Gadolinium modifies the cell membrane to inhibit permeabilization by nanosecond electric pulses

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Highlights

  • Gd3+ causes a lasting modification of the cell membrane to inhibit electroporation.

  • Gd3+ does not clog electropores.

  • Modification by Gd3+ occurs promptly and independently of electropore presence.

  • Gd3+ makes the plasma membrane less prone to electropermeabilization.

  • Gd3+ reduces the size and/or lifetime of formed electropores.

Abstract

Lanthanide ions are the only known blockers of permeabilization by electric pulses of nanosecond duration (nsEP), but the underlying mechanisms are unknown. We employed timed applications of Gd3+ before or after nsEP (600-ns, 20 kV/cm) to investigate the mechanism of inhibition, and measured the uptake of the membrane-impermeable YO-PRO-1 (YP) and propidium (Pr) dyes. Gd3+ inhibited dye uptake in a concentration-dependent manner. The inhibition of Pr uptake was always about 2-fold stronger. Gd3+ was effective when added after nsEP, as well as when it was present during nsEP exposure and removed afterward. Pores formed by nsEP in the presence of Gd3+ remained quiescent unless Gd3+ was promptly washed away. Such pores resealed (or shrunk) shortly after the wash despite the absence of Gd3+. Finally, a brief (3 s) Gd3+ perfusion was equally potent at inhibiting dye uptake when performed either immediately before or after nsEP, or early before nsEP. The persistent protective effect of Gd3+ even in its absence proves that inhibition by Gd3+ does not result from simple pore obstruction. Instead, Gd3+ causes lasting modification of the membrane, occurring promptly and irrespective of pore presence; it makes the membrane less prone to permeabilization and/or reduces the stability of electropores.

Introduction

The phenomenon that high-voltage intense electric pulses of nanosecond duration (nsEP) cause cell membrane permeabilization, or electroporation, has been well studied in recent decades [1], [2]. In addition to plasma membrane permeabilization, nsEP can lead to cell swelling and blebbing [3], [4] and activation of necrotic and apoptotic cell death pathways [5], [6], [7]. An important quality of pores formed by nsEP is that the diameter is thought to be no larger than approximately 1–1.5 nm (“nanopores”). This size parameter was determined experimentally by the selective uptake of smaller dye molecules, such as YO-PRO-1 (YP) and ions (such as Tl+) versus larger dye molecules, such as propidium (Pr) [8], [9], as well as by the blockage of cell swelling using solutes that are too large to pass through the pores [3]. Finally, pores formed by nsEP are stable with a lifetime as long as several minutes [1], [8], [10], [11].

To date, lanthanide ions, such as gadolinium (Gd3+), are the only known inhibitors of electropermeabilization [4], [12]. Gd3+ was shown to attenuate the effects of nsEP, reducing cell swelling and blebbing, and increased cell survival [12]. However, the details and mechanism of Gd3+ block of electropermeabilization are poorly understood.

Lanthanide ions are well-known nonspecific inhibitors of several types of voltage-gated and mechanosensitive ion channels, as well as ion transporters and membrane-bound receptors [13], [14], [15]. Though the mechanism of inhibition by lanthanide ions is not clear, it is thought to be due to their similar cationic radii with that of calcium (Ca2+) ions [16]. On the other hand, there is evidence that lanthanide ions can bind to phospholipids with high affinity and affect the physical properties of the lipid bilayer, altering the function of membrane-bound proteins [17], [18], [19]. Therefore, it remains a controversy how lanthanide ions are such potent nonspecific inhibitors of ion channels, transporters, and receptors.

Understanding the inhibitory effect of Gd3+ on electropermeabilization can help to uncover a broader mechanism of Gd3+’s action on the cell membrane and, in turn, on membrane-bound proteins. It is unclear whether Gd3+ clogs pores that are formed by nsEP or if it alters the plasma membrane to interfere with pore formation and/or stability. In the present study, we sought to investigate the inhibitory mechanism of Gd3+ on electropermeabilization. Our results revealed a persistent and sustained protection of cells from nsEP by Gd3+, even when Gd3+ ions were absent in the solution. These findings prove that Gd3+ does not clog pores that are formed by nsEP. Instead, Gd3+ causes a lasting modification of the plasma membrane, which occurs independently of pore presence. Thus, the membrane becomes less susceptible to permeabilization, and pores that are formed are less stable.

Section snippets

Cell culture

Chinese hamster ovary cells (CHO-K1) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in culture at 37 °C, 5% CO2 in Ham’s F12K Media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The media and its components were purchased from Mediatech Cellgro (Herndon, VA), except for the serum which was purchased from Atlanta Biologicals (Norcross, GA). One day prior to experiments, cells were passaged and transferred onto

Gd3+ inhibits nsEP-induced uptake of YP and Pr in a concentration-dependent manner

The first series of experiments investigated the concentration range over which Gd3+ protects cells from the effects of nsEP-induced membrane permeabilization. CHO-K1 cells were bathed in a physiological solution containing varying concentrations of Gd3+, ranging from 0 to 1000 μM. Following nsEP exposure (10 pulses), we observed a significant increase in both YP and Pr emission in cells bathed in 0 μM Gd3+ (“control”, Fig. 1A and B). Incrementally increasing the concentration of Gd3+ up to 1000 

Discussion

In this study, we investigated how Gd3+ protects cells from the effects of nsEP-induced permeabilization. Does Gd3+ clog nsEP-induced pores formed in the plasma membrane? Or does it modify the plasma membrane to interfere with pore formation and/or stability? Here we show that Gd3+ inhibits uptake of both YP and Pr in a dose-dependent manner, with a ∼2-fold greater inhibition of Pr uptake. We found that Gd3+ inhibition of dye uptake is persistant, being sustained even when Gd3+ is absent in the

Acknowledgments

This work was supported by R01GM088303 from the National Institute of General Medical Sciences (to A.G.P.). We thank Dr. S. Xiao (Old Dominion University, Norfolk, VA) for continued help with nsEP exposure and dosimetry.

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