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)|
|Number of pages||6|
|Journal||Biochemical and Biophysical Research Communications|
|State||Published - Dec 9 2016|
Bibliographical noteFunding 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.
© 2016 Elsevier Inc.
Copyright 2017 Elsevier B.V., All rights reserved.
- Biomimetic membrane
- Calcium regulation
- Drug discovery
- Electrostatic fusion
- Lipid reconstitution
- Transport proteins
University Imaging Centers