Elsevier

Analytical Biochemistry

Volume 480, 1 July 2015, Pages 58-66
Analytical Biochemistry

Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes

https://doi.org/10.1016/j.ab.2015.04.005Get rights and content

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–Mg2+. 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 Ca2+-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.

Section snippets

Protein purification

Escherichia coli SecA from BL21(λDE3)/pT7-SecA was purified as described previously [28]. SecA homologous from other bacteria was purified similarly from BL21.19 as described previously [10], [28], [29]. Purified proOmpA, an outer membrane protein precursor, was prepared as described previously [30], and membrane complexes of SecYEG and SecDF•YajC were purified as described previously [26].

Mouse gap junction protein Cx26 was expressed in insect cell line Sf9 using the Bac-to-Bac baculovirus

Bacterial protein-conducting channels with SecA–liposomes in oocytes

As described previously [23], by injecting oocytes with proteoliposomes together with essential factors such as ATP–Mg2+ and precursor proOmpA, we were able to detect increased outward currents with E. coli SecA–liposomes (Fig. 1 and Fig. 2A; the recording activities are presented in Fig. 1, upper left panel). Liposomes injected with purified SecA homologs from other bacteria, including Bacillus subtilis (Bs SecA), Bacillus anthracis (Ba SecA1), Staphylococcus aureus (Sa SecA1), Streptococcus

Discussion

In this article, we have combined several previously described methodologies for a simple and efficient system to correlate results with in vitro assembled protein complexes in liposomes to their in-cell functionality. This method employs direct injection of preassembled/folded proteins into proteoliposomes (Fig. 1) instead of cDNA or mRNA into Xenopus oocytes. Le Caherec and coworkers [35] described the injection of the reconstituted proteoliposomes into oocytes in order to monitor aquaporin

Acknowledgments

We thank D. Oliver, A. Driessen, F. Duong, and E. Lin for plasmids; R. Veenstra and G. Sosinsky for their Cx26 collaboration; J. L. Ingraham, J. Houghton, and C. Alston for comments and editing; and C. Alexander for technical assistance. Y-H.H., and J-S.J., were fellows of the Molecular Basis of Disease Program, and J.Z. was a fellow of the Brain and Behavior Program, at Georgia State University. The core facility is supported in part by the Georgia Research Alliance. This work was supported in

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