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

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• 作者：Tengfei Bian^a ; ^b ; Joseph M. Autry^c ; ^d ; Denise Casemore^a ; Ji Li^c ; ^d ; David D. Thomas^c ; ¹ ; Gaohong He^b ; ¹ ; Chengguo Xing^a ; ¹ ; ² ; ^{chengguoxing@cop.ufl.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Biomimetic membrane ; Calcium regulation ; Drug discovery ; Electrostatic fusion ; Lipid reconstitution ; Transport proteins
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2016
• 出版时间：9 December 2016
• 年：2016
• 卷：481
• 期：3-4
• 页码：206-211
• 全文大小：1088 K

文摘

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.