PMID- 27815070
OWN - NLM
STAT- MEDLINE
DCOM- 20170522
LR  - 20181113
IS  - 1090-2104 (Electronic)
IS  - 0006-291X (Print)
IS  - 0006-291X (Linking)
VI  - 481
IP  - 3-4
DP  - 2016 Dec 9
TI  - Direct detection of SERCA calcium transport and small-molecule inhibition in 
      giant unilamellar vesicles.
PG  - 206-211
LID - S0006-291X(16)31702-8 [pii]
LID - 10.1016/j.bbrc.2016.10.096 [doi]
AB  - We have developed a charge-mediated fusion method to reconstitute the 
      sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) in giant unilamellar vesicles 
      (GUV). Intracellular Ca(2+) 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(2+) 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 
      Ca(2+) transport by SERCA in single GUV was detected directly using confocal 
      fluorescence microscopy with the Ca(2+) indicator Fluo-5F. The GUV reconstitution 
      system was validated for functional screening of Ca(2+) 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 Ca(2+) 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.
CI  - Copyright A(c) 2016 Elsevier Inc. All rights reserved.
FAU - Bian, Tengfei
AU  - Bian T
AD  - 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.
FAU - Autry, Joseph M
AU  - Autry JM
AD  - 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.
FAU - Casemore, Denise
AU  - Casemore D
AD  - Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 
      2231 6th St SE, Minneapolis, MN 55455, United States.
FAU - Li, Ji
AU  - Li J
AD  - 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.
FAU - Thomas, David D
AU  - Thomas DD
AD  - Department of Biochemistry, Molecular Biology, and Biophysics, University of 
      Minnesota, 321 Church St SE, Minneapolis, MN 55455, United States.
FAU - He, Gaohong
AU  - He G
AD  - State Key Laboratory of Fine Chemicals, R&D Center of Membrane Science and 
      Technology, Dalian University of Technology, Dalian 116024, China.
FAU - Xing, Chengguo
AU  - Xing C
AD  - 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.
LA  - eng
GR  - R01 CA163864/CA/NCI NIH HHS/United States
GR  - R01 GM027906/GM/NIGMS NIH HHS/United States
GR  - R01 HL129814/HL/NHLBI NIH HHS/United States
PT  - Journal Article
DEP - 20161101
PL  - United States
TA  - Biochem Biophys Res Commun
JT  - Biochemical and biophysical research communications
JID - 0372516
RN  - 0 (Lipids)
RN  - 0 (Oleic Acids)
RN  - 0 (Phosphatidylcholines)
RN  - 0 (Small Molecule Libraries)
RN  - 0 (Unilamellar Liposomes)
RN  - 0 (o-ethyldioleoylphosphatidylcholinium)
RN  - 67526-95-8 (Thapsigargin)
RN  - EC 3.6.3.8 (Sarcoplasmic Reticulum Calcium-Transporting ATPases)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Animals
MH  - Calcium/*metabolism
MH  - Ion Transport/drug effects
MH  - Lipids/chemistry
MH  - Microscopy, Fluorescence
MH  - Oleic Acids/chemistry
MH  - Phosphatidylcholines/chemistry
MH  - Rabbits
MH  - Sarcoplasmic Reticulum Calcium-Transporting ATPases/*metabolism
MH  - Small Molecule Libraries/*pharmacology
MH  - Static Electricity
MH  - Thapsigargin/pharmacology
MH  - Unilamellar Liposomes/*metabolism
PMC - PMC5123963
MID - NIHMS828333
OTO - NOTNLM
OT  - Biomimetic membrane
OT  - Calcium regulation
OT  - Drug discovery
OT  - Electrostatic fusion
OT  - Lipid reconstitution
OT  - Transport proteins
EDAT- 2016/11/07 06:00
MHDA- 2017/05/23 06:00
PMCR- 2017/12/09
CRDT- 2016/11/06 06:00
PHST- 2016/10/06 00:00 [received]
PHST- 2016/10/12 00:00 [accepted]
PHST- 2016/11/07 06:00 [pubmed]
PHST- 2017/05/23 06:00 [medline]
PHST- 2016/11/06 06:00 [entrez]
PHST- 2017/12/09 00:00 [pmc-release]
AID - S0006-291X(16)31702-8 [pii]
AID - 10.1016/j.bbrc.2016.10.096 [doi]
PST - ppublish
SO  - Biochem Biophys Res Commun. 2016 Dec 9;481(3-4):206-211. doi: 
      10.1016/j.bbrc.2016.10.096. Epub 2016 Nov 1.