Evaluation of molecularity of rate-limiting step of pore formation by antimicrobial peptides studied using mitochondria as a biosensor

Toxic agents, derived from bee or hornet venoms and from fungi - melittin, mastoparan, and alamethicin are able to permeabilize biological membranes. We studied the initial steps of pore formation by these peptides in rat liver mitochondria preparations (RLM) generating transmembrane potential (ΔΨ). RLM has been used as a potassium transmembrane current (PTC) sensor. The PTC induced in RLM depends linearly on the degree of steady-state activation of RLM respiration. The concentration order of such activation by melittin in a “potassium” incubation medium containing 6 mM Mg2+ was 2.01 ± 0.15. In the case of mastoparan, the reaction order was 1.83 ± 0.23. The first steady-state phase of activation of RLM respiration by alamethicin was not detected in “Tris” incubation medium; it appeared only after addition of KCl. The order of the reaction limiting such activation was 1.92 ± 0.07. It is suggested that PTC in this phase is determined by the channels with the lowest degree of oligomerization formed by “dimers”. The ratio of equally active membrane concentrations of peptides obviously reflects the ratio of average lifetimes (ALT) for corresponding “dimers” (alamethicin and melittin, 38.5; mastoparan and melittin, 0.32). It is concluded that the results of this investigation may be useful for comparative testing of perspective pharmaceuticals.

► Rat liver mitochondria based biosensor of potassium transmembrane current. ► Molecularity of melittin and mastoparan “pre-pore” formation in mitochondria is two. ► The ratio of average lifetimes of mastoparan and melittin “pre-pores” is 0.32. ► Lowest oligomerization alamethicin channel contains two peptide molecules.