognized amino acid residues 9-17 of PLB had no significant effect on Ca2+ transport by AT-1 SR vesicles, but it increased Ca2+ transport into mouse ventricular SR vesicles greater than 10-fold by reversing PLB inhibition of SERCA 2 at low ionized Ca2+ concentration. To further explore the domains of PLB responsible for SERCA 2 inhibition, we examined the effect of a PLB synthetic peptide consisting of amino acid residues 2-25 on Ca2+ uptake by AT-1 SR vesicles. Even at concentrations as high as 0.44 mM, no significant effect of the peptide was observed. Based on these results, we conclude that the cytoplasmic domain of PLB, containing the phosphorylation sites, by itself is insufficient to inhibit the Ca2+ pump and that the transmembrane region, which stabilizes the pentamer, is also essential for Ca2+ transport regulation.