Characteristics of Ca2+ transport by corn mitochondria

Mitochondria isolated from corn (Zea mays L.) coleoptiles by an improved procedure which yields functionally intact preparations are much more active in respiration-coupled Ca²⁺ accumulation than those employed in most earlier studies. Ca²⁺ uptake by these mitochondria is phosphate-dependent and is accompanied by decrease in Δψ, H⁺ extrusion and increase in the rate of respiration. A sigmoidal plot with a Hill coefficient of 2.22 was obtained when the rates of Ca²⁺ uptake were plotted as a function of free Ca²⁺ concentration. The K_0.5 for Ca²⁺ influx was about 31 μM and a V_max of 140 nmol Ca²⁺ per min per mg was attained at a free-Ca²⁺ concentration of about 120 μM. Ca²⁺ uptake is sensitive to inhibition by ruthenium red and Mg²⁺. The external free-Ca²⁺ concentration maintained at steady state was about 2 μM and was independent of the respiratory substrate and of external Na⁺, but was increased by exogenous Mg²⁺. In addition, this preparation of corn mitochondria has shown a much higher ability for Ca²⁺ retention in the presence of phosphate and NAD(P)H oxidants than liver mitochondria.     

A partial sequence of ionic changes associated with the acrosome reaction of Strongylocentrotus purpuratus

Relationships among several of the ion movements associated with the acrosome reaction of S. purpuratus were investigated. Egg jelly initiates ⁴⁵Ca²⁺ and ²²Na⁺ uptake, and K⁺ and H⁺ efflux. H⁺ efflux and ²²Na⁺ uptake occur with approximately equivalent stoichiometries as rapidly as the appearance of acrosomal rods, perhaps reflecting a linked process. Most K⁺ loss, as measured either by ⁴²K⁺ efflux or K⁺-ion-selective electrodes, occurs after the acrosome reaction is complete. Since an elevation of seawater K⁺ (from 10 to 15 mM) or the addition of 0.5 mM tetraethylammonium (TEA), an inhibitor of K⁺ channels, inhibits the acrosome reaction half-maximally, K⁺ movements or alterations of K⁺-dependent membrane potentials may regulate the triggering by jelly. Most, but not all, of the ⁴⁵Ca²⁺ influx is inhibited with a mixture of 10 μM FCCP, 1 mM CN^?, and 2 μg/ml oligomycin, suggesting that the mitochondria store most of the Ca²⁺. The extracellular Na⁺ concentration affects Ca²⁺ fluxes: sperm placed into 5 mM Na⁺ seawater have enhanced ⁴⁵Ca²⁺ uptake, but do not undergo the acrosome reaction, unless 30 mM Na⁺ is also added. Low Na⁺ concentrations lead to spontaneous triggering, by allowing for both Ca²⁺ influx and Na⁺-dependent H⁺ efflux. At least one early Ca²⁺ requirement precedes the Na⁺ and H⁺ movements, as inferred from attempts at reversing the inhibitors of jelly induction of the acrosome reaction. When sperm are incubated with jelly in the absence of Ca²⁺, then washed and incubated with jelly in the presence of Ca²⁺, the acrosome reaction is triggered only upon the second incubation. However, when sperm are mixed with jelly in the presence of the other inhibitors (verapamil, TEA, 5 mM Na⁺ seawater, low pH, or elevated K⁺), they are altered so that even upon subsequent washing, jelly-mediated triggering is no longer possible. This suggests the existence of an intermediate state in the reaction pathway, that follows an event for which Ca²⁺ is required, but that precedes the Na⁺ and H⁺ movements, which are inhibited by all inhibitors of the acrosome reaction. These data are used to develop a partial sequence of ionic changes associated with the triggering mechanism.     

Energy-dependence exchange of K+ in heart mitochondria. K+ efflux.

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 $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchanger (which responds to the transmembrane pH differential and which is the pathway for net K⁺ efflux).     

Inhibition by Sr2+ of specific mitochondrial Ca2+-efflux pathways

The effect of Sr²⁺ on the set point for external Ca²⁺ was studied in rat heart and liver mitochondria with the aid of a Ca²⁺-sensitive electrode. In respiring mitochondria the set point is determined by the rates of Ca²⁺ influx on the Ca²⁺ uniporter and efflux by various mechanisms. We studied the Ca²⁺-Na⁺ exchange pathway in heart mitochondria and the Δψ-modulated efflux pathway in liver mitochondria. Prior accumulation of Sr²⁺ was found to shift the set points towards lower external Ca²⁺ both in heart mitochondria under conditions of Ca²⁺-Na⁺ exchange and in liver mitochondria under conditions that should promote opening of the Δψ-modulated pathway. The effect on the set point was found to be due to inhibition of Ca²⁺ efflux by Sr²⁺ taken up by the mitochondria, while Sr²⁺ efflux was too slow to be measurable.     

Unidirectional Na(+) and Ca (2+) fluxes in two euryhaline teleost fishes, Fundulus heteroclitus and Oncorhynchus mykiss, acutely submitted to a progressive salinity increase

Na⁺ and Ca²⁺ regulation were compared in two euryhaline species, killifish (normally estuarine-resident) and rainbow trout (normally freshwater-resident) during an incremental salinity increase. Whole-body unidirectional fluxes of Na⁺ and Ca²⁺, whole body Na⁺ and Ca²⁺, and plasma concentrations (trout only), were measured over 1-h periods throughout a total 6-h protocol of increasing salinity meant to simulate a natural tidal flow. Killifish exhibited significant increases in both Na⁺ influx and efflux rates, with efflux slightly lagging behind efflux up to 60% SW, but net Na⁺ balance was restored by the time killifish reached 100% SW. Whole body Na⁺ did not change, in agreement with the capacity of this species to tolerate daily salinity fluctuations in its natural habitat. In contrast, rainbow trout experienced a dramatic increase in Na⁺ influx (50-fold relative to FW values), but not Na⁺ efflux between 40 and 60% SW, resulting in a large net loading of Na⁺ at higher salinities (60–100% SW), and increases in plasma Na⁺ and whole body Na⁺ at 100% SW. Killifish were in negative Ca²⁺ balance at all salinities, whereas trout were in positive Ca²⁺ balance throughout. Ca²⁺ influx rate increased two- to threefold in killifish at 80 and 100% SW, but there were no concomitant changes in Ca²⁺ efflux. Ca²⁺ flux rates were affected to a larger degree in trout, with twofold increases in Ca²⁺ influx at 40% SW and sevenfold increases at 100% SW. Again, there was no change in Ca²⁺ efflux with salinity, so plasma Ca²⁺ concentration increased in 100% SW. As the killifish is regularly submitted to increased salinity in its natural environment, it is able to rapidly activate changes in unidirectional fluxes in order to ensure ionic homeostasis, in contrast to the trout.     

Stimulation of K+ flux into mitochondria by phenylarsine oxide

The dithiol-reactive reagent phenylarsine oxide causes a pH-dependent stimulation of unidirectional K⁺ flux into respiring rat liver mitochondria. This stimulation is diminished by subsequent addition of either the dithiol 2,3-dimercaptopropanol or the monothiol 2-mercaptoethanol. In contrast, uncoupling by phenylarsine oxide is reversed by 2,3-dimercaptopropanol but not by 2-mercaptoethanol. The data suggest separate sites of interaction of phenylarsine oxide with mechanisms of K⁺ entry and ATP synthesis. Stimulatory effects of mersalyl and phenylarsine oxide on K⁺ influx are not additive. Thus PheASO and mersalyl may affect K⁺ influx at a common site. Pretreatment of the mitochondria with DCCD, which inhibits K⁺ influx, fails to alter sensitivity to PheAsO or mersalyl. Thus the DCCD binding site associated with the K⁺ influx mechanism appears to be separate from and independent of the sulfhydryl group(s) which mediate stimulation of K⁺ influx by PheAsO and mersalyl.PheAsO, like mersalyl, also increases the rate of unidirectional K⁺ efflux from respiring mitochondria. The combined presence of PheAsO plus mersalyl causes a greater stimulation of K⁺ efflux than is observed with either reagent alone.Abbreviations used: BAL, British AntilLewisite or 2,3-dimercaptopropanol; DCCD, dicyclohexylcarbodiimide; DBCT, dibutylchloromethyltin chloride; 2-ME, 2-mercaptoethanol; PheAsO, phenylarsine oxide.     

Controls on na influx in corn roots

We have investigated the effects of hyperpolarization and depolarization, and the presence of K⁺ and/or Ca²⁺, on ²²Na⁺ influx into corn (Zea mays L.) root segments. In freshly excised root tissue which is injured, Na⁺ influx is unaffected by hyperpolarization with fusicoccin, or depolarization with uncoupler (protonophore), or by addition of K⁺. However, added Ca²⁺ suppresses Na⁺ influx by 60%. In washed tissue which has recovered, Na⁺ influx is doubled over that of freshly excised tissue, and the influx is increased by fusicoccin and suppressed by uncoupler. This energy-linked component of Na⁺ influx is completely eliminated by low concentrations of K⁺, leaving the same level and kind of Na⁺ influx seen in freshly excised roots. The K⁺-sensitive energy linkage appears to be by the carrier for active K⁺ influx. Calcium is equally inhibitory to Na⁺ influx in washed as in fresh tissue. Other divalent cations are only slightly less effective. Net Na⁺ uptake was about 25% of ²²Na⁺ influx, but proportionately the response to K⁺ and Ca²⁺ was about the same.

The constancy of K⁺-insensitive Na⁺ influx under conditions known to hyperpolarize and depolarize suggests that if Na⁺ transport is by means of a voltage-sensitive channel, the rise or fall of channel resistance must be proportional to the rise or fall in potential difference. The alternative is a passive electroneutral exchange of ²²Na⁺ for endogenous Na⁺. The data suggest that an inwardly directed Na⁺ current is largely offset by an efflux current, giving both a small net uptake and isotopic exchange.

     

Retention of Ca2+ by rat liver and rat heart mitochondria: Effect of phosphate,Mg2+, and NAD(P) redox state

Parallel measurements of Ca²⁺ uptake, oxygen consumption, endogenous Mg²⁺ efflux, and swelling in rotenone-poisoned rat liver and rat heart mitochondria showed that heart mitochondria is much more resistant to uncoupling by Ca²⁺ in the presence of phosphate than rat liver mitochondria. The extent of Mg²⁺ efflux and swelling induced by Ca²⁺ accumulation are much less pronounced in heart mitochondria. Uncoupling and swelling in liver mitochondria seem to result from the loss of membrane-bound Mg²⁺ as a consequence of Ca²⁺ recycling across the membrane as induced by phosphate. Exogenous Mg²⁺ protects liver mitochondria against the deleterious effects of Ca²⁺ by inhibiting a ruthenium red-insensitive Ca²⁺ efflux induced by phosphate. Phosphate does not induce recycling of Ca²⁺ in heart mitochondria. On the other hand, heart mitochondria respiring on NAD-linked substrates or with succinate in the absence of rotenone behave like liver mitochondria with respect to the alterations caused by Ca²⁺ recycling. In heart mitochondria the recycling of Ca²⁺ is related to the redox state of pyridine nucleotides, which suggests that the ruthenium red-insensitive efflux of Ca²⁺ is subject to metabolic control. In addition it has been observed that Sr²⁺does not undergo cyclic movements across the membrane. The data indicate that the efflux pathway is more specific for Ca²⁺ than the ruthenium red-sensitive influx transporter.     

Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ efflux rates through Na⁺-dependent and Na⁺-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Ionic regulation of the cardiac sodium-calcium exchanger

The Na⁺-Ca²⁺ exchanger (NCX) links transmembrane movements of Ca²⁺ ions to the reciprocal movement of Na+ ions. It normally functions primarily as a Ca²⁺ efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca²⁺ influx under certain conditions. Na⁺ and Ca²⁺ ions exert complex regulatory effects on NCX activity. Ca²⁺ binds to two regulatory sites in the exchanger's central hydrophilic domain, and this interaction is normally essential for activation of exchange activity. High cytosolic Na⁺ concentrations, however, can induce a constitutive activity that by-passes the need for allosteric Ca²⁺ activation. Constitutive NCX activity can also be induced by high levels of phopshotidylinositol-4,5-bisphosphate (PIP₂) and by mutations affecting the regulatory calcium binding domains. In addition to promoting constitutive activity, high cytosolic Na⁺ concentrations also induce an inactivated state of the exchanger (Na⁺-dependent inactivation) that becomes dominant when cytosolic pH and PIP₂ levels fall. Na+-dependent inactivation may provide a means of protecting cells from Ca²⁺ overload due to NCX-mediated Ca²⁺ influx during ischemia.     

The ascidian sperm reaction: Ca2+ uptake in relation to H+ efflux

During the ascidian sperm reaction the single large cylindrical mitochondrion which lies next to the nucleus in the head swells, becomes spherical, and migrates along the tail to be lost when it reaches the end. This sequence is initiated by eggs, egg water, high pH, low Na⁺, or the ionophore X537A. Accompanying the sperm reaction induced by low Na⁺ are H⁺ efflux and Ca²⁺ influx in a ratio of near 100:1 as determined by ⁴⁵Ca²⁺ and atomic absorption analysis. Simultaneous pH and Ca²⁺ electrode measurements suggest that the movement of H⁺ begins 10–13 sec before the movement of Ca²⁺. Ca²⁺ uptake can be inhibited by verapamil without affecting H⁺ efflux or the sperm reaction. Acid release and Ca²⁺ uptake are proportional to the initial pH of the medium when the reaction is triggered by high pH. Acid release initiated by low Na⁺ is proportional to Ca²⁺ concentrations above 2 mM. H⁺ and Ca²⁺ movements differ in magnitude, kinetics, and inhibition by verapamil, thus suggesting that H⁺ is probably not exchanged for Ca²⁺. Instead we propose that loss of H⁺ triggers the uptake of Ca²⁺, which initiates the sperm reaction.     

The effect of taurine on the efflux of calcium from locust mitochondria

The effect of taurine has been studied on ⁴⁵Ca²⁺ efflux from mitochondria obtained from the flight muscle and thoracic ganglia of the desert locust. Mitochondria from both tissues readily accumulated ⁴⁵Ca²⁺ and this uptake was stimulated by the presence of phosphate ⁴⁵Ca²⁺ accumulation was abolished by ruthenium red (5 μ M). Only in the presence of 10 mM Na⁺ and either ruthenium red (5 μ M) or EGTA (500 μ M), was an efflux of ⁴⁵Ca²⁺ observed. Taurine (20 mM) abolished the Na⁺-dependent ⁴⁵Ca²⁺ efflux but had no effect in the absence of Na⁺. These results suggest that taurine may contribute to the control of the concentration of intracellular free calcium.     

Reduced external calcium or sodium stimulates calcium influx in Pelvetia eggs

The effect of external calcium and sodium ion concentrations on the calcium fluxes on the Pelvetia fastigiata De Toni egg was measured. Decreasing external [Ca²⁺] greatly increased the permeability of the eggs to Ca²⁺; at 1 mM external Ca²⁺ this permeability was 60 times as great as it was at the normal [Ca²⁺] of 10 mM. Lowering the external [Na⁺] also increased Ca²⁺ influx; at 2 mM Na⁺, the Ca²⁺ influx was 2–3 times as great as it was at the normal [Na⁺] if choline was used as a Na⁺ substitute. Lithium was less effective as a Na⁺ substitute in increasing Ca²⁺ influx. The extra Ca²⁺ influx in low [Na⁺] seemed to be dependent on internal [Na⁺]. The Ca²⁺ efflux increased transiently and then declined in low Na⁺ media.     

Improved measurements of Na+ fluxes in plants using calixarene-based microelectrodes

Ion-selective microelectrodes are a powerful tool in studying adaptive responses of plant cells and tissues to various abiotic stresses. However, application of this technique in Na⁺ flux measurements was limited due to poor selectivity for Na⁺ ions of commercially available Na⁺ cocktails. Often, these cocktails cannot discriminate between Na⁺ and other interfering ions such as K⁺ and Ca²⁺, leading to inaccurate measurements of Na⁺ concentration and, consequently, inaccurate Na⁺ flux calculations. To overcome this problem, three Na⁺-selective cocktail mixtures were prepared using tetramethoxyethyl ester derivative of p-t-butyl calix[4]arene. These cocktail mixtures were compared with commercially available ETH 227-based Na⁺ cocktail for selectivity for Na⁺ ions over other ions (particularly K⁺ and Ca²⁺). Among the three calixarene-based Na⁺ cocktails tested, cocktail 2 [in % w/w: Na⁺ ionophore (4-tert-butylcalix[4]arene-tetra acetic acid tetraethyl ester) 3.5, the plasticizer (2-nitrophenyl octyl ether) 95.9 and lipophilic anion (potassium tetrakis (4-chlorophenyl) borate) 0.6] showed the best selectivity for Na⁺ ions over K⁺ and Ca²⁺ ions and was highly stable over time (up to 10 h). Na⁺ flux measurements under a wide range of NaCl concentrations (25-150 mM) using Na⁺ cocktail 2 established a clear dose-response relationship between severity of salt stress and magnitude of Na⁺ influx at the distal elongation and mature zones of Arabidopsis thaliana roots. Furthermore, Na⁺ cocktail 2 was compared with commercially available ETH 227-based Na⁺ cocktail by measuring Na⁺ fluxes at the two Arabidopsis root zones in response to 100 mM NaCl treatment. With calixarene-based Na⁺ cocktail 2, a large decreasing Na⁺ influx (0-15 min) followed by small Na⁺ influx (15-45 min) was measured, whereas with ETH-based Na⁺ cocktail Na⁺ influx was short-lived (1-3 min) and was followed by Na⁺ efflux (3-45 min) that might have been due to K⁺ and Ca²⁺ efflux measured together with Na⁺ influx. In conclusion, Na⁺-selective calixarene-based microelectrodes have excellent potential to be used in real-time Na⁺ flux measurements in plants.     

Reactions of corn root tissue to calcium

Washing corn (Zea mays L.) root tissue in water causes loss of about one-third of the exchangeable Ca²⁺ over the first 10 to 15 minutes. Upon transfer to K⁺-containing solutions, the tissue shows a short period of rapid K⁺ influx which subsequently declines. Addition of 0.1 millimolar Ca²⁺ decreases the initial rapid K⁺ influx, but increases the sustained rate of K⁺ and Cl⁻ uptake. It was confirmed (Elzam and Hodges 1967 Plant Physiol 42: 1483-1488) that 0.1 millimolar Ca²⁺ is more effective than higher concentrations for the initial inhibition, and that Mg²⁺ will substitute.

The inhibition arises from a mild shock affect of restoring Ca²⁺. With 0.1 millimolar Ca²⁺ net H⁺ efflux is blocked for 10 to 15 minutes and the cells are depolarized by about 30 millivolts. However, 1 millimolar Ca²⁺ rapidly produces increased K⁺ influx and blocks net H⁺ efflux for only a few minutes; blockage is preceded by a brief net H⁺ influx which may restore and increase ion transport by reactivating the plasmalemma H⁺-ATPase.

Stimulation of electrogenic H⁺-pumping with fusicoccin eliminates the shock responses and minimizes Ca²⁺ effects on K⁺ influx. Fusicoccin also strongly decreases Ca²⁺ influx, but has no effect on Ca²⁺ efflux. Ice temperatures and high pH decreased Ca²⁺ efflux, but uncoupler and chlorpromazine did not.

It is suggested that the inhibitory and promotive actions of Ca²⁺ are manifested through decreases or increases in the protonmotive force.

     

Glutamate-supported calcium movements in rat liver mitochondria effects of anions and pH

Ca²⁺ efflux from rat liver mitochondria in the presence of glutamate is stimulated by a decrease in pH from 7.3 to 6.8 and the rate is dependent on the phosphate concentration. During Ca²⁺ (13 μm) uptake and release at low pH (+ phosphate), swelling is minimal, but a large oxidation of pyridine nucleotides and sustained membrane depolarization occurs. The depolarization (but not Ca²⁺ efflux) is reversed by ruthenium red. An absolute requirement for phosphate to support Ca²⁺ efflux is demonstrated by using acetate or lactate to support Ca²⁺ uptake (efflux is depressed at pH 6.8). Preincubation with mersalyl, to block phosphate movements, with subsequent phosphate addition preceeding Ca²⁺ uptake also inhibits efflux. β-Mercaptoethanol then stimulates efflux concomittent with membrane repolarization. Ca²⁺ efflux is not a simple result of collapse of ΔpH since nigericin inhibits phosphate transport and Ca²⁺ release. Following Ca²⁺ uptake at pH 6.8, respiratory inhibition occurs, but oxygen consumption coupled to ATP synthesis can be stimulated by succinate (+ rotenone). Addition of succinate allows reuptake of Ca²⁺, reduction of pyridine nucleotides, and repolarization of the membrane potential. Respiratory inhibition is also seen with nigericin, but no Ca²⁺ efflux is observed. Coupled respiration with glutamate is seen at pH 6.8 following Ca²⁺ uptake in the presence of lactate with subsequent addition of phosphate to promote Ca²⁺ efflux. We conclude that Ca²⁺ efflux is not a consequence of respiratory inhibition, but is mediated solely by phosphate movements. The inhibitory effect of Mg²⁺ on Ca²⁺ efflux is probably due to Mg²⁺-dependent inhibition of the Ca²⁺ diffusion potential so that the compensatory increase in ΔpH due to membrane depolarization does not occur and phosphate entry is slowed.     

Biochemical studies of the excitable membrane of Paramecium aurelia. I. 45Ca2+ fluxes across resting and excited membrane

The characteristics of Ca²⁺ transport across the excitable membrane of Paramecium aurelia were studied by measuring ⁴⁵Ca²⁺ influx and efflux. The intracellular concentration of free Ca²⁺ in resting P. aurelia was at least ten times less than the extracellular concentration. Ca²⁺ influx was easily measurable at 0°C, but not at 23°C. The influx of ⁴⁵Ca²⁺ was stimulated by the same conditions which cause membrane depolarization and ciliary reversal. Addition of Na⁺ and K⁺ (which stimulate ciliary reversal) resulted in a 10-fold increase in the rate of Ca²⁺ influx. An externally applied, pulsed, electric field (1–2 mA/cm² of electrode surface), caused the rate of Ca²⁺ influx to increase 3–5 times, with the extent of stimulation dependent on the current density and the pulse width Ca²⁺ influx had the characteristics of a passive transport system and was associated with the chemically or electrically triggered Ca²⁺ “gating” mechanism, which has been studied electrophysiologically. In contrast, Ca²⁺ efflux appeared to be catalyzed by an active transport system. With cells previously loaded at 0°C with ⁴⁵Ca²⁺, Ca²⁺ efflux was rapid at 23°C, but did not occur at 0°C. This active Ca²⁺ efflux mechanism is probably responsible for maintaining the low internal Ca²⁺ levels in unstimulated 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.     

The dynamics of mitochondrial Ca fluxes

We have investigated the kinetics of mitochondrial Ca²⁺ influx and efflux and their dependence on cytosolic [Ca²⁺] and [Na⁺] using low-Ca²⁺-affinity aequorin. The rate of Ca²⁺ release from mitochondria increased linearly with mitochondrial [Ca²⁺] ([Ca²⁺]_M). Na⁺-dependent Ca²⁺ release was predominant al low [Ca²⁺]_M but saturated at [Ca²⁺]_M around 400 μM, while Na⁺-independent Ca²⁺ release was very slow at [Ca²⁺]_M below 200 μM, and then increased at higher [Ca²⁺]_M, perhaps through the opening of a new pathway. Half-maximal activation of Na⁺-dependent Ca²⁺ release occurred at 5-10 mM [Na⁺], within the physiological range of cytosolic [Na⁺]. Ca²⁺ entry rates were comparable in size to Ca²⁺ exit rates at cytosolic [Ca²⁺] ([Ca²⁺]_c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca²⁺]_c. As a consequence, the presence of [Na⁺] considerably reduced the rate of [Ca²⁺]_M increase at [Ca²⁺]_c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca²⁺]_c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca²⁺]_M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca²⁺ buffering, and comparison of our results with data on total mitochondrial Ca²⁺ fluxes indicate that the mitochondrial Ca²⁺ bound/Ca²⁺ free ratio is around 10- to 100-fold for most of the observed [Ca²⁺]_M range and suggest that massive phosphate precipitation can only occur when [Ca²⁺]_M reaches the millimolar range.     

Mitochondrial localization of NCXs: Balancing calcium and energy homeostasis

It is well established that mitochondria are the main source of ATP production within cells. However, mitochondria have other remarkable functions, serving as important modulators of cellular Ca²⁺ signaling, and it is now generally recognized that control over Ca²⁺ homeostasis is intrinsically interwoven with mitochondrial abilities to adjust and tune ATP production. In this review, we describe the mechanisms that mitochondria use to balance Ca²⁺ homeostasis maintenance and cell energy metabolism. In recent years, the knowledge on the molecular machinery mediating Ca²⁺ influx/efflux has been improved and, albeit still open to further investigations, several lines of evidence converge on the hypothesis that plasma membrane Na⁺/Ca²⁺ exchanger (NCX) isoforms are also expressed at the mitochondrial level, where they contribute to the Ca²⁺ and Na⁺ homeostasis maintenance. In particular, the connection between mitochondrial NCX activity and metabolic substrates utilization is further discussed here. We also briefly focus on the alterations of both mitochondrial Ca²⁺ handling and cellular bioenergetics in neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease.