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Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles.


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.

SUBMITTER: Bian T 

PROVIDER: S-EPMC5123963 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles.

Bian Tengfei T   Autry Joseph M JM   Casemore Denise D   Li Ji J   Thomas David D DD   He Gaohong G   Xing Chengguo C  

Biochemical and biophysical research communications 20161101 3-4


We have developed a charge-mediated fusion method to reconstitute the sarco/endoplasmic reticulum Ca<sup>2+</sup>-ATPase (SERCA) in giant unilamellar vesicles (GUV). Intracellular Ca<sup>2+</sup> 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+</sup> dysregulation in human diseases including cancer, diabetes, and heart failure. Here we report the developm  ...[more]

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