Repetitive transient depolarizations of the inner mitochondrial membrane induced by proton pumping

Abstract

Single mitochondria show the spontaneous fluctuations of DeltaPsim. In this study, to examine the mechanism of the fluctuations, we observed DeltaPsim in single isolated heart mitochondria using time-resolved fluorescence microscopy. Addition of malate, succinate, or ascorbate plus TMPD to mitochondria induced polarization of the inner membrane followed by repeated cycles of rapid depolarizations and immediate repolarizations. ADP significantly decreased the frequency of the rapid depolarizations, but the ADP effect was counteracted by oligomycin. On the other hand, the rapid depolarizations did not occur when mitochondria were polarized by the efflux of K(+) from the matrix. The rapid depolarizations became frequent with the increase in the substrate concentration or pH of the buffer. These results suggest that the rapid depolarizations depend on the net translocation of protons from the matrix. The frequency of the rapid depolarizations was not affected by ROS scavengers, Ca(2+), CsA, or BA. In addition, the obvious increase in the permeability of the inner membrane to calcein (MW 623) that was entrapped in the matrix was not observed upon the transient depolarization. The mechanisms of the spontaneous oscillations of DeltaPsim are discussed in relation to the matrix pH and the permeability transitions.

FIGURE 1

Time-resolved TMRE fluorescence of single mitochondria in response to addition of malate. TMRE fluorescence was monitored in the absence of ADP. (A and B) ΔΨm-dependent fluorescence images of TMRE in single mitochondria. The time interval between images is 3 s. (A) The arrow marks the addition of malate (5 mM). (B) The arrow marks the rapid depolarization observed after addition of malate. (C) The typical responses of ΔΨm observed in single mitochondria to addition of malate. The vertical axis represents the fluorescence intensity of TMRE in single mitochondria, normalized by the TMRE fluorescence in buffer. At t = 0, 5 mM malate was added (arrow). The fluctuations of ΔΨm significantly depend on individual mitochondria. Mitochondria 1–3 are fluctuating, whereas mitochondrion 4 does not show the fluctuation of ΔΨm.

FIGURE 2

Electron transfer and oscillations of ΔΨm. (A–C) Rotenone (1 μM) was added to mitochondria that were bathed in KCl buffer 10 min before the measurements. (A) The response of ΔΨm observed in single mitochondria. At t = 0, malate was added to accumulate NADH in mitochondria. At t = 120, valinomycin was added to polarize the inner membrane without the pumping of protons. The vertical axis is the same as in Fig. 1 C. (B) Autofluorescence of NAD(P)H in the mitochondria suspended in KCl buffer containing 0.5 mM ADP. At t = 0, malate was added. (C) The time course of succinate-induced polarization of a single mitochondrion. At t = 0, malate was added. At t = 120, succinate was added to polarize the inner membrane by proton pumping. The vertical axis is the same as in Fig. 1 C. (D) Changes in ΔΨm upon addition of ascorbate + TMPD in the presence of antimycin A. (E) Frequencies of the rapid depolarizations of the inner membrane. Frequencies were measured in sucrose buffer in the presence of malate (5 mM), succinate (5 mM) + rotenone (1 μM), or ascorbate (2.5 mM) + TMPD (0.25 mM) + antimycin A (2 μM).

FIGURE 3

Frequencies of the rapid depolarizations of the inner membrane. (A) Effects of malate, ADP (0.5 mM) or oligomycin (1 μM) on the frequencies of the rapid depolarizations. Frequencies were measured in sucrose buffer at pH 7.4. Values represent the mean ± SE (n > 100). *, P < 0.05 vs. 5 mM malate without ADP and oligomycin. (B) Effects of pH on the frequencies. The frequencies were measured in the presence of 5 mM malate with neither ADP nor oligomycin. Values represent the mean ± SE (n > 100). *, P < 0.05 vs. pH 7.4. (C) Effects of CsA (1 μM) and BA (2 μM) on the frequencies. The control was measured in the presence of 5 mM malate with neither ADP nor oligomycin at pH 7.4. Values represent the mean ± SE (n > 100).

FIGURE 4

Effects of Ca²⁺ and Mg²⁺ on ΔΨm. The changes in ΔΨm were measured in the presence of 5 mM malate. The concentrations of Ca²⁺ and Mg²⁺ were adjusted with 1 mM EGTA and 1 mM EDTA, respectively. (A) Effects of Ca²⁺ and Mg²⁺ on the frequencies of the oscillations. Values represent the mean ± SE (n > 100). *, P < 0.05 vs. 0 μM Mg²⁺. (B) Effects of Ca²⁺ and Mg²⁺ on the percentages of mitochondria polarized upon addition of 5 mM malate. *, P < 0.05 vs. 0 μM Ca²⁺.

FIGURE 5

ROS effects on oscillations of ΔΨm. (A) The effects of catalase, GSH, and TMPyP on oscillations of ΔΨm evoked by 5 mM malate. (B) The effects of H₂O₂ on oscillations of ΔΨm evoked by 1 mM malate. Values represent the mean ± SE (n > 100).

FIGURE 6

Calcein fluorescence inside and outside mitochondria. (A) Time-resolved TMRE (1, 3) and calcein (2, 4) fluorescence in single mitochondria. TMRE and calcein fluorescence was simultaneously monitored in the same mitochondrion. Typical behaviors observed for two different mitochondria (Mitochondrion 1 and 2) are shown. The vertical axes for TMRE fluorescence are the same as those of Fig. 1 C. The vertical axes for calcein fluorescence are shown in arbitrary unit. Malate was added at t = 0. Arrows show the time when the rapid depolarizations occurred. (B) Release of calcein under the various conditions. The vertical axis represents the calcein release from mitochondria to the medium. The detailed explanation of the vertical axis is described in the text. Values represent the mean ± SE (n > 3). *, P < 0.05 versus the sample without ADP, oligomycin, CsA, BA, Ca²⁺, and Mg²⁺. (C) Fluorescence images of calcein entrapped in individual mitochondria in the same microscopic field. Bar, 5 μm. (1) Before addition of malate; (2) after incubation with malate in the absence of ADP.

FIGURE 7

Schematic illustration of the proposed model for transient and repetitive depolarizations. (a) Proton pumps in the electron transfer chain translocate protons from the matrix to the intermembrane space. (b) The pH beneath the inner membrane is increased by proton pumping. (c) Opening of a pH sensitive and proton conductive channel is induced by the increase in the pH. (d) Reentry of protons through the channel decreases the pH, resulting in closing of the channel.