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

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.