Real-Time Fluorimetric Analysis of Gramicidin D- and Alamethicin-Induced K⁺Efflux from Sf9 and Cf1 Insect Cells

Abstract

Gramicidin D and alamethicin are pore-forming peptides which exhibit lethal properties against a large spectrum of cells. Despite a wealth of experimental data from artificial membranes, the time course and quantitative analysis of the activity of these ionophores are not well described in living cells. In the present study, the newly described fluorescent dye CD-222 was used to monitor extracellular potassium ion concentration and report the effects of these antibiotics on the K+ permeability of the plasma membrane of Spodoptera frugiperda (Sf9) and Choristoneura fumiferana (Cf1) insect cells. Both peptides induced a rapid efflux of intracellular K+ as a consequence of ion channel formation in the cell membrane. K+ efflux began without any measurable delay. While the final extracellular K+ concentration was unaffected by ionophore concentration, the rate of K+ efflux was dose dependent. Using a model describing the partition of the peptides in lipid membranes, the K+ efflux kinetic parameters were determined for both cell types and both pore formers. The proposed stoichiometry for the channel formed by gramicidin in living cells is in good agreement with the two-monomers model based on data from artificial membrane systems. The K+-permeable channel formed by alamethicin in insect cells appears to involve three monomers.

<title>Lipid bilayers suspended on microfabricated supports</title>

“…This is consistent with measurements upon gramicidin A made by other workers [11][12] and shows that the membrane must be a true bilayer since gramicidin does not conduct in lipid multilayers. …”

Membrane Permeabilization of a Mammalian Neuroendocrine Cell Type (PC12) by the Channel‐Forming Peptides Zervamicin, Alamethicin, and Gramicidin

“…For example, upon intraperitoneal administration of 4 mg ZER/kg, the maximum safe dosage to show neurotropic effects in mice [17], the concentration of ZER in the brain was found to be 0.1 mm [18], thus much lower than the cell-damaging doses found in our patch-clamp recordings. The following EC 50 (50% effective concentration) values were previously reported for ALA and ZER in permeabilization and lysis studies with procaryotic and eukaryotic cells: EC 50 ¼ 16 -30 mm (ALA, red blood cells and chromaffin cells) [9] [10], EC 50 % 50 mm (ALA, leukocytes) [11], EC 50 % 2.4 mm (ALA, insect cells) [14], EC 50 ¼ 3 mm (ZER, Gram-positive bacteria) [8]. The permeabilizing activity of both peptaibols is much lower compared to GRAM which caused hemolysis at concentrations of 3 -10 nm [36] and formed channels in insect cells at 0.02 -0.5 mm (EC 50 % 0.2 mm) [14].…”

“…The following EC 50 (50% effective concentration) values were previously reported for ALA and ZER in permeabilization and lysis studies with procaryotic and eukaryotic cells: EC 50 ¼ 16 -30 mm (ALA, red blood cells and chromaffin cells) [9] [10], EC 50 % 50 mm (ALA, leukocytes) [11], EC 50 % 2.4 mm (ALA, insect cells) [14], EC 50 ¼ 3 mm (ZER, Gram-positive bacteria) [8]. The permeabilizing activity of both peptaibols is much lower compared to GRAM which caused hemolysis at concentrations of 3 -10 nm [36] and formed channels in insect cells at 0.02 -0.5 mm (EC 50 % 0.2 mm) [14]. These concentrations did not have acute (within minutes) toxic effects on our PC12 cultures, judged from the unaltered morphology of the cells.…”

“…Furthermore, an inflow of Ca 2 þ into chromaffin cells has been observed in the presence of ALA [13], which induced catecholamine secretion [10]. A fast depletion of intracellular K þ from insect cells was found upon treatment with ALA [14]. Another peptaibol, ampullosporin A, was found to induce hypothermia in mice, suggesting a neuroleptic type of activity [15] [16].…”

The Coumarin Moiety as Chromophore of Fluorescent Ion Indicators in Biological Systems

“…While a detailed account on the application of the aforementioned probes is beyond the scope of this review, a few examples are given which indicate the potential of these synthetic fluorescent probes as ion indicators: The use of BTC and MCC-type probes in investigations of Ca 2+ -dependent exocytosis [19], and rates of K + transport across biological membranes [54], respectively. The monitoring of K + efflux from Sf 9 and Cf1 insect cells using CD-222 [55], the applicability of crown ether derivatives such as C343-crown in ion detection [56], the potential use of 4-(monoaza-18-crown-6-methyl)-7-octadecanoylaminocoumarin in optical fiber fluorescence sensor for the detection of saxitoxin [57], the real-time detection of phosphodiesterase 3':5'-cyclic mononucleotide activity by the Cd 2+ -cylen type anion probe [58], and the demonstration of Mg 2+ buffering mechanisms in PC12 cells using KMG-20 [59].…”