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 Gd(3+) 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. Gd(3+) inhibited dye uptake in a concentration-dependent manner. The inhibition of Pr uptake was always about 2-fold stronger. Gd(3+) 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 Gd(3+) remained quiescent unless Gd(3+) was promptly washed away. Such pores resealed (or shrunk) shortly after the wash despite the absence of Gd(3+). Finally, a brief (3s) Gd(3+) 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 Gd(3+) even in its absence proves that inhibition by Gd(3+) does not result from simple pore obstruction. Instead, Gd(3+) 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.