Dennis W. Jung

Properties of a Cyclosporin-insensitive Permeability Transition Pore in Yeast Mitochondria

Yeast mitochondria (Saccharomyces cerevisiae) contain a permeability transition pore which is regulated differently than the pore in mammalian mitochondria. In a mannitol medium containing 10 mm Pi and ethanol (oxidizable substrate), yeast mitochondria accumulate large amounts of Ca2+ (>400 nmol/mg of protein) upon the addition of an electrophoretic Ca2+ ionophore (ETH129). Pore opening does not occur following Ca2+ uptake, even though ruthenium red-inhibited rat liver mitochondria undergo rapid pore opening under analogous conditions. However, a pore does arise in yeast mitochondria when Ca2+ and Pi are not present, as monitored by swelling, ultrastructure, and matrix solute release. Pore opening is slow unless a respiratory substrate is provided (ethanol or NADH) but also occurs rapidly in response to ATP (2 mm) when oligomycin is present. Pi and ADP inhibit pore opening (EC50 ∼1 and 4 mm, respectively), however, cyclosporin A (7 μg/ml), oligomycin (20 μg/ml), or carboxyatractyloside (25 μm) have no effect. The pore arising during respiration is also inhibited by nigericin or uncoupler, indicating that an acidic matrix pH antagonizes the process. Pi also inhibits pore opening by lowering the matrix pH (Pi/OH− antiport). However, inhibition of the ATP-induced pore by Pi is seen in the presence of mersalyl, suggesting a second mechanism of action. Since pore induction by ATP is not sensitive to carboxyatractyloside, ATP appears to act at an external site and Pi may antagonize the interaction. Isoosmotic polyethylene glycol-induced contraction of yeast mitochondria swollen during respiration, or in the presence of ATP, is 50% effective at a solute size of 1.0–1.1 kDa. This suggests that the same pore is induced in both cases and is comparable in size with the permeability transition pore of heart and liver mitochondria.

Energy-dependent exchange of K+ in heart mitochondria K+ influx

The efflux 42K+ from isolated beef heart mitochondria under conditions of near steadystate K+ is increased by repiration and is sensitive to uncouplers and to exogenous Mg2 The respiration-dependent efflux is strongly activated by inorganic phosphate in the presence of external K+, but not Na+, and is inhibited by oxidative phosphorylation. Low concentrations of mersalyl also activate respiration-dependent efflux of 42K+ in the absence of net alteration in matrix K+. Acetate in the presence of mersalyl brings about net accumulation of K+ with retention of internal 42K+. The results are consistent with a model in which nearly constant matrix K+ is maintained by the regulated interplay between a K+ uniport (which is responsive to membrane potential and which is the pathway for K+ influx) and a K+H+ exchanger (which responds to the transmembrane pH differential and which is the pathway for net K+ efflux).

Estimation of intramitochondrial pCa and pH by fura-2 and 2,7 biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) fluorescence

Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the fluorescent pH indicator biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The free acid forms of both probes are retained in the matrix and their fluorescence can be used to monitor the pCa and pH, respectively, of this compartment. When fura-2 loaded rat heart myocytes are lysed with digitonin, a portion of the dye is retained in the mitochondrial fraction and its fluorescence reports the uptake and release of Ca2+ by the mitochondria. It is concluded that fura-2 and BCECF may report mitochondrial as well as cytosol parameters when the probes are used in intact cells.

Cation transport systems in mitochondria: Na+ and K+ uniports and exchangers

It is now well established that mitochondria contain three antiporters that transport monovalent cations. A latent, allosterically regulated K+/H+ antiport appears to serve as a cation-extruding device that helps maintain mitochondrial volume homeostasis. An apparently unregulated Na+/H+ antiport keeps matrix [Na+] low and the Na+-gradient equal to the H+-gradient. A Na+/Ca2+ antiport provides a Ca2+-extruding mechanism that permits the mitochondrion to regulate matrix [Ca2+] by balancing Ca2+ efflux against influx on the Ca2+-uniport. All three antiports have well-defined physiological roles and their molecular properties and regulatory features are now being determined. Mitochondria also contain monovalent cation uniports, such as the recently described ATP- and glibenclamide-sensitive K+ channel and ruthenium red-sensitive uniports for Na+ and K+. A physiological role of such uniports has not been established and their properties are just beginning to be defined.

Regulation of the mitochondrial Na+Ca2+ antiport by matrix pH☆

The effect of matrix pH (pHi) on the activity of the mitochondrial Na+/Ca2+ antiport has been studied using the fluorescence of SNARF-1 to monitor pHi and Na(+)-dependent efflux of accumulated Ca2+ to follow antiport activity. Heart mitochondria respiring in a KCl medium maintain a large delta pH (interior alkaline) and show optimal Na+/Ca2+ antiport only when the pH of the medium (pH0) is acid. Addition of nigericin to these mitochondria decreases delta pH and increases the membrane potential (delta psi). Nigericin strongly activates Na+/Ca2+ antiport at values of pH0 near 7.4 but inhibits antiport activity at acid pH0. When pHi is evaluated in these protocols, a sharp optimum in Na+/Ca2+ antiport activity is seen near pHi 7.6 in the presence or absence of nigericin. Activity falls off rapidly at more alkaline values of pHi. The effects of nigericin on Na+/Ca2+ antiport are duplicated by 20 mM acetate and by 3 mM phosphate. In each case the optimum rate of Na+/Ca2+ antiport is obtained at pHi 7.5 to 7.6 and changes in antiport activity do not correlate with changes in components of the driving force of the reaction (i.e., delta psi, delta pH, or the steady-state Na+ gradient). It is concluded that the Na+/Ca2+ antiport of heart mitochondria is very sensitive to matrix [H+] and that changes in pHi may contribute to the regulation of matrix Ca2+ levels.

Osmotic swelling of heart mitochondria in acetate and chloride salts. Evidence for two pathways for cation uptake.

Abstract Swelling of nonenergized heart mitochondria suspended in acetate salts appears to depend on the activity of an endogenous cation/H + exchanger. Passive swelling in acetate shows a characteristic cation selectivity sequence of Na + >Li + >K + , Rb + , Cs + , or tetramethylammonium, a sharp optimum at pH 7.2–7.3, activation by Ca 2+ , and loss of activity on aging which can be related to loss of endogenous K + . The reaction is nearly insensitive to either addition of exogenous Mg 2+ or removal of membrane Mg 2+ with EDTA. Each of these characteristics of passive swelling in acetate salts is duplicated in chloride media when tripropyltin is added to induce Cl − /OH − exchange. In contrast to nonenergized mitochondria, swelling of respiring mitochondria has been postulated to depend on electrophoretic uptake of cations in response to an interior negative membrane potential. Respiration-dependent swelling in acetate shows an indistinct cation selectivity sequence with Li + and Na + supporting higher rates of swelling at higher efficiency than K + , Rb + , and Cs + . The high rates of respiration-dependent swelling in Li + and Na + are inhibited by low levels of exogenous Mg 2+ ( K i of 5–10 μ m ), but a significant swelling with almost no cation selectivity persists in the presences of 2 m m Mg 2+ . Removal of membrane Mg 2+ by addition of EDTA strongly activates the rate of respiration-dependent swelling and converts a sigmoid dependency of swelling rate on Li + concentration to a hyperbolic one with a Km of about 14 m m Li + . The cation selectivity and Mg 2+ dependence of the reaction induced in chloride salts by tripropyltin are identical to these properties in acetate. Energy-dependent swelling in acetate shows optimum activity at pH 6.5 which appears related to the availability of free acetic acid, since the corresponding reaction induced in chloride shows a broad optimum at about pH 7.5. These studies support the concept that monovalent cations enter nonenergized mitochondria by electroneutral exchange with protons but penetrate respiring mitochondria by electrophoretic movement through one or more uniport pathways. They further suggest that both a Mg 2+ -sensitive uniport with high activity for Na + and Li + and a Mg 2+ -insensitive pathway with little cation discrimination are available in the membrane.

Ruthenium red-sensitive and -insensitive release of Ca2+ from uncoupled heart mitochondria.

The uncoupler-induced release of accumulated Ca2+ from heart mitochondria can be separated into two components, one sensitive and one insensitive to ruthenium red. In mitochondria maintaining reduced NAD(P)H pools and adequate levels of endogenous adenine nucleotides, the release of Ca2+ following addition of an uncoupler is virtually all inhibited by ruthenium red and can be presumed to occur via reversal of the Ca2+ uniporter. When ruthenium red is added to block efflux via this pathway, high rates of Ca2+ efflux can still be induced by an uncoupler, provided either NADH is oxidized or mitochondrial adenine nucleotide pools are depleted by prior treatment. This ruthenium red-insensitive Ca2+-efflux pathway is dependent on the level of Ca2+ accumulated and is accompanied by swelling of the mitochondria and loss of endogenous Mg2+. Loss of Ca2+ by this relatively nonspecific pathway is strongly inhibited by Sr2+ and by nupercaine, as well as by oligomycin and exogenous adenine nucleotides. The loss of Ca2+ from uncoupled heart mitochondria occurs via a combination of these two mechanisms except under conditions chosen specifically to limit efflux to one or the other pathway.

Magnesium transport by mitochondria

The pathways for the uptake and extrusion of Mg2+ by mitochondria are not well defined. the present evidence suggests that uptake occurs by nonspecific diffusive pathways in response to elevated membrane potential. There is disagreement as to some of the properties of Mg2+ efflux from mitochondria, but the reaction resembles K+ efflux in many ways and may occur in exchange for H+. Matrix free magnesium ion concentration, [Mg2+], can be measured using fluorescent probes and is set very close to cytosol [Mg2+] by a balance between influx and efflux and by the availability of ligands, such as Pi. There are indications that matrix [Mg2+] may be under hormonal control and that it contributes to the regulation of mitochondrial metabolism and transport reactions.

Respiration-dependent uptake and extrusion of Mg2+ by isolated heart mitochondria

It has been known for some time that isolated heart mitochondria can both take up and extrude Mg2+ by respiration-dependent, uncoupler-sensitive processes. A re-examination of these reactions reveals that the respiration-dependent uptake of Mg2+ can be quite rapid and efficient and is apparently preceded by a passive binding to the inner membrane. The rate of Mg2+ uptake can exceed 30 ng ion/min/mg protein at an efficiency of about 1 ng ion Mg2+ accumulated per ng atom O2 consumed. Passive binding and respiration-dependent accumulation of Mg2+ are strongly inhibited by K+ and other monovalent cations and the uptake reaction is further decreased by the presence of ATP or ADP. Under conditions approaching those faced by mitochondria in situ (state 3 respiration in a KCl medium) the rate of Mg2+ uptake, as estimated from 28Mg2+ distribution, is no more than 0.25 ng ion/min/mg. When heart mitochondria are suspended in a Mg2+-free medium, a slow, respiration-dependent Mg2+ efflux is seen. This reaction is quite insensitive to external K+ and otherwise shows an inhibitor profile markedly different from that of the Mg2+ accumulation reaction. Neither the uptake nor the loss of Mg2+ is inhibited by ruthenium red or diltiazem. These reactions therefore appear unrelated to those involved in the uptake and release of Ca2+. It is concluded that heart mitochondria have separate pathways available for Mg2+ uptake and release.

On the relationship between matrix free Mg2+ concentration and total Mg2+ in heart mitochondria

The matrix free magnesium ion concentration, [Mg2+]m, estimated using the fluorescent probe furaptra, averaged 0.67 mM in 15 preparations of beef heart mitochondria containing an average of 21 nmol total Mg2+ per mg protein. [Mg2+]m was compared with total Mg2+ during respiration-dependent uptake and efflux of Mg2+ and during osmotic swelling. In the absence of external Pi these mitochondria contain about 32 nmol/mg non-diffusible Mg-binding sites with an apparent Kd of 0.34 mM. [Mg2+]m depends on both the size of the total Mg2+ pool and the ability of matrix anions to provide Mg-ligands. Pi interacts strongly with Mg2+ to decrease [Mg2+]m and, in the absence of external Mg2+, promotes respiration-dependent Mg2+ efflux and a decrease in [Mg2+]m to very low levels. The uptake of Pi by respiring mitochondria converts delta pH to membrane potential (delta psi) and provides additional Mg-binding sites. This permits large accumulations of Mg2+ and Pi with little change in [Mg2+]m. Nigericin also converts delta pH to delta psi in respiring mitochondria and induces a large and rapid increase in both total Mg2+ and [Mg2+]m. Mersalyl increases the permeability of the mitochondrial membrane to cations and this also induces a marked increase in both total Mg2+ and [Mg2+]m. These results suggest that mitochondria take up Mg2+ by electrophoretic flux through membrane leak pathways, rather than via a specific Mg2+ transporter. Mitochondria swollen by respiration dependent uptake of potassium phosphate show decreased [Mg2+]m, whereas those swollen to the same extent in potassium acetate do not. This suggests that [Mg2+]m is well-buffered during osmotic volume changes unless there is also a change in ligand availability.