Development of Simple and Rapid Evaluation for Channel Proteins Embedded in Cell-Derived Vesicles Based on Dielectrophoretic Manipulation
Authors: Eguchi Ryu , Isozaki Yushi , Suzuki Masato , Yasukawa Tomoyuki 
Published: December 2024

Introduction

Channel proteins in the plasma membrane play important roles in regulating the transport of specific molecules and ions in response to concentration differences between inside and outside cells. Therefore, the simple and rapid analysis of channel function and screening of agents for regulating the channel functions are desired in the research fields for pharmacology and drug discovery. Patch-clamp technique provides a unique and precise analysis for channel function. However, it has low measurement throughput to requires precise manipulation of a glass electrode to contact with a single cell. In this study, we applied the manipulation technique by dielectrophoresis (DEP) for easy and rapid evaluation of the opening and closing of channels embedded in Giant Plasma Membrane Vesicles (GPMVs). The time dependence of the fluorescence intensity of the internal dye of GPMVs, which were ordered well on the microwell array electrode by DEP was used to investigate the open/closed state of connexin, that is a channel protein expressed on the membrane of GPMVs. Furthermore, we revealed that the DEP behavior of GPMVs depends on the applied frequency and the open/closed state of connexins on GPMVs.

Methods

HeLa cells were transfected to express connexin 43 fused with FusionRed at the C-terminal (Cx-HeLa). The Cx-HeLa cells stained with Calcein-AM were incubated for 6 hours in an active buffer (10 mM HEPES, 2 mM CaCl2, 150 mM NaCl, 25 mM paraformaldehyde, 2 mM dithiothreitol, 125 mM glycine, pH 7.4). The active buffer containing GPMVs was exchanged to DEP medium (270 mM sucrose, 2 mM CaCl2, 50 mS m-1 in conductivity) by dialysis. The DEP device consisted of a bottom indium-tin-oxide (ITO) substrate with 10,000 microwells (16 µm in diameter and 10 µm in height) and a top ITO substrate mounted on the bottom substrate via a double adhesion tape (15 µm in thickness) (Fig. A). After GPMVs suspension was introduced in the DEP device, sine waves were applied to the top and bottom ITO substrates to induce DEP force to GPMVs.

Results and Conclusion

GPMVs were isolated from Cx-HeLa cells, and the mean diameter of GPMVs was 5.6 µm (S.D. 2.1 µm). It is known that connexin channels maintain the close state in the solution containing Ca2+ [1]. When an AC voltage of 800 kHz was applied, the GPMVs with the connexin closed were quickly trapped in the microwells. This indicates the attractive force to higher electric field regions, called positive dielectrophoresis (p-DEP), acted on the GPMVs. The outer solution of GPMVs arrayed by p-DEP was exchanged to the buffer without Ca2+ for opening connexins. The fluorescence intensity of GPMVs in the open connexin state decreased rapidly compared to that in the closed connexin state (Fig. B). This result indicates that the fluorescent dye diffused outside through the opened connexin.

Next, the dielectrophoretic behavior of the GPMVs introduced into the device was investigated. When an AC voltage with 500 kHz was applied, the GPMVs with closed connexins moved to the center of four microwells (Fig. C). This result indicates that negative-DEP (n-DEP), the repulsive force from the higher electric field region was induced to GPMVs. The critical factor for identifying the electric properties of GPMVs is the cross-over frequency at which the n-DEP and p-DEP switch. The cross-over frequency was found to be between 500 kHz and 700 kHz for GPMVs with closed channels, while it was shifted to the region between 700 kHz and 800 kHz with open channels. The shift of cross-over frequency could be attributed to the decrease in membrane capacitance caused by the opening of the connexin channels. In conclusion, DEP manipulation of GPMVs could be useful for tracking single vesicles and easily discriminating the open/closed state of connexins embedded in the membrane.

References

[1] J. Thimm, A. Mechler, H. Lin, S. Rhee, R. Lal, J. Biol. Chem.
2005, 280 (11), 10646–10654.

Figure 1