Minocycline chelates Ca2+, binds to membranes, and depolarizes mitochondria by formation of Ca2+-dependent ion channels

Abstract

Minocycline (an anti-inflammatory drug approved by the FDA) has been reported to be effective in mouse models of amyotrophic lateral sclerosis and Huntington disease. It has been suggested that the beneficial effects of minocycline are related to its ability to influence mitochondrial functioning. We tested the hypothesis that minocycline directly inhibits the Ca(2+)-induced permeability transition in rat liver mitochondria. Our data show that minocycline does not directly inhibit the mitochondrial permeability transition. However, minocycline has multiple effects on mitochondrial functioning. First, this drug chelates Ca(2+) ions. Secondly, minocycline, in a Ca(2+)-dependent manner, binds to mitochondrial membranes. Thirdly, minocycline decreases the proton-motive force by forming ion channels in the inner mitochondrial membrane. Channel formation was confirmed with two bilayer lipid membrane models. We show that minocycline, in the presence of Ca(2+), induces selective permeability for small ions. We suggest that the beneficial action of minocycline is related to the Ca(2+)-dependent partial uncoupling of mitochondria, which indirectly prevents induction of the mitochondrial permeability transition.

Fig. 1

Structural formula of Minocycline

Fig. 2

Determination of isosbestic points for the minocycline spectrum under different experimental conditions. (A Panels) Ca²⁺-dependent changes in the spectrum of minocycline at three different concentrations (Black, 60 µM; Red, 120 µM; Green, 200 µM) in sucrose buffer at three different [Ca²⁺]. (B Panels) Determination of the Ca²⁺-independent isosbestic points for minocycline spectra in sucrose buffer at three concentrations of minocycline (Mino) at four [Ca²⁺] (Black, no Ca²⁺; Red, 50 µM; Green, 100 µM; Yellow, 150 µM; Blue, 200 µM). (C Panels) Dependence of the minocycline spectra on the experimental buffer and [Ca²⁺]. The Hepes buffer contained 3 mM Hepes, pH 7.4; the Sucrose buffer consisted of 300 mM sucrose, 3 mM Hepes, pH 7.4; and the PBS*Tween buffer contained 10 mM phosphate-buffered saline, 0.5 % Tween, pH 7.4. The minocycline concentration was 100 and 200 µM ([Ca²⁺]: Black, no Ca²⁺; Red, 20 µM; Green, 50 µM; Yellow, 100 µM). (D Panels) pH dependence (Black, pH 6.6; Red, pH 7.4; Green, pH 8.2) of the minocycline (80 µM) spectrum in sucrose buffer supplemented with 40 and 60 µM Ca²⁺

Fig. 3

Calculation of the ratio of the complex formed between minocycline and Ca²⁺ (Mino*Ca) to free minocycline (Mino) in sucrose buffer. (Panel A) Concentration dependence of the minocycline (Mino) spectra. (Panel B) Calibration curve for Mino absorbance at 340 nm versus concentration, showing the equation and coefficients of the linear regression. (Panel C) Ca²⁺-dependent changes in the spectrum of 100 µM minocycline. The values of the absorbance for each Ca²⁺ concentration at 340 nm in Panel C were used to calculate the concentration of Mino using the equation of the linear regression from Panel B. [Mino*Ca] was calculated as 100 µM minus [Mino]. (Panel D) The sigmoidal dependence of the Mino*Ca/Mino ratio over a wide range of [Ca²⁺]. (Panel E) Linearity of Mino*Ca/Mino versus [Ca²⁺] in the low [Ca²⁺] range of Panel D. The binding constant (K_Ca2+) found from Panel E for Ca²⁺ in the presence of 100 µM minocycline is ∼65 µM as determined from the value of the Mino*Ca/Mino ratio equal to 1

Fig. 4

(Panel A) Decrease of minocycline (200 µM) concentration in sucrose buffer (1 ml) after incubation with RLM (1 mg/ml) for 5 min on ice in the absence or presence of 50 µM Ca²⁺. N=4. (Panel B) Ca²⁺-dependence of minocycline (100 µM) binding to mitochondria (1 mg/ml/3min). Gray diamond symbols with error bars are the average ± standard deviation. N=4. White and black diamond symbols represent values from two experiments. The sucrose buffer contained 300 mM sucrose, 3 mM Hepes, 2.5 mM KH₂PO₄, and 5 mM succinate plus 1 µM rotenone, pH 7.4

Fig. 5

(Panel A) Time-dependence of minocycline (100 µM) binding to RLM (0.5 mg of protein/ml). N=4. (Panel B) Minocycline (200 µM) binding (5 min on ice) to RLM is dependent on RLM concentration and the presence of Ca²⁺. The values are the average ± standard deviation. N=4, except for a single measurement indicated as ‘$’. Individual sets of experiments in Panels A and B were carried out four months apart, which may have introduced some variability. The experimental buffer conditions were the same as those described in the legend to Fig. 2 and the total volume of the experimental mixture was 1 ml

Fig. 6

Effects of different treatments on four parameters measured simultaneously in RLM. Traces ‘a’, MPT induction by Ca²⁺; traces ‘b’, effect of minocycline; traces ‘c’, inhibitory effect of Ru360 on MPT. Experimental conditions: sucrose buffer contained 300 mM sucrose, 3 mM Hepes, 2.5 mM KH₂PO₄, and 5 mM succinate plus 1 µM rotenone, pH 7.4. The RLM concentration was 0.45 mg/ml. Additions of Ca²⁺, minocycline (M) and Ru360 (Ru) were 60, 100 µM and 100 µg/ml, respectively. Panels A–D: O₂ uptake, Ca²⁺ flux, ΔΨ, and swelling, respectively. (Panel A) The decrease in O₂ concentration reflects O₂ uptake by mitochondria. (Panel B) The increase in Ca²⁺ concentration indicates either Ca²⁺ efflux from mitochondria or absence of Ca²⁺ influx into mitochondria. (Panel C) The increase in TPP⁺ concentration reflects ΔΨ dissipation. (Panel D) The decrease in absorbance indicates mitochondrial swelling. The traces are representative of at least four experiments obtained with different mitochondrial preparations

Fig. 7

Effects of minocycline on ΔΨ and swelling of mitochondria upon addition of Ca²⁺. (Panels A and B) Sucrose-(black traces) and 150 mM KCl buffer (gray traces), respectively. (Panels C and D) The effects of minocycline on ΔΨ and swelling of RLM under slightly acidic (pH 6.8, black traces) and slightly basic (pH 8.0, gray traces) conditions in Sucrose buffer, respectively. Additions: RLM, rat liver mitochondria 0.5 mg/ml; M, minocycline 200 µM; Ca²⁺ 60 µM (enough to induce the MPT-trace not shown)

Fig. 8

(Panel A) Minocycline (20 µM) in the presence of 3 mM Ca²⁺ induces an electrical current in BLM made from DPhPC. The experimental buffer was 10 mM Tris, 10 mM MES, 100 mM KCl, pH 8.5; the voltage was 30 mV. (Panels B, C) Ion channel activity induced by minocycline (8 µM) in the presence of Ca²⁺ (3 mM in panel B and 20 µM in panel C) at 50 mV in BLM made from DPhPC. The experimental buffer was 10 mM Tris, 10 mM MES, 1 M KCl, pH 9.0. (A current of 0.7 pA at 50 mV corresponds to a conductance of 14 pS)

Fig. 9

(Panel A) The dependence of the BLM electrical current on the concentration of minocycline at V=50 mV. (Panel B) I–V curves for minocycline (50 µM)-mediated electrical current under symmetrical (100 mM:100 mM KCl; closed circles) and asymmetrical (190 mM:100 mM KCl; open circles) conditions. BLM was made from DPhPC in the presence of Ca²⁺ (3 mM). The experimental buffer conditions were the same as described for Fig. 8, Panel A

Fig. 10

Dependence of the BLM conductance on the voltage applied in the presence of 20 µM minocycline and 3 mM CaCl₂. The BLM was made from an E. coli total lipid fraction. The experimental buffer conditions were the same as those described for Fig. 8, Panel A

Fig. 11

(Panel A) Minocycline induces proton permeability in liposomes loaded with pyranine in the presence of Ca²⁺ (5 mM). Addition of nigericin was used as a positive control. The lipid concentration was 20 µg/ml. Excitation, 455 nm; emission, 505 nm. (Panel B) Minocycline does not induce carboxyfluorescein (CF) leakage from dye-loaded liposomes in the presence or absence of Ca²⁺ (5 mM). Addition of melittin was used as a positive control. The lipid concentration was 5 µg/ml. Excitation, 488 nm; emission, 520 nm; a.u., arbitrary units