Influence Of Potassium, Sodium And Calcium Ions On The Interaction Between Amphotericin B And Lipid Membrane

Polyene macrolide antibiotics has strong antifungal activities,which is used against mycotic infections.Particularly,amphotericin B（AmB）is the most potent antifungal drug available.It consists of a heptaene macrolide skeleton,with a functional carboxyl group at position C16 and a mycosamine sugar appendage at position C19.It is estimated that AmB antifungal activity depends on the presence of sterols in the cellular membrane.AmB molecule and ergosterol form complexes,which aggregate and form pores（or channels）inside the cellular membrane,leading to the deaths of fungal cells.However,at the same time,AmB molecules combine with the cholesterol to form pores,which produces toxicity to human cells.The adverse reaction of AmB to human body greatly limits its use in clinical effect.So it is necessary for discussing how to reduce its toxicity to cells,so as to improve the effect of clinical use of AmB.In addition,these pores induce considerable effects on physiological ion transport,especially K⁺ ions.And AmB may inhibit the ATP（Na⁺-K⁺）activity in animal cells.On the other hand,it is reported that intake（>4 mEQ/kg）per day of a certain amount of sodium salt may improve the toxicity of AmB to the patients.It was observed that the enrichment of fungal cell cultures with K⁺ ions exhibits protective properties against the toxic activity of AmB.The K⁺ ions have an influence on the aggregation levels of AmB molecules on the membrane.It has been reported that calcium ions play a notable role in the formation of AmB ion channels.Therefore,the research on the influence of the potassium,sodium and calcium ions on the AmB-lipid membrane system has a great significance for reference to understand the reduction of cell toxicity.Therefore,in order to know the effect of potassium,sodium and calcium ion on amphotericin B,we have studied the influence of the three kinds of metal cations on AmB/DPPC membrane and AmB/Chol/DPPC membrane using the Langmuir monolayer and liposome bilayer models.For Langmuir monolayer model,we have analysed the thermodynamic parameters and elastic modulus of the membrane to understand the influence of metal cations to the molecular interaction.Thus for liposome bilayer model,the fluorescence lipid probes have been used to tab the hydrophilic and hydrophobic domain of membrane,which can monitor the influence of metal cations on the different membrane domain.Conclusions are as follows:（1）Obviously,different cations can influence the phase transition of AmB-DPPC monolayer.When X_AmB= 0.7,in the presence of Ca²⁺ ions,the surface pressure and elasticity modulus of the phase transition increased apparently compared with those in the absence of cations,which is different from that in the presence of K⁺ or Na⁺ ions.The phase transition of the monolayer needs a higher concentration of AmB in the sequence Na⁺>pure buffer>K⁺>Ca²⁺.（2）At the same surface pressure,these metal cations cause different change in expansion of the π-A curve,which suggests that they could alter the area per molecule of the Chol/DPPC monolayer in liquid phase.In water,after addition of AmB,the area per molecule of the lipid monolayer decreases at above a certain surface pressure thus increases at below the certain surface pressure,which is the surface pressure at the point of transition of the AmB/Chol/DPPC monolayer from liquid expanded to liquid condensed phase.And the addition of AmB induce the change of physical state of the lipid monolayer from liquid-gel state to liquid state.These metal cations have a mainly effect on phase transition of the AmB-lipid monolayer.The K⁺ions and Ca²⁺ ions make the obvious point of phase transition disappeared.But the presence of Na⁺ ions has little influence on phase transition of the AmB-lipid monolayer.The addtion of AmB and the presence of these metal cations weaken the attractive force between the molecules on the monolayers.And the degree of weakening are difference in the presence of different metal ions.The drug molecules may induce the change of phase state and order of cell membrane.（3）The AmB-cholesterol complex could be formed,which results in the appearance of pore（ion channel）in membrane of liposome.For hydrophilic areas of membrane in liposomes without Chol,the presence of potassium,sodium and calcium ions make the ordering of headgroup in membrane enhanced,and the sequence is Ca²⁺>Na⁺>K⁺.But for hydrophobic areas,the K⁺ ions strengthen the packing ability of acyl chain.On the contrary,Ca²⁺ and Na⁺ ions make the acyl chain loop up to the membrane interface,leading to the weakening of its packing ability.Differently,Na⁺ions reduce the ordering of hydrophilic areas and weaken the ability of packing of acyl chain in membrane of the liposomes with Chol,which is just the opposite of the effect of Ca²⁺ and K⁺ ions.（4）On the other hand,these metal cations have an influence on the effect of Chol.In AmB/Chol/DPPC liposome system,some cholesterol molecules combine with AmB to form complex,and others interact with acyl chain.Cholesterol induce the NBD group to locate a deeper domain,which suggests cholesterol strengthen the ability of packing of membrane.The phenomenon may be influenced by potassium,sodium and calcium ions.For the effect of Chol on hydrophilic head,the K⁺ ions has no clear role.The Na⁺ ions restrain slightly but the Ca²⁺ ions promote the effect.However,for hydrocarbon chain,the K⁺ ions restrain the effect of Chol while the Na⁺ and Ca²⁺ ions promote it.It is reported that intake of high sodium salt may improve the toxicity of AmB to the patients,which may be relative to that the Na⁺ ions can promote the effect of cholesterol to strengthen the ability of packing of membrane.In other words,the great ability of packing of membrane may influence on the pores formed by AmB and reduce its toxicity to membrane.