Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles.
Biochem Biophys Res Commun, 2016/12/09;481(3-4):206-211.
Bian T[1], Autry JM[2], Casemore D[3], Li J[2], Thomas DD[4], He G[5], Xing C[6]
Affiliations
PMID: 27815070DOI: 10.1016/j.bbrc.2016.10.096
Impact factor: 3.322
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.
Keywords: Biomimetic membrane; Calcium regulation; Drug discovery; Electrostatic fusion; Lipid reconstitution; Transport proteins
MeSH terms
Animals; Calcium; Ion Transport; Lipids; Microscopy, Fluorescence; Oleic Acids; Phosphatidylcholines; Rabbits; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Small Molecule Libraries; Static Electricity; Thapsigargin; Unilamellar Liposomes
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