Relative Affinity of Calcium Pump Isoforms for Phospholamban Quantified by Fluorescence Resonance Energy Transfer

Hou, Zhanjia; Robia, Seth L.

doi:10.1016/j.jmb.2010.07.023

Cited by 20 publications

(44 citation statements)

References 25 publications

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“…The data suggest that neither PLB nor SERCA is immobilized in the sarcoplasmic reticulum membrane. FRET between Cer-SERCA and YFP-PLB was quantified in these myocytes with the E-FRET method (17,29). FRET increased with protein concentration toward a maximum level (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Cer (17). PLB interacts similarly with SERCA1a (skeletal muscle isoform) and SERCA2a (cardiac isoform) (20,21), but with a slightly higher affinity for SERCA1a (17).…”

Section: Methodsmentioning

confidence: 99%

“…PLB interacts similarly with SERCA1a (skeletal muscle isoform) and SERCA2a (cardiac isoform) (20,21), but with a slightly higher affinity for SERCA1a (17). Adenoviral vectors of canine Cer-SERCA2a and canine YFP-PLB were produced using the AdEasy system (Stratagene, La Jolla, CA).…”

Section: Methodsmentioning

confidence: 99%

“…population of cells, as described previously (5,16,17,18,25,26). Observed FRET was compared cell-by-cell to YFP fluorescence, which was taken as an index of protein concentration (5, 16 -18, 26 …”

Section: E-fret Quantification and High Throughputmentioning

confidence: 99%

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Phospholamban Binds with Differential Affinity to Calcium Pump Conformers

Bidwell¹,

Blackwell²,

Hou

et al. 2011

Journal of Biological Chemistry

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133

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To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca 2؉ -ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. is a P-type ion pump that maintains the Ca 2ϩ gradient across the endoplasmic reticulum. In cardiac muscle cells, calcium (Ca 2ϩ ) sequestration by SERCA is critical for muscle relaxation during the cardiac cycle, and disordered Ca 2ϩ handling is associated with cardiac dysfunction (1-3). SERCA activity is regulated by phospholamban (PLB), a helical transmembrane peptide that reduces the apparent affinity of the pump for Ca 2ϩ . Inhibition of SERCA by PLB is partially relieved through phosphorylation of PLB by protein kinase A and Ca 2ϩ /calmodulindependent protein kinase (4), which alters the structure of the PLB-SERCA regulatory complex (5) and increases PLB oligomerization into non-inhibitory pentamers (5, 6). It is widely recognized that PLB inhibition of SERCA is also relieved by elevated Ca 2ϩ , but the mechanism of this functional effect is unclear. One possibility is that PLB binds selectively to the Ca 2ϩ -free "E2" conformation of SERCA and cannot bind to the Ca 2ϩ -bound "E1" conformation (Fig. 1A, Dissociation Model). This model is supported by the observation that elevated Ca 2ϩ abolishes chemical cross-linking of the PLB transmembrane domain to reactive residues on SERCA (7-11) and reduces coimmunoprecipitation (12). Cross-linking was also prevented by the SERCA inhibitor thapsigargin (Tg) (7, 9 -11). Another recent study showed that oligomerization of a PLB-SERCA fusion construct was increased by micromolar Ca 2ϩ (13), consistent with the idea that Ca 2ϩ causes displacement of PLB from the inhibitory cleft, permitting PLB self-association into pentamers. Overall, these data suggest that only certain conformational substates of SERCA interact with PLB. The dissociation model predicts that PLB unbinds from SERCA during the period of systole (cardiac contraction) when Ca 2ϩ is high, and the regulatory complex reforms during diastole (cardiac relaxation) when the cytoplasmic Ca 2ϩ concentration is low. An alternative theory was generated from in vitro measurements of fluorescence resonance energy transfer (FRET) between SERCA and PLB. Mueller et al. (14) showed that FRET was decreased, but not abolished, by Ca 2ϩ . This result suggested that relief of inhibition was accomplished by a conformational change of the regulatory complex, rather than unbinding of PLB from the pump. This study estimated a dissociation constant significantly lower than the expected in vivo concentrations of PLB and SERCA. Li et al. (15) also provided evidence from fluorescence spectroscopy that suggests that the binding of PLB and Ca 2ϩ is not...

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Section: Resultsmentioning

confidence: 99%

“…Cer (17). PLB interacts similarly with SERCA1a (skeletal muscle isoform) and SERCA2a (cardiac isoform) (20,21), but with a slightly higher affinity for SERCA1a (17).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: E-fret Quantification and High Throughputmentioning

confidence: 99%

See 2 more Smart Citations

Phospholamban Binds with Differential Affinity to Calcium Pump Conformers

Bidwell¹,

Blackwell²,

Hou

et al. 2011

Journal of Biological Chemistry

Self Cite

133

View full text Add to dashboard Cite

show abstract

“…PLB is a homopentameric, integral sarcoplasmic reticulum membrane protein that upon deoligomerization into active monomers reversibly inhibits sarco/ endoplasmic reticulum calcium ATPase (SERCA) (13)(14)(15), thereby directly regulating cardiac Ca 2ϩ kinetics and contractility (16 -21). PLB has a "helix-loop-helix" tertiary structure consisting of the N-terminal cytosolic domain IA (residues 1-16), flexible linker (residues [17][18][19][20][21][22], domain IB (residues [23][24][25][26][27][28][29][30], and C-terminal transmembrane (TM) domain II (residues 31-52) (22,23). The C-terminal TM domain is highly conserved among species (13) and may be important for PLB oligomerization (24,25) and SERCA regulation (26).…”

mentioning

confidence: 99%

Phospholamban C-terminal Residues Are Critical Determinants of the Structure and Function of the Calcium ATPase Regulatory Complex

Abrol

Smolin

Armanious

et al. 2014

Journal of Biological Chemistry

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Background: Loss of phospholamban (PLB) C-terminal residues causes cardiomyopathy in humans. Results: Deletion or mutation of C-terminal residues significantly altered PLB structure and the structure of the PLB-SERCA regulatory complex. Conclusion: PLB C-terminal residues determine the localization of the N terminus of PLB on SERCA. Significance: PLB C terminus is an important structural determinant that affects localization of PLB N terminus at a remote location on SERCA.

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Molecular Modeling of Fluorescent SERCA Biosensors

2016

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Molecular modeling and simulation are useful tools in structural biology, allowing the formulation of functional hypotheses and interpretation of spectroscopy experiments. Here, we describe a method to construct in silico models of a fluorescent fusion protein construct, where a cyan fluorescent protein (CFP) is linked to the actuator domain of the Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCA). This CFP-SERCA construct is a biosensor that can report on structural dynamics in the cytosolic headpiece of SERCA. Molecular modeling and FRET experiments allow us to generate new structural and mechanistic models that better describe the conformational landscape and regulation of SERCA. The methods described here can be applied to the creation of models for any fusion protein constructs and also describe the steps needed to simulate FRET results using molecular models.

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Relative Affinity of Calcium Pump Isoforms for Phospholamban Quantified by Fluorescence Resonance Energy Transfer

Cited by 20 publications

References 25 publications

Phospholamban Binds with Differential Affinity to Calcium Pump Conformers

Phospholamban Binds with Differential Affinity to Calcium Pump Conformers

Phospholamban C-terminal Residues Are Critical Determinants of the Structure and Function of the Calcium ATPase Regulatory Complex

Molecular Modeling of Fluorescent SERCA Biosensors

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