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论文类型：期刊论文

发表时间：2016-12-09

发表刊物：BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

收录刊物：SCIE、PubMed、Scopus

卷号：481

期号：3-4

页面范围：206-211

ISSN号：0006-291X

关键字：Biomimetic membrane; Calcium regulation; Drug discovery; Electrostatic fusion; Lipid reconstitution; Transport proteins

摘要：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. (C) 2016 Elsevier Inc. All rights reserved.