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Author Bian, Tengfei ♦ Autry, Joseph M. ♦ Casemore, Denise ♦ Li, Ji ♦ Thomas, David D. ♦ He, Gaohong ♦ Xing, Chengguo
Source United States Department of Energy Office of Scientific and Technical Information
Content type Text
Language English
Subject Keyword APPLIED LIFE SCIENCES ♦ ANIMAL CELLS ♦ CALCIUM IONS ♦ CLINICAL TRIALS ♦ ENDOPLASMIC RETICULUM ♦ HEART FAILURE ♦ INHIBITION ♦ MEMBRANE TRANSPORT
Abstract 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.
ISSN 0006291X
Educational Use Research
Learning Resource Type Article
Publisher Date 2016-12-09
Publisher Place United States
Journal Biochemical and Biophysical Research Communications
Volume Number 481
Issue Number 3-4


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