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

Research output: Contribution to journalArticle

4 Scopus citations

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 languageEnglish (US)
Pages (from-to)206-211
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume481
Issue number3-4
DOIs
StatePublished - Dec 9 2016

Keywords

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

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