Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles

Tengfei Bian; Joseph M. Autry; Denise Casemore; Ji Li; David D. Thomas; Gaohong He; Chengguo Xing

doi:10.1016/j.bbrc.2016.10.096

Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles

Tengfei Bian, Joseph M. Autry, Denise Casemore, Ji Li, David D. Thomas, Gaohong He, Chengguo Xing

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Abstract

We have developed a charge-mediated fusion method to reconstitute the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) in giant unilamellar vesicles (GUV). Intracellular Ca²⁺ transport by SERCA controls key processes in human cells such as proliferation, signaling, and contraction. Small-molecule effectors of SERCA are urgently needed as therapeutics for Ca²⁺ dysregulation in human diseases including cancer, diabetes, and heart failure. Here we report the development of a method for efficiently reconstituting SERCA in GUV, and we describe a streamlined protocol based on optimized parameters (e.g., lipid components, SERCA preparation, and activity assay requirements). ATP-dependent Ca²⁺ transport by SERCA in single GUV was detected directly using confocal fluorescence microscopy with the Ca²⁺ indicator Fluo-5F. The GUV reconstitution system was validated for functional screening of Ca²⁺ transport using thapsigargin (TG), a small-molecule inhibitor of SERCA currently in clinical trials as a prostate cancer prodrug. The GUV system overcomes the problem of inhibitory Ca²⁺ accumulation for SERCA in native and reconstituted small unilamellar vesicles (SUV). We propose that charge-mediated fusion provides a widely-applicable method for GUV reconstitution of clinically-important membrane transport proteins. We conclude that GUV reconstitution is a technological advancement for evaluating small-molecule effectors of SERCA.

Original language	English (US)
Pages (from-to)	206-211
Number of pages	6
Journal	Biochemical and Biophysical Research Communications
Volume	481
Issue number	3-4
DOIs	https://doi.org/10.1016/j.bbrc.2016.10.096
State	Published - Dec 9 2016

Bibliographical note

Funding Information:
This research was funded by a graduate fellowship from the China Scholarship Council to T.B., and research grants from the United States National Institutes of Health to C.X. ( CA163864 ) and D.D.T. ( GM27906 ). SERCA purification was performed at the Biophysical Technology Center, University of Minnesota Department of Biochemistry, Molecular Biology, and Biophysics ( https://cbs.umn.edu/btc ). Jesse McCaffrey assisted with SERCA purification, Bengt Svensson assisted with molecular modeling, and Octavian Cornea assisted with manuscript preparation. Dynamic light scattering was performed in the laboratory of Jayanth Panyam, Department Head of Pharmaceutics, University of Minnesota. Fluorescence microscopy was performed using the Nikon NiE C2 upright confocal imaging system at the University of Minnesota Imaging Centers ( http://uic.umn.edu ), directed by Mark Sanders.

Publisher Copyright:
© 2016 Elsevier Inc.

Keywords

Biomimetic membrane
Calcium regulation
Drug discovery
Electrostatic fusion
Lipid reconstitution
Transport proteins

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This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.bbrc.2016.10.096

http://europepmc.org/articles/pmc5123963

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title = "Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles",

abstract = "We have developed a charge-mediated fusion method to reconstitute the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in giant unilamellar vesicles (GUV). Intracellular Ca2+ transport by SERCA controls key processes in human cells such as proliferation, signaling, and contraction. Small-molecule effectors of SERCA are urgently needed as therapeutics for Ca2+ dysregulation in human diseases including cancer, diabetes, and heart failure. Here we report the development of a method for efficiently reconstituting SERCA in GUV, and we describe a streamlined protocol based on optimized parameters (e.g., lipid components, SERCA preparation, and activity assay requirements). ATP-dependent Ca2+ transport by SERCA in single GUV was detected directly using confocal fluorescence microscopy with the Ca2+ indicator Fluo-5F. The GUV reconstitution system was validated for functional screening of Ca2+ transport using thapsigargin (TG), a small-molecule inhibitor of SERCA currently in clinical trials as a prostate cancer prodrug. The GUV system overcomes the problem of inhibitory Ca2+ accumulation for SERCA in native and reconstituted small unilamellar vesicles (SUV). We propose that charge-mediated fusion provides a widely-applicable method for GUV reconstitution of clinically-important membrane transport proteins. We conclude that GUV reconstitution is a technological advancement for evaluating small-molecule effectors of SERCA.",

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note = "Funding Information: This research was funded by a graduate fellowship from the China Scholarship Council to T.B., and research grants from the United States National Institutes of Health to C.X. ( CA163864 ) and D.D.T. ( GM27906 ). SERCA purification was performed at the Biophysical Technology Center, University of Minnesota Department of Biochemistry, Molecular Biology, and Biophysics ( https://cbs.umn.edu/btc ). Jesse McCaffrey assisted with SERCA purification, Bengt Svensson assisted with molecular modeling, and Octavian Cornea assisted with manuscript preparation. Dynamic light scattering was performed in the laboratory of Jayanth Panyam, Department Head of Pharmaceutics, University of Minnesota. Fluorescence microscopy was performed using the Nikon NiE C2 upright confocal imaging system at the University of Minnesota Imaging Centers ( http://uic.umn.edu ), directed by Mark Sanders. Publisher Copyright: {\textcopyright} 2016 Elsevier Inc.",

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AU - He, Gaohong

AU - Xing, Chengguo

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KW - Drug discovery

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Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles

Abstract

Bibliographical note

Keywords

UN SDGs

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Nikon C2 Upright Spectral Confocal and Widefield Imaging System

University Imaging Centers

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Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles

Abstract

Bibliographical note

Keywords

UN SDGs

Access

OpenUrl availability

Other files and links

Fingerprint

Equipment

Nikon C2 Upright Spectral Confocal and Widefield Imaging System

University Imaging Centers

Cite this