Oligomycin inhibits store-operated channels by a mechanism independent of its effects on mitochondrial ATP.

Inhibitors of mitochondrial oxidative metabolism have been proposed to interfere with Ca2+ influx mediated by store-operated channels (SOC), secondary to their effects on ATP production. We assessed SOC activity by 45Ca2+ influx and fluorimetric measurements of free Ca2+ or Mn2+ quench in thapsigargin-treated Chinese hamster ovary cells and Jurkat T-cells, and additionally by electrophysiological measurements of the Ca2+-release-activated Ca2+ current (Icrac) in Jurkat T-cells. Various mitochondrial antagonists were confirmed to inhibit SOC. However, the following evidence supported the proposal that oligomycin, in particular, exerts an inhibitory effect on SOC in addition to its known actions on mitochondria and Na+-pump activity: (i) the concentrations of oligomycin required to inhibit SOC-mediated Ca2+ influx or Icrac (half-inhibitory concentration approximately 2 microM) were nearly 50-fold higher than the concentrations that blocked mitochondrial ATP production; (ii) the rank order of potency of oligomycins A, B and C for decreasing SOC-mediated Ca2+ influx or Icrac differed from that known for inhibition of mitochondrial function; (iii) oligomycin blocked Icrac under voltage clamp and with intracellular Na+ and K+ concentrations fixed by dialysis from the patch pipette, arguing that the effect was not secondary to membrane polarization or pump activity; and (iv) fixing the cytosolic ATP concentration by dialysis from the patch pipette attenuated rotenone- but not oligomycin-mediated inhibition of Icrac. Oligomycin also blocked volume-activated Cl- currents, a profile common to some other known blockers of SOC that are not known mitochondrial inhibitors. These findings raise the possibility that oligomycin interacts directly with SOC, and thus may extend the known pharmacological profile for this type of Ca2+-influx pathway.

Barium influx mediated by the cardiac sodium-calcium exchanger in transfected Chinese hamster ovary cells.

We examined Ba2+ influx using isotopic and fura-2 techniques in transfected Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells). Ba2+ competitively inhibited exchange-mediated 45Ca2+ uptake with a Ki approximately 3 mM. Ba2+ uptake was stimulated by pretreating the cells with ouabain and by removing extracellular Na+, as expected for Na+/Ba2+ exchange activity. The maximal velocity of Ba2+ accumulation was estimated to be 50% of that for Ca2+. When the monovalent cation ionophore gramicidin was used to equilibrate internal and external concentrations of Na+, Ba2+ influx was negligible in the absence of Na+ and increased to a maximum at 20-40 mM Na+. At higher Na+ concentrations, Ba2+ influx declined, presumably due to the competition between Na+ and Ba2+ for transport sites on the exchanger. Unlike Ca2+, Ba2+ did not appear to be taken up by intracellular organelles. Thus, 133Ba2+ uptake in ouabain-treated cells was not reduced by mitochondrial inhibitors such as-Cl-CCP or oligomycin-rotenone. Moreover, intracellular Ca2+ stores that had been depleted of Ca2+ by pretreatment of the cells with ionomycin (a Ca2+ ionophore) remained empty during a subsequent period of Ba2+ influx. Ca2+ uptake or release by intracellular organelles secondarily regulated exchange activity through alterations in [Ca2+]i. Exchange-mediated Ba2+ influx was inhibited when cytosolic [Ca2+] was reduced to 20 nM or less and was accelerated at cytosolic Ca2+ concentrations of 25-50 nM We conclude that (a) Ba2+ substitutes for Ca2+ as a transport substrate for the exchanger, (b) cytosolic Ba2+ does not appear to be sequestered by intracellular organelles, and (c) exchange-mediated Ba2+ influx is accelerated by low concentrations of cytosolic Ca2+.

Sodium-calcium exchange and calcium homeostasis in transfected Chinese hamster ovary cells.

Our experiments with transfected cells provide new insights into the role of Na-Ca exchange activity in Ca homeostasis and emphasize the role of local interactions in determining exchanger function. Thus, the effects of ATP depletion and cytochalasin D highlight the influence of the actin cytoskeleton in regulating exchange activity. Cytoskeletal interactions could provide a mechanism for modulating exchange activity by mechanical stretch and might constitute a novel feedback mechanism for regulating contractile activity in the heart. The effects of Na on Ca entry during SDCI in the transfected cells suggest that local gradients of [Ca]i are important determinants of exchanger function. The surface distribution of exchanger proteins in relation to that of Ca channels therefore represents another area in which interactions with the cytoskeleton may be a central element in understanding the physiological function(s) of the exchange activity. At present, it seems likely that the exchanger's central hydrophilic domain mediates the connection between the exchanger and the cytoskeleton. This provides a rationale for understanding the importance of tissue-specific alterations in the exchanger's hydrophilic domain, which appear to have little affect on the kinetic behavior of the exchanger. Future work in our laboratory will be directed toward clarifying the role of cytoskeletal interactions in exchanger function.

Sodium-calcium exchange and store-dependent calcium influx in transfected chinese hamster ovary cells expressing the bovine cardiac sodium-calcium exchanger. Acceleration of exchange activity in thapsigargin-treated cells.

The effects of extracellular Na+ on store-dependent Ca2+ influx were compared for transfected Chinese hamster ovary cells expressing the bovine cardiac Na+-Ca2+ exchanger (CK1.4 cells) and vector-transfected control cells. Store-dependent Ca2+ influx was elicited by depletion of intracellular Ca2+ stores with ionomycin, thapsigargin, or extracellular ATP, a purinergic agonist. In each case, the rise in [Ca2+]i upon the addition of extracellular Ca2+ was reduced in CK1.4 cells compared with control cells at physiological [Na+]o. When Li+ or NMDG was substituted for Na+, the CK1.4 cells showed a greater rise in [Ca2+]i than control cells over the subsequent 3 min after the addition of Ca2+o. Under Na+-free conditions, SK&F 96365 (50 microM), a blocker of store-operated Ca2+ channels, nearly abolished the thapsigargin-induced rise in [Ca2+]i in the control cells but only partially inhibited this response in the CK1.4 cells. We conclude that in the CK1.4 cells, Ca2+ entry through store-operated channels was counteracted by Na+o-dependent Ca2+ efflux at physiological [Na+]o, whereas Ca2+ entry was enhanced through Na+i-dependent Ca2+ influx in the Na+-free medium. We examined the effects of thapsigargin on Ba2+ entry in the CK1.4 cells because Ba2+ is transported by the Na+-Ca2+ exchanger, but it enters these cells only poorly through store-operated channels, and it is not sequestered by intracellular organelles. Thapsigargin treatment stimulated Ba2+ influx in a Na+-free medium, consistent with an acceleration of Ba2+ entry through the Na+-Ca2+ exchanger. We conclude that organellar Ca2+ release induces a regulatory activation of Na+-Ca2+ exchange activity.

ATP-dependent regulation of sodium-calcium exchange in Chinese hamster ovary cells transfected with the bovine cardiac sodium-calcium exchanger.

Chinese hamster ovary cells expressing the bovine cardiac Na/Ca exchanger were treated with ouabain to increase [Na+]i and stimulate Ca2+ influx by Na/Ca exchange. Depletion of cellular ATP inhibited 45Ca uptake by 40% or more and reduced the half-maximal Na+ concentration for inhibition of 45Ca uptake from 90 to 55 mM. ATP depletion also reduced the rate of rise in [Ca2+]i when [Na+]o was reduced and inhibited the decline in [Ca2+]i when high [Na+]o was restored. The effects of ATP depletion were either absent or reduced in cells expressing a mutant exchanger missing most of the cytosolic hydrophilic domain. We were unable to detect a phosphorylated form of the exchanger in immunoprecipitates from 32P-labeled cells. ATP depletion caused a breakdown in the actin cytoskeleton of the cells. Treatment of the cells with cytochalasin D mimicked the effects of ATP depletion on the [Na+] inhibition profile for 45Ca uptake. Thus, ATP depletion inhibits both the Ca2+ influx and Ca2+ efflux modes of Na/Ca exchange, and may alter the competitive interactions of extracellular Na+ and Ca2+ with the transporter. The latter effect appears to be related to changes in the actin cytoskeleton.

Na+/Ca2+ antiport in the mammalian heart.

The cardiac Na+/Ca2+ antiporter moves 3 Na+ across the plasma membrane in exchange for a single Ca2+ moving in the opposite direction. It is the principal Ca2+ efflux mechanism in myocardial cells; however, it also contributes to Ca2+ influx under certain conditions. It is particularly abundant in the heart, but is also expressed in other tissues such as smooth and skeletal muscle, the kidney and the brain. The cardiac antiporter itself is a protein of 938 amino acids, with a cleaved NH2-terminal signal sequence, 11 putative transmembrane segments and a large hydrophilic domain of 520 amino acids between the fifth and sixth transmembrane segments. Alternative mRNA splicing mechanisms generate tissue-specific isoforms in a limited region within the hydrophilic domain. Most of the hydrophilic domain can be deleted without altering the kinetics of the transport reaction; the regulatory properties of the antiporter are markedly affected by this deletion however. Two different modes of regulation of antiport activity have been characterized and appear to involve two different inactive states of the carrier. The first is promoted by the presence of cytosolic Na+ in the absence of ATP and the second is promoted by the absence of cytosolic Ca2+. ATP-dependent regulation of antiport activity may involve interactions with the cellular cytoskeleton, since the effects of ATP depletion can be mimicked by cytochalasin D. Ca(2+)-dependent regulation of antiport activity appears to involve the interaction of cytosolic Ca2+ with two acidic amino acid sequences within a limited region of the hydrophilic domain.