Forward operation of adenine nucleotide translocase during F₀F₁‐ATPase reversal: critical role of matrix substrate‐level phosphorylation

Abstract

In pathological conditions, F(0)F(1)-ATPase hydrolyzes ATP in an attempt to maintain mitochondrial membrane potential. Using thermodynamic assumptions and computer modeling, we established that mitochondrial membrane potential can be more negative than the reversal potential of the adenine nucleotide translocase (ANT) but more positive than that of the F(0)F(1)-ATPase. Experiments on isolated mitochondria demonstrated that, when the electron transport chain is compromised, the F(0)F(1)-ATPase reverses, and the membrane potential is maintained as long as matrix substrate-level phosphorylation is functional, without a concomitant reversal of the ANT. Consistently, no cytosolic ATP consumption was observed using plasmalemmal K(ATP) channels as cytosolic ATP biosensors in cultured neurons, in which their in situ mitochondria were compromised by respiratory chain inhibitors. This finding was further corroborated by quantitative measurements of mitochondrial membrane potential, oxygen consumption, and extracellular acidification rates, indicating nonreversal of ANT of compromised in situ neuronal and astrocytic mitochondria; and by bioluminescence ATP measurements in COS-7 cells transfected with cytosolic- or nuclear-targeted luciferases and treated with mitochondrial respiratory chain inhibitors in the presence of glycolytic plus mitochondrial vs. only mitochondrial substrates. Our findings imply the possibility of a rescue mechanism that is protecting against cytosolic/nuclear ATP depletion under pathological conditions involving impaired respiration. This mechanism comes into play when mitochondria respire on substrates that support matrix substrate-level phosphorylation.

Assessing mitochondrial dysfunction in cells

“…However, the simplicity is deceptive, since the correct design and interpretation of experiments is not trivial. A welldesigned experiment will give much the same information as with isolated mitochondria, with the proviso that although accurate relative values of potential can be obtained [78,[80][81][82], absolute calibration has only recently been achieved ( [83,84] [85,86] and only the key factors will be summarized below.…”

“…The two independent signals from the cationic and anionic indicators allow changes in ψ p and Δψ m to be estimated independently [78]. More sophisticated models allow fully quantitative calculation of both potentials using this approach ( [83,84] If only a single technique is to be used, then respiration, i.e. proton current, is generally the method of choice for subtle changes, since the steep relationships between pmf and the fluxes through oxidative phosphorylation ( Figure 2B) mean that small changes in pmf are associated with large changes in respiration.…”

Cancer as a Metabolic Disease: Implications for Novel Therapeutics

“…It is now recognized from numerous studies that oxygen consumption rates are not always linked to a normally coupled oxidative phosphorylation (83)(84)(85)(86). It can be difficult to determine the degree to which mitochondrial ATP production arises from coupled respiration or from TCA cycle substrate level phosphorylation (87)(88)(89)(90). The origin of mitochondrial ATP production in tumor cells requires further clarification in light of these issues.…”

“…It would therefore be important for investigators to exclude the influence of a Crabtree effect on the assessment of energy measurements in cultured cells. Although a Crabtree effect might suppress OxPhos, the TCA cycle should remain functional and produce ATP through substrate level phosphorylation (87)(88)(89)(90). Under certain conditions (hypoxia), the tumor TCA cycle can work in both forward and reverse (reductive) directions (129,130).…”

Complex Contribution of Cyclophilin D to Ca2+-induced Permeability Transition in Brain Mitochondria, with Relation to the Bioenergetic State

“…A major difference among prerequisites for the manifestation of necrosis versus apoptosis is energy availability; a sufficient decline in energy reserves, primarily in ATP concentration, is a switch for a cell to die by necrosis rather than apoptosis (7,8). Such an extensive decrease in ATP is invariably associated with loss of mitochondrial membrane potential, ⌬⌿m (9,10). Mindful of the large increases in intracellular Ca 2ϩ during cell injury (11) and the loss of ⌬⌿m preceding cell death (12), the conundrum appears that excessive Ca 2ϩ induces PTP under conditions unfavorable for electrophoretic Ca 2ϩ uptake by mitochondria (13).…”