title: Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes.
abstract: Establishing recordable channels in membranes of oocytes formed by expressing exogenous complementary DNA (cDNA) or messenger RNA (mRNA) has contributed greatly to understanding the molecular mechanisms of channel functions. Here, we report the extension of this semi-physiological system for monitoring the channel activity of preassembled membrane proteins in single cell oocytes by injecting reconstituted proteoliposomes along with substrates or regulatory molecules. We build on the observation that SecA from various bacteria forms active protein-conducting channels with injection of proteoliposomes, protein precursors, and ATP-Mg(2+). Such activity was enhanced by reconstituted SecYEG-SecDF•YajC liposome complexes that could be monitored easily and efficiently, providing correlation of in vitro and intact cell functionality. In addition, inserting reconstituted gap junction Cx26 liposomes into the oocytes allowed the demonstration of intracellular/extracellular Ca(2+)-regulated hemi-channel activities. The channel activities can be detected rapidly after injection, can be monitored for various effectors, and are dependent on specific exogenous lipid compositions. This simple and effective functional system with low endogenous channel activity should have broad applications for monitoring the specific channel activities of complex interactions of purified membrane proteins with their effectors and regulatory molecules.
authors: Department of Biology, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, USA.
authors: Ying-Hsin
authors: YH
authors: Hsieh
authors: Department of Chemistry, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, USA.
authors: Juan
authors: J
authors: Zou
authors: Department of Biology, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, USA.
authors: Jin-Shan
authors: JS
authors: Jin
authors: Department of Biology, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, USA.
authors: Hsiuchin
authors: H
authors: Yang
authors: Department of Chemistry, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, USA.
authors: Yanyi
authors: Y
authors: Chen
authors: Department of Biology, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, USA.
authors: Chun
authors: C
authors: Jiang
authors: Department of Chemistry, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, USA. Electronic address: jyang@gsu.edu.
authors: Jenny
authors: J
authors: Yang
authors: Department of Biology, Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, USA. Electronic address: biopct@gsu.edu.
authors: Phang C
authors: PC
authors: Tai
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002460:Cell Line; D000072259:Connexin 26; D017630:Connexins / Q000378:metabolism*; D007313:Insecta; D026901:Membrane Transport Proteins / Q000378:metabolism*; D051379:Mice; D009865:Oocytes / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D059010:Single-Cell Analysis; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D017630:Connexins; C000607045:GJB2 protein, Xenopus; D026901:Membrane Transport Proteins; D011510:Proteolipids; D000069816:SEC Translocation Channels; C020485:proteoliposomes; D000072259:Connexin 26; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins
keywords: Channel activity; Cx26; Oocytes; Proteoliposomes; SecA; SecA–SecYEG complex