Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption

Abstract

We have previously described a fluorometric method to measure ADP-ATP exchange rates in mitochondria of permeabilized cells, in which several enzymes that consume substantial amounts of ATP and other competing reactions interconverting adenine nucleotides are present. This method relies on recording changes in free extramitochondrial Mg(2+) with the Mg(2+)-sensitive fluorescent indicator Magnesium Green (MgGr)™, exploiting the differential affinity of ADP and ATP for Mg(2+). In particular, cells are permeabilized with digitonin in the presence of BeF3(-) and Na3VO4, inhibiting all ATP- and ADP-utilizing reactions but mitochondrial exchange of ATP with ADP catalyzed by the adenine nucleotide translocase. The rate of ATP appearing in the medium upon the addition of ADP to energized mitochondria is then calculated from the rate of change in free extramitochondrial Mg(2+) using standard binding equations. Here, we describe a variant of this method involving an improved calibration step. This step minimizes errors that may be introduced during the conversion of the MgGr™ signal into free extramitochondrial [Mg(2+)] and ATP. Furthermore, we describe an approach for combining this methodology with the measurement of mitochondrial membrane potential and oxygen consumption in the same sample. The method described herein is useful for the study of malignant cells, which are known to thrive in hypoxic environments and to harbor mitochondria with profound functional alterations.

Keywords: ADP–ATP exchange; Adenine nucleotide carrier; Adenine nucleotide translocase; Bioenergetics; Oxidative phosphorylation; Systems biology.

Figure 17.1. Magnesium Green fluorescence calibration and estimation of K_d of ATP and ADP for Mg²⁺

(A) Reconstructed time recordings of MgGr raw fluorescence traces in permeabilized HEK293 cells as a function of extramitochondrial [Mg²⁺] (left part of the traces), and as a function of extramitochondrial ADP or ATP (right parts of the traces). (B) MgGr fluorescence changes are dependent on extramitochondrial [Mg²⁺]. (C) Calibrated time recordings of extramitochondrial [Mg²⁺] (left part of the traces), and as a function of extramitochondrial ADP and ATP (right parts of the traces) are shown. (D) Calibrated extramitochondrial Mg²⁺ plots as a function of ADP or ATP are shown, from which we estimated K_d of ADP and ATP for Mg²⁺ using the least squares method to fit the data.

Figure 17.2. Determination of extramitochondrial [Mg²⁺] and conversion to ATP

(A) Reconstructed time recording of MgGr raw fluorescence in permeabilized HEK293 cells upon addition of 2 mM ADP (where indicated), followed by incremental 10 nM additions of the uncoupler SF 6847. (B) Calibrated time recording of extramitochondrial [Mg²⁺] obtained from panel (A). (C) Corrected calibrated trace of panel (B), as described in the text. (D) Calculated amount of ATP appearing in the medium converted from panel (C). The rate of ATP appearing in the medium is indicated in µmol min⁻¹ mg protein⁻¹.

Figure 17.3. Mitochondrial membrane potential and oxygen consumption determination in permeabilized cells

(A) Reconstructed time recording of safranin O raw fluorescence in permeabilized HEK293 cells upon addition of 2 mM ADP (where indicated), followed by incremental 10 nM additions of the uncoupler SF 6847. (B) Calibrated time recording of ΔΨm obtained from panel (A). (C) Reconstructed time recording of oxygen concentration in the medium (black trace) and oxygen flux (gray trace) recorded simultaneously with either MgGr signal (panel A) or safranin O signal (panel A).