Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Public Library of Science full text link Public Library of Science Free PMC article
Full text links

Actions

Share

doi: 10.1371/journal.pone.0041919. Epub 2012 Jul 24.

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

Affiliations

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

Alexandra I Sorochkina et al. PLoS One.2012.

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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of different peptides on the membrane potential of rat liver mitochondria measured by safranine O.
PanelA. 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. PanelB. Dose dependence of the effect of [Glu1]gA on mitochondrial membrane potential.
Figure 2
Figure 2. Effect of [Glu1]gA on mitochondrial membrane potential in renal cells.
Confocal images of cultured renal cells loaded with TMRE in control (A) and after treatment with 0.01 mg/ml [Glu1]gA (B). Diagram (C) presents the mean intensity of TMRE fluorescence through 10 confocal images for each [Glu1]gA (solid bars) or gA (hatched bars) concentration.
Figure 3
Figure 3. Viability of cultured renal cells according to the MTT test after 24 h treatment with appropriate concentration of [Glu1]gA or gA in culture medium without serum.
Experiments were performed 2 times in which 10 wells per each concentration were analyzed.
Figure 4
Figure 4. Micrographs of rat kidney cell culture loaded with Lysotracker Green in the absence (A), and in the presence of 0.1 mg/ml (B) of [Glu1]gA.
Figure 5
Figure 5. Dissipation of the pH gradient on membranes of pyranine-loaded liposomes by gA and [Glu1]gA (both 2 µg/ml).
Inner liposomal pH was estimated from pyranine fluorescence intensity measured at 505 nm upon excitation at 455 nm. Nigericin (1 µM) was added at 400–420 s to equilibrate the pH. Control, a record without peptides.
Figure 6
Figure 6. Single-channel recordings (A, D) and the corresponding current histograms (B, E) of gramicidin A (picogram/ml, A, B, and C) and [Glu1]gA (2 nanogram/ml, D, E, and F) at a voltage of 100 mV applied to the DPhPC/decane membrane.
The solution was 100 mM HCl. (C,F) Open state duration histograms fitted by a single exponential with a time constant of 116 ms (C) and 16 ms (F). The records were filtered at 100 Hz (A,B, andC) or 1000 Hz (D,E, andF).
Figure 7
Figure 7. Effect of liposomes (1 µg/ml DPhPC) on the induction of electrical current on planar lipid bilayer by gA (2 nanogram/ml, panel A) and [Glu1]gA (200 nanogram/ml, panel B).
The peptides and liposomes were added at one side of the membrane. The solution was 100 mM KCl, 10 mM Tris, 10 mM MES, pH = 7.0, BLM voltage was 50 mV.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mitchell P. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biol Rev. 1966;41:445–502. - PubMed
    1. Decoursey TE. Voltage-gated proton channels and other proton transfer pathways. Physiol Rev. 2003;83:475–579. - PubMed
    1. Skulachev VP. Uncoupling: new approaches to an old problem of bioenergetics. Biochim Biophys Acta 1363: 100–124. S0005–2728(97)00091–1 [pii] 1998. - PubMed
    1. Stuart JA, Brindle KM, Harper JA, Brand MD. Mitochondrial proton leak and the uncoupling proteins. J Bioenerg Biomembr. 1999;31:517–525. - PubMed
    1. Korshunov SS, Skulachev VP, Starkov AA. High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 1997;416:15–18. - PubMed

Publication types

MeSH terms

Substances

Related information

LinkOut - more resources

Full text links
Public Library of Science full text link Public Library of Science Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp