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.
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 2231 6th St SE, Minneapolis, MN 55455, United States; State Key Laboratory of Fine Chemicals, R&D Center of Membrane Science and Technology, Dalian University of Technology, Dalian 116024, China.
Tengfei
T
Bian
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 321 Church St SE, Minneapolis, MN 55455, United States; Biophysical Technology Center, Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 321 Church St SE, Minneapolis, MN 55455, United States.
Joseph M
JM
Autry
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 2231 6th St SE, Minneapolis, MN 55455, United States.
Denise
D
Casemore
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 321 Church St SE, Minneapolis, MN 55455, United States; Biophysical Technology Center, Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 321 Church St SE, Minneapolis, MN 55455, United States.
Ji
J
Li
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 321 Church St SE, Minneapolis, MN 55455, United States.
David D
DD
Thomas
State Key Laboratory of Fine Chemicals, R&D Center of Membrane Science and Technology, Dalian University of Technology, Dalian 116024, China.
Gaohong
G
He
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 2231 6th St SE, Minneapolis, MN 55455, United States. Electronic address: chengguoxing@cop.ufl.edu.
Chengguo
C
Xing
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D017136:Ion Transport / Q000187:drug effects; D008055:Lipids / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009829:Oleic Acids / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000494:pharmacology*; D055672:Static Electricity; D019284:Thapsigargin / Q000494:pharmacology; D053835:Unilamellar Liposomes / Q000378:metabolism*
D008055:Lipids; D009829:Oleic Acids; D010713:Phosphatidylcholines; D054852:Small Molecule Libraries; D053835:Unilamellar Liposomes; C120779:o-ethyldioleoylphosphatidylcholinium; D019284:Thapsigargin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium
Biomimetic membrane; Calcium regulation; Drug discovery; Electrostatic fusion; Lipid reconstitution; Transport proteins