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.
['Bian|Tengfei|T|', 'Autry|Joseph M|JM|', 'Casemore|Denise|D|', 'Li|Ji|J|', 'Thomas|David D|DD|', 'He|Gaohong|G|', 'Xing|Chengguo|C|']
D000818:Animals D002118:Calcium D017136:Ion Transport D008055:Lipids D008856:Microscopy, Fluorescence D009829:Oleic Acids D010713:Phosphatidylcholines D011817:Rabbits D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases D054852:Small Molecule Libraries D055672:Static Electricity D019284:Thapsigargin D053835:Unilamellar Liposomes
Transport proteins Biomimetic membrane Calcium regulation Drug discovery Electrostatic fusion Lipid reconstitution