Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles
- Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 2231 6th St SE, Minneapolis, MN 55455 (United States)
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 321 Church St SE, Minneapolis, MN 55455 (United States)
- State Key Laboratory of Fine Chemicals, R&D Center of Membrane Science and Technology, Dalian University of Technology, Dalian 116024 (China)
We have developed a charge-mediated fusion method to reconstitute the sarco/endoplasmic reticulum Ca{sup 2+}-ATPase (SERCA) in giant unilamellar vesicles (GUV). Intracellular Ca{sup 2+} 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 Ca{sup 2+} 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 Ca{sup 2+} transport by SERCA in single GUV was detected directly using confocal fluorescence microscopy with the Ca{sup 2+} indicator Fluo-5F. The GUV reconstitution system was validated for functional screening of Ca{sup 2+} 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 Ca{sup 2+} 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.
- OSTI ID:
- 22696728
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 481, Issue 3-4; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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