Derivatives of Rhodamine 19 as Mild Mitochondria-targeted Cationic Uncouplers

Yuri N Antonenko,Armine V Avetisyan,Dmitry A Cherepanov,Dmitry A Knorre,Galina A Korshunova,Olga V Markova,Silvia M Ojovan,Irina V Perevoshchikova,Antonina V Pustovidko,Tatyana I Rokitskaya,Inna I Severina,Ruben A Simonyan,Ekaterina A Smirnova,Alexander A Sobko,Natalia V Sumbatyan,Fedor F Severin,Vladimir P Skulachev

doi:10.1074/jbc.m110.212837

Abstract

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.

Highlights

Transport of electrons along the mitochondrial respiratory chain is accompanied by the formation of an electrochemical gradient of hydrogen ions (⌬␮Hϩ)3 at the inner mitochondrial membrane. ⌬␮Hϩ is used for ATP production and other energyconsuming processes
It has recently been shown that mitochondria-targeted antioxidants (SkQ1 and 10-(6Јubiquinonyl) decyltriphenylphosphonium) as well as SkQ1 lacking quinone exhibit protonophorous action when added together with free fatty acids [7]
Because a certain amount of free fatty acids is usually present in living cells, SkQ1 may be considered as a mitochondriatargeted protonophore

Summary

Introduction

Transport of electrons along the mitochondrial respiratory chain is accompanied by the formation of an electrochemical gradient of hydrogen ions (⌬␮Hϩ) at the inner mitochondrial membrane. ⌬␮Hϩ is used for ATP production and other energyconsuming processes. The strategy of “mild uncoupling” [2] relies on the fact that partial decrease in ⌬␮Hϩ can be beneficial for cells especially under some pathological conditions, suggesting that uncouplers are good candidates for therapeutic uses. Such applications are hindered by high toxicity, as in the case of 2,4-dinitrophenol (DNP), which was temporarily used at the beginning of the 20th century as an obesitypreventing drug [5]. Rhodamine 19 Derivatives as Mild Cationic Uncouplers phorous activity of DNP It turned out, that the conjugate was substantially less active than the parent DNP. Because a certain amount of free fatty acids is usually present in living cells, SkQ1 may be considered as a mitochondriatargeted protonophore

Methods

Results

Conclusion