Low levels of CdZ+ (1 -5 pM) produce rapid swelling of mitochondria, which is respiration-dependent and uncoupler-sensitive. No cation requirement is apparent, since the swelling occurs in a medium containing only sucrose and the respiratory substrate. The swelling is inhibited by ruthenium red, suggesting that this effect of Cd2+ requires its entry into mitochondria. In medium containing 9 mM K + , addition of Cd2+ along with ruthenium red increases the rate of K' influx threefold. In the presence of K + , Rb+ or Li', but not of Na', addition of Cd2+ produces first efflux of H + into the medium followed by discharge of the pH gradient or uncoupling. Only the latter effect is inhibited by ruthenium red, showing that the efflux and influx of H f are independent reactions. The H' efflux appears to be an antiport response to the induced K + entry. Its activation by CdZ+ is similar to the known effect ofp-chloromercuriphenyl sulfonate. The H + influx or uncoupling appears to result from binding of Cd2+ to some matrix-facing membrane site, perhaps the dithiol group on coupling factor B, and may relate to apparent permeability changes associated with Cd2 +-induced swelling.Sulfhydryl-group reagents are known to activate mitochondrial monovalent cation transport [l -41. Studies in our laboratory have provided evidence for the presence of at least two types of dithiol groups in rat liver mitochondria that react with cadmium ion (Cd") and phenylarsine oxide. On addition of Cd2+ to respiring rat liver mitochondria, a transient K f -dependent H + efflux occurs, followed by reentry of H + or uncoupling [5]. The initial response is similar to the effect of mercurial reagents on mitochondria [l, 21. Presently available data suggest that mitochondrial accumulation of cation may be due to the interplay between a passive cation uniporter and a cation/proton exchanger [6, 71. Alternatively the possibility of direct involvement of K + transport in the energy transduction process has been considered [4, 8, 91. The existence of a specific K f influx mechanism is indicated by the saturability of K + influx with respect to external K + [lo, 211. An 82-kDa protein, which may function as a K + / H + antiporter, has recently been identified [I21 on the basis of its reactivity with N,N'-dicyclohexyl carbodiimide, a known inhibitor of K + influx and efflux [7, The present communication documents two main events happening in rat liver mitochondria following addition of Cd2+ : (a) activation of cation/proton exchange with considerable specificity towards K + , and (b) subsequent discharge of the proton gradient. 12-141. MATERIALS AND METHODSRat liver mitochondria were prepared by a conventional procedure in 0.25 M sucrose [15] and the protein content was determined by the biuret method [16]. Cation permeability Abbreviation. CIHgPhSO,, p-chloromercuriphenyl sulfonate. was determined by the decrease in absorbance at 546nm, which results from mitochondria1 swelling following uptake of the ions from the medium [l] or by the pH change...

A protein fraction has been obtained from detergent-~lubili~ mitochondrial membranes by its affinity for quinine, an inhibitor of K+ transport. A peptide derived from the predominant 53 kDa protein in this fraction is found to be identical in sequence to a portion of aldehyde dehydrogenase. Antigenically unrelated bands at 97,77, 57, and 31 kDa are also seen on polyacrylamide gels. Observations utilizing a fluorescent probe entrapped in the lumen of membrane vesicles indicate that the reconstituted protein fraction imparts permeability to the K+ analog TV. These and other findings suggest that the aflinity purified fraction includes a cation transport catalyst.

Unidirectional fluxes of Mg2+ across the limiting membranes of rat liver mitochondria have been measured in the presence of the respiratory substrate succinate by means of the radioisotope 28Mg. Rates of both influx and efflux of Mg2+ are decreased when respiration is inhibited. A linear dependence of the reciprocal of the Mg2+ influx rate on the reciprocal of the Mg2+ concentration is observed. The apparent Km for Mg2+ averages about 0.7 mM. N-Ethyl-maleimide, an inhibitor of transmembrane phosphate-hydroxyl exchanges, enhances the observed pH dependence of Mg2+, influx. In the presence of MalNEt, the apparent Vmax of Mg2+ influx is greater at pH 8 than at pH 7, and there is a linear dependence of the Mg2+ influx rate on the external OH- concentration. The K+ analogue Tl+ inhibits Mg2+ influx, while La3+, an inhibitor of mitochondrial Ca2+ transport, has no effect on Mg2+ influx. Mg2+ competitively inhibits the flux of K+ into rat liver mitochondria. The mechanism(s) mediating mitochondrial Mg2+ and K+ fluxes appear to be similar in their energy dependence, pH dependence, sensitivity to Tl+, and insensitivity to La3+.

No abstract

Based on published evidence that cation transport in mitochondria is not significantly dependent on a membrane potential, it is suggested that the process of mitochondrial cation transport may be nonelectrogenic. These experiments focused on the possibility that K+ flux into rat liver mitochondria may be directly coupled, via an energy-linked carrier mechanism, to OH- influx or H+ efflux. The dependence of the unidirectional K+ influx on the external K+ concentration indicates involvement of a saturable mechanism. Increasing the external pH from 7.0 to 8.0 increases the apparent V max of the K+ influx without significantly altering the apparent Km for K+. The pH dependence is greater in the presence of N-ethyl maleimide, a known inhibitor of the mitochondrial Pi/OH- exchange mechanism. N-Ethyl maleimide decreases the apparent V max at pH 7.0 and increases it at pH 8.0. Evidence indicates that both N-ethyl maleimide and a high external Pi concentration may stimulate the K+ influx at alkaline external pH (8.0) by preventing net exchanges between endogenous Pi and external OH-. An apparent first-order dependence of the K+ influx on the external OH- concentration is observed in the presence of N-ethyl maleimide. These results are consistent with a possible role of external OH- as a cosubstrate of the K+ transport mechanism.