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N-terminally glutamate-substituted analogue of gramicidin A as protonophore and selective mitochondrial uncoupler.

Sorochkina AI, Plotnikov EY, Rokitskaya TI, Kovalchuk SI, Kotova EA, Sychev SV, Zorov DB, Antonenko YN - PLoS ONE (2012)

Bottom Line: On the contrary, [Glu1]gA was much less potent in forming proton channels in planar lipid bilayers than gA.The difference in the behavior of [Glu1]gA and gA in natural and artificial membranes could be ascribed to increased capability of [Glu1]gA to permeate through membranes and/or redistribute between different membranes.Based on the protective role of mild uncoupling, [Glu1]gA and some other proton-conducting gA analogues may be considered as prototypes of prospective therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

ABSTRACT
Limited uncoupling of oxidative phosphorylation could be beneficial for cells by preventing excessive generation of reactive oxygen species. Typical uncouplers are weak organic acids capable of permeating across membranes with a narrow gap between efficacy and toxicity. Aimed at designing a nontoxic uncoupler, the protonatable amino acid residue Glu was substituted for Val at the N-terminus of the pentadecapeptide gramicidin A (gA). The modified peptide [Glu1]gA exhibited high uncoupling activity in isolated mitochondria, in particular, abolishing membrane potential at the inner mitochondrial membrane with the same or even larger efficacy as gA. With mitochondria in cell culture, the depolarizing activity of [Glu1]gA was observed at concentrations by an order of magnitude lower than those of gA. On the contrary, [Glu1]gA was much less potent in forming proton channels in planar lipid bilayers than gA. Remarkably, at uncoupling concentrations, [Glu1]gA did not alter cell morphology and was nontoxic in MTT test, in contrast to gA showing high toxicity. The difference in the behavior of [Glu1]gA and gA in natural and artificial membranes could be ascribed to increased capability of [Glu1]gA to permeate through membranes and/or redistribute between different membranes. Based on the protective role of mild uncoupling, [Glu1]gA and some other proton-conducting gA analogues may be considered as prototypes of prospective therapeutic agents.

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Effect of different peptides on the membrane potential of rat liver mitochondria measured by safranine O.Panel A. Shown are traces of fluorescence in the medium described in “Materials and Methods.” In all traces, 5 mM of succinate and 1 µM of rotenone were supplemented about 150 s before the addition of a peptide at t = 0 s. Control, no other additions. gA, [Glu1]gA, [Glu3]gA, [Lys1]gA, and [Lys3]gA show traces after the addition of 5 nM (i.e. about 10 nanogram/ml) of a corresponding peptide at t = 0 s. Trace “excess [Glu1]gA” was measured with 1 µg/ml peptide. Panel B. Dose dependence of the effect of [Glu1]gA on mitochondrial membrane potential.
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pone-0041919-g001: Effect of different peptides on the membrane potential of rat liver mitochondria measured by safranine O.Panel A. Shown are traces of fluorescence in the medium described in “Materials and Methods.” In all traces, 5 mM of succinate and 1 µM of rotenone were supplemented about 150 s before the addition of a peptide at t = 0 s. Control, no other additions. gA, [Glu1]gA, [Glu3]gA, [Lys1]gA, and [Lys3]gA show traces after the addition of 5 nM (i.e. about 10 nanogram/ml) of a corresponding peptide at t = 0 s. Trace “excess [Glu1]gA” was measured with 1 µg/ml peptide. Panel B. Dose dependence of the effect of [Glu1]gA on mitochondrial membrane potential.

Mentions: Protonophores are known to collapse the difference of electric potentials across the inner mitochondrial membrane [38]–[43] that causes uncoupling of oxidation and phosphorylation [44]–[46]. To assess the effect of gramicidin derivatives on the mitochondrial membrane potential, we assayed the latter with the potential-dependent dye safranine O. The fluorescence of this dye (at a high concentration) is known to drop with increasing the mitochondrial membrane potential and rise upon the addition of protonophores [42], [47]. Figure 1A displays time courses of fluorescence intensity of safranine O added to a suspension of isolated rat liver mitochondria which were energized by the addition of succinate in combination with rotenone about 150 s before being supplemented with low concentration of peptides (5 nM or about 10 nanogram/ml) at t = 0 s. It can be seen that the glutamate-substituted gA analogue [Glu1]gA and the parent gA were most efficient in decreasing the mitochondrial membrane potential. Figure 1B shows dose dependence of the effect of [Glu1]gA. The concentration dependence of the relative drop in the membrane potential (measured at 300 s after the peptide addition) showed the following series of the uncoupling potency of the analogs: [Glu1]gA > = gA > [Glu3]gA > [Lys3]gA > [Lys1]gA. Notably, the concentrations of the cationic gA analogues [Lys3]gA and [Lys1]gA required to decrease the membrane potential were similar to those of the mitochondrial presequence and other cationic peptides [48]. At concentrations higher than 100 nM (200 nanogram/ml), [Glu1]gA stimulated mitochondrial respiration up to the level of fully uncoupled respiration (data not shown).


N-terminally glutamate-substituted analogue of gramicidin A as protonophore and selective mitochondrial uncoupler.

Sorochkina AI, Plotnikov EY, Rokitskaya TI, Kovalchuk SI, Kotova EA, Sychev SV, Zorov DB, Antonenko YN - PLoS ONE (2012)

Effect of different peptides on the membrane potential of rat liver mitochondria measured by safranine O.Panel A. Shown are traces of fluorescence in the medium described in “Materials and Methods.” In all traces, 5 mM of succinate and 1 µM of rotenone were supplemented about 150 s before the addition of a peptide at t = 0 s. Control, no other additions. gA, [Glu1]gA, [Glu3]gA, [Lys1]gA, and [Lys3]gA show traces after the addition of 5 nM (i.e. about 10 nanogram/ml) of a corresponding peptide at t = 0 s. Trace “excess [Glu1]gA” was measured with 1 µg/ml peptide. Panel B. Dose dependence of the effect of [Glu1]gA on mitochondrial membrane potential.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3404012&req=5

pone-0041919-g001: Effect of different peptides on the membrane potential of rat liver mitochondria measured by safranine O.Panel A. Shown are traces of fluorescence in the medium described in “Materials and Methods.” In all traces, 5 mM of succinate and 1 µM of rotenone were supplemented about 150 s before the addition of a peptide at t = 0 s. Control, no other additions. gA, [Glu1]gA, [Glu3]gA, [Lys1]gA, and [Lys3]gA show traces after the addition of 5 nM (i.e. about 10 nanogram/ml) of a corresponding peptide at t = 0 s. Trace “excess [Glu1]gA” was measured with 1 µg/ml peptide. Panel B. Dose dependence of the effect of [Glu1]gA on mitochondrial membrane potential.
Mentions: Protonophores are known to collapse the difference of electric potentials across the inner mitochondrial membrane [38]–[43] that causes uncoupling of oxidation and phosphorylation [44]–[46]. To assess the effect of gramicidin derivatives on the mitochondrial membrane potential, we assayed the latter with the potential-dependent dye safranine O. The fluorescence of this dye (at a high concentration) is known to drop with increasing the mitochondrial membrane potential and rise upon the addition of protonophores [42], [47]. Figure 1A displays time courses of fluorescence intensity of safranine O added to a suspension of isolated rat liver mitochondria which were energized by the addition of succinate in combination with rotenone about 150 s before being supplemented with low concentration of peptides (5 nM or about 10 nanogram/ml) at t = 0 s. It can be seen that the glutamate-substituted gA analogue [Glu1]gA and the parent gA were most efficient in decreasing the mitochondrial membrane potential. Figure 1B shows dose dependence of the effect of [Glu1]gA. The concentration dependence of the relative drop in the membrane potential (measured at 300 s after the peptide addition) showed the following series of the uncoupling potency of the analogs: [Glu1]gA > = gA > [Glu3]gA > [Lys3]gA > [Lys1]gA. Notably, the concentrations of the cationic gA analogues [Lys3]gA and [Lys1]gA required to decrease the membrane potential were similar to those of the mitochondrial presequence and other cationic peptides [48]. At concentrations higher than 100 nM (200 nanogram/ml), [Glu1]gA stimulated mitochondrial respiration up to the level of fully uncoupled respiration (data not shown).

Bottom Line: On the contrary, [Glu1]gA was much less potent in forming proton channels in planar lipid bilayers than gA.The difference in the behavior of [Glu1]gA and gA in natural and artificial membranes could be ascribed to increased capability of [Glu1]gA to permeate through membranes and/or redistribute between different membranes.Based on the protective role of mild uncoupling, [Glu1]gA and some other proton-conducting gA analogues may be considered as prototypes of prospective therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

ABSTRACT
Limited uncoupling of oxidative phosphorylation could be beneficial for cells by preventing excessive generation of reactive oxygen species. Typical uncouplers are weak organic acids capable of permeating across membranes with a narrow gap between efficacy and toxicity. Aimed at designing a nontoxic uncoupler, the protonatable amino acid residue Glu was substituted for Val at the N-terminus of the pentadecapeptide gramicidin A (gA). The modified peptide [Glu1]gA exhibited high uncoupling activity in isolated mitochondria, in particular, abolishing membrane potential at the inner mitochondrial membrane with the same or even larger efficacy as gA. With mitochondria in cell culture, the depolarizing activity of [Glu1]gA was observed at concentrations by an order of magnitude lower than those of gA. On the contrary, [Glu1]gA was much less potent in forming proton channels in planar lipid bilayers than gA. Remarkably, at uncoupling concentrations, [Glu1]gA did not alter cell morphology and was nontoxic in MTT test, in contrast to gA showing high toxicity. The difference in the behavior of [Glu1]gA and gA in natural and artificial membranes could be ascribed to increased capability of [Glu1]gA to permeate through membranes and/or redistribute between different membranes. Based on the protective role of mild uncoupling, [Glu1]gA and some other proton-conducting gA analogues may be considered as prototypes of prospective therapeutic agents.

Show MeSH
Related in: MedlinePlus