Presentation Information
[P04-496]Quantitative evaluation of cation permeability in lipid membranes containing gramicidin A using pH-sensitive dyes
○Natsumi Hiratsuka1, Kota Ito2, Daisuke Saeki1,2, Yukihisa Okumura1,2 (1. Graduate School of Science and Technology, Shinshu University, (Japan), 2. Graduate School of Medicine, Science and Technology, Shinshu University, (Japan))
Keywords:
Gramicidin A,Liposome
Introduction:
Biological membranes possess selective permeability, which is primarily driven by biomolecules embedded within the lipid bilayer. It is important to determine the ion permeability to understand the basic function of the cell membrane and the mechanism of related phenomena such as the cytotoxicity shown by certain types of antibiotics. In our previous study, we quantitatively evaluated the cation permeability of lipid membranes containing amphotericin B using liposomes encapsulating a pH-sensitive dye, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS)1). In the present study, we applied this evaluation method to gramicidin A (GA), which forms monovalent cation-selective pores and exhibits toxicity at extremely low concentration.
Experimental:
A suspension of liposomes approximately 0.1 μm in diameter, was prepared by the extrusion method using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as lipids. A pH 7.0 MES buffer containing HPTS and the chloride salt of a target cation, LiCl, NaCl, KCl, or CaCl2, was used as the aqueous phase. Unencapslated HPTS was removed by a gel filtration, and the suspension was diluted to a total lipid concentration of 1 mmol/L. Subsequently, a solution of GA in 2,2,2-trifluoroethanol was added to the above suspension at 5, 1, or 0.1 mol% relative to POPC. The liposome suspension and an NaOH solution were rapidly mixed using a stopped-flow spectrophotometer, and the initial slope of the fluorescence intensity change over time was obtained. The cation permeable rate per GA molecule, PGA (cations · s-1 · molecule-1), and the permeability per unit membrane area, PS (cations · s-1 · m-2), were then determined.
Results and discussion:
A rapid increase in the fluorescence intensity was observed exclusively for monovalent cations while that was not observed for divalent Ca2+, suggesting that the GAs formed pores that selectively permeated monovalent cations in the lipid membrane. The time required to reach a plateau decreased with increasing the GA concentration, indicating the number of the GA pores rose with the GA concentration. The orders of both PGA and PS were same, Li+ < Na+ < K+, consistent with those of the permeability coefficient ratios evaluated by the electrophysiological measurements of membrane potential2). PGA decreased with increasing the GA concentration, indicating an increased fraction of GA molecules that did not contribute to the pore formation. PS became larger with increasing the GA concentration.
References
1) Ito, K., Saeki, D and Okumura, Y. Quantitative evaluation of the effect of sterol species on the cation permeability of lipid membranes containing amphotericin B. The Society of Chemical Engineers, Japan 54th, PB176 (2023).
2) Myers, V. B. and Haydon, D. A. Ion transfer across lipid membranes in the presence of gramicidin A: II. The ion selectivity. Biophys. Acta 274, 313–322 (1972).
Biological membranes possess selective permeability, which is primarily driven by biomolecules embedded within the lipid bilayer. It is important to determine the ion permeability to understand the basic function of the cell membrane and the mechanism of related phenomena such as the cytotoxicity shown by certain types of antibiotics. In our previous study, we quantitatively evaluated the cation permeability of lipid membranes containing amphotericin B using liposomes encapsulating a pH-sensitive dye, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS)1). In the present study, we applied this evaluation method to gramicidin A (GA), which forms monovalent cation-selective pores and exhibits toxicity at extremely low concentration.
Experimental:
A suspension of liposomes approximately 0.1 μm in diameter, was prepared by the extrusion method using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as lipids. A pH 7.0 MES buffer containing HPTS and the chloride salt of a target cation, LiCl, NaCl, KCl, or CaCl2, was used as the aqueous phase. Unencapslated HPTS was removed by a gel filtration, and the suspension was diluted to a total lipid concentration of 1 mmol/L. Subsequently, a solution of GA in 2,2,2-trifluoroethanol was added to the above suspension at 5, 1, or 0.1 mol% relative to POPC. The liposome suspension and an NaOH solution were rapidly mixed using a stopped-flow spectrophotometer, and the initial slope of the fluorescence intensity change over time was obtained. The cation permeable rate per GA molecule, PGA (cations · s-1 · molecule-1), and the permeability per unit membrane area, PS (cations · s-1 · m-2), were then determined.
Results and discussion:
A rapid increase in the fluorescence intensity was observed exclusively for monovalent cations while that was not observed for divalent Ca2+, suggesting that the GAs formed pores that selectively permeated monovalent cations in the lipid membrane. The time required to reach a plateau decreased with increasing the GA concentration, indicating the number of the GA pores rose with the GA concentration. The orders of both PGA and PS were same, Li+ < Na+ < K+, consistent with those of the permeability coefficient ratios evaluated by the electrophysiological measurements of membrane potential2). PGA decreased with increasing the GA concentration, indicating an increased fraction of GA molecules that did not contribute to the pore formation. PS became larger with increasing the GA concentration.
References
1) Ito, K., Saeki, D and Okumura, Y. Quantitative evaluation of the effect of sterol species on the cation permeability of lipid membranes containing amphotericin B. The Society of Chemical Engineers, Japan 54th, PB176 (2023).
2) Myers, V. B. and Haydon, D. A. Ion transfer across lipid membranes in the presence of gramicidin A: II. The ion selectivity. Biophys. Acta 274, 313–322 (1972).
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