Membrane potentials and resistances of giant mitochondria. Metabolic dependence and the effects of valinomycin.

Abstract

The membrane potentials and resistances of giant mitochondria from mice fed cuprizone have been studied. They were found to correspond approx. 10-20 mV, positive inside, and 2 M~, respectively. These properties were found to be independent of the metabolic state. The microelectrodes were in the inner mitochondrial space since (a) the potentials in the presence of valinomycin depended on the K § concentration of the medium and the magnitude of the K § diffusion potentials was consistent with the presence of a high internal concentration of K § (b) almost identical results were obtained with mitochondria from which the external membrane had been removed and the cristae were evaginated, and (c) punch-through experiments, in which the microelectrodes were advanced until they emerged through the other side of the mitochondria, showed an identical membrane potential both in the presence and in the absence of valinomycin. The potentials were stable under a variety of conditions and showed no sign of decay or membrane leakiness.Detailed evidence that the impaled mitochondria are metabolically viable will be presented in a separate publication. KEY WORDS mitochondrial potentials microelectrodes oxidative phosphorylationThe Mitchell chemiosmotic hypothesis proposes that the electrochemical potential gradient of H § across membranes (the so-called protonmotive force) plays the major role in mitochondrial phosphorylation and the transport of ions. This hypothesis has been described in detail in a series of publications (e.g., references 28-34). The electrochemical potential gradient of H +, which is the result of proton pumps (in the form of the socalled loops of Mitchell), is constituted of the H § concentration gradient and the membrane potential.Tupper and Tedeschi (50, 52-54) were able to measure membrane potentials and membrane resistances directly in Drosophila giant mitochondria by means of microelectrodes driven by a piezoelectric device. The membrane potential was found to range from 10 to 20 mV, positive inside, and the resistance was 1-4 f~cm ~. Metabolism was not found to affect either the potentials or resistances significantly (53). The specific resistances were J. CELL BIOLOGY 9 The Rockefeller University Press 9 0021-9525/78

Assays of the metabolic viability of single giant mitochondria. Experiments with intact and impaled mitochondria.

“…As shown in Tables I1 and IV, the metabolic state and the presence or absence of inhibitors had no effect on the membrane potential (E) or the resistance (R), in agreement with our previous results (14,15). Therefore, no metabolic dependence of the mitochondrial membrane potential can be demonstrated.…”

“…In interpretation that the impaled mitochondria are functional since they accumulate calcium salts in the appropriate medium and, in addition, they phosphorylate ADP to ATP which causes myofibrillar contraction. Our accompanying paper (15) presents evidence that the microelectrode is in the inside space of the mitochondria since (a) the membrane potential shows the predicted dependence on the external concentration of K § in the presence of valinomycin, (b) the electrical parameters remain unchanged by the removal of the outer membrane and the evagination of the inner mitochondrial membrane, and (c) punch-through experiments, in which the microelectrode traverses through the mitochondrion, reveal a single membrane potential in the presence of valinomycin or under either energized or inhibited conditions. In these and other experiments using the giant mouse mitochondria, the evidence does not support the Mitchell chemiosmotic hypothesis which proposes a direct role of metabolism in the membrane potential and predicts a potential at least one order of magnitude higher and with an opposite sign.…”

“…Details of the preparation of the mitochondria, and those referring to the preparation and manipulations carried out with the microelectrodes, have been presented previously (15). For the experimental determinations, the temperature was maintained between 19 ~ and 25~…”

“…The potentials and resistances were unaffected by concomitant calcium phosphate accumulation or oxidative phosphorylation. KEY WORDS mitochondrial potentials microelectrodes calcium phosphate transport oxidative phosphorylation Large mitochondria are produced in the livers of cuprizone fed mice (14,15,20,21). Most of the mitochondria are approximately equal in size to those isolated from normal mice.…”

Electrophysiology clarifies the megariddles of the mitochondrial permeability transition pore

“…This obviously fed the ongoing discussion. Tedeschi and co-workers responded to criticism with some brilliant experiments, employing now giant mitochondria obtained from the livers of cuprizone-fed mice [11][12][13][14][15]. They showed that myofibrils adjacent to impaled mitochondria supplied with ADP would contract, thus indicating the production of ATP.…”