Derivatives of rhodamine 19 as mild mitochondria-targeted cationic uncouplers

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.

FIGURE 1.

Chemical structures of rhodamine 19 derivatives (SkQR1, C_nR1) and rhodamine B derivatives (SkQR4, C₁₂R4) used in the present work.

FIGURE 2.

A, stimulation of rat liver mitochondria respiration by SkQR1. Shown are traces of the rate of respiration in the medium described under “Materials and Methods.” Additions were as follows: 5 mm succinate, 2 μm rotenone, 2 μm SkQR1, 2 μm SkQR4, 1 μm FCCP (trace with SkQR1), and two additions of FCCP (0.5 μm) in the trace with SkQR4. B, dose dependence of respiratory stimulation by SkQR1, SkQR4, C₁₂R1, C₁₂R4, and FCCP. C, the increase in the respiration rate caused by C₁₂R1 was insensitive to the addition of carboxyatractilate (2 μm). Error bars, S.E.

FIGURE 3.

A, effect of SkQR1 and SkQR4 on the membrane potential of rat liver mitochondria measured by DiS-C3-(5). Shown are traces of fluorescence in the medium described under “Materials and Methods.” In all traces, 5 mm succinate and 1 μm rotenone were added at 300 s, and 100 μm DNP was added at the end of each trace. Control, no other additions. Gray trace, 2 μm SkQR1 was added at 380 s. Dashed trace, 2 μm SkQR4 was added at 500 s. B, dose dependence of the effect of SkQR1 on the potential of mitochondria. a.u., arbitrary units.

FIGURE 4.

A, dissipation of pH gradient on membranes of pyranine-loaded liposomes by SkQR1, SkQR4, and FCCP. Inner liposome pH was estimated from the ratio (I₄₅₅/I₄₁₀) of pyranine fluorescence intensities measured at 505 nm upon excitation at 455 and 410 nm, respectively. Nigericin (1 μm) was added at 550 s to equilibrate the pH. The control curve is with FCCP but without valinomycin. Concentrations were as follows: SkQR1 (100 nm), SkQR4 (100 nm), FCCP (100 nm), valinomycin (10 nm), and lipid (75 μm). B, dose dependence of the effect of SkQR1 and FCCP. The control curve corresponded to 10 nm valinomycin without protonophores.

FIGURE 5.

A, dissipation of pH gradient on membranes of pyranine-loaded liposomes by C₁₂R1 and C₁₂R4 (both 150 nm). Inner liposome pH was estimated from the ratio (I₄₅₅/I₄₁₀) of pyranine fluorescence intensities measured at 505 nm upon excitation at 455 nm and 410 nm, respectively. Nigericin (1 μm) was added at 550 s to equilibrate the pH. Other conditions were as described in the legend to Fig. 2. B, protonophorous action of rhodamine 19 alkyl esters (C_nR1, 150 nm). Lipid concentration was 200 μm.

FIGURE 6.

A, C₁₂R1 but not C₁₂R4 induced formation of H⁺ diffusion potential on planar bilayer lipid membrane made from diphytanoylphosphatidylcholine. Concentrations of C₁₂R1 and C₁₂R4 were 0.5 μm. pH_cis = 7, and pH_trans = 8. B, formation of diffusion potential of penetrating cations C₁₂R4 and C₁₂R1 (both 1 μm at one side and 0.1 μm at the other) added at the cis side of the planar membrane at symmetrical pH 7.

FIGURE 7.

C₁₂R1, unlike anionic uncouplers, affects yeast cells, depending on their mitochondrial membrane potential. Shown are the dependences of stimulation of yeast cell respiration rate (V/V₀) and growth on the concentration of FCCP (A) and C₁₂R1 (B). C, C₁₂R1 (10 μm; dark gray) or DNP (500 μm; light gray) were added to yeast cells (grande or petite) growing on non-fermentable (glycerol) or fermentable (glucose) carbon sources. The growth rate without uncouplers corresponds to 100%. Error bars, S.E.

FIGURE 8.

Snapshots of SkQR1 permeation through a bilayer lipid membrane driven by the biasing potential (umbrella sampling method in molecular dynamics simulations). Shown are the lipid carbon tails (cyan thin sticks), nitrogen atoms of choline and phosphorus atoms of phosphate groups (blue and brown spheres), water molecules (small red spheres), and SkQR1 (aromatic rings (yellow), dodecyl residue (green), carbon (cyan), oxygen (red), and hydrogen (white)). A, equilibrium structure obtained after 100 ns of unperturbed molecular dynamics simulation (parallel position of rhodamine ring relative to the membrane surface). B, structure after 590 ns of umbrella sampling simulation (orthogonal conformation). C, structure after 940 ns of simulation (parallel conformation).

FIGURE 9.

Scheme of functioning of the cationic protonophores SkQR1 and C₁₂R1. A deprotonated neutral form (R) and a singly protonated cationic form (RH⁺) can cross the membrane, whereas a doubly protonated form (RH₂²⁺) cannot. The scheme shows the following three steps of the carrier cycling: a voltage-dependent stage of RH⁺ translocation from cytoplasmic to the matrix side (step 2); RH⁺ deprotonation on the matrix side and movement of the neutral form R across the membrane along its concentration gradient (step 3); and protonation of R on the outer side of the membrane. Proton pumping by respiratory chains is designated by step 1.