Altered Ionic Selectivity of the Sodium Channel Revealed by Cysteine Mutations within the Pore

To explore the role of pore-lining amino acids in Na⁺ channel ion-selectivity, pore residues were  replaced serially with cysteine in cloned rat skeletal muscle Na⁺ channels. Ionic selectivity was determined by measuring permeability and ionic current ratios of whole-cell currents in Xenopus oocytes. The rSkM1 channels displayed an ionic selectivity sequence Na⁺>Li⁺>NH₄ ⁺>>K⁺>>Cs⁺ and were impermeable to divalent cations.  Replacement of residues in domain IV showed significantly enhanced current and permeability ratios of NH₄ ⁺ and K⁺, and negative shifts in the reversal potentials recorded in the presence of external Na⁺ solutions when compared to cysteine mutants in domains I, II, and III (except K1237C). Mutants in domain IV showed altered selectivity sequences: W1531C (NH₄ ⁺>K⁺>Na⁺≥Li⁺≈Cs⁺), D1532C, and G1533C (Na⁺>Li⁺≥NH₄ ⁺>K⁺>Cs⁺). Conservative replacement of the aromatic residue in domain IV (W1531) with phenylalanine or tyrosine retained Na⁺ selectivity of the channel while the alanine mutant (W1531A) reduced ion selectivity. A single mutation within the third pore forming region (K1237C) dramatically altered the selectivity sequence of the rSkM1 channel (NH₄ ⁺>K⁺>Na⁺≥Li⁺≈Cs⁺) and was permeable to divalent cations having the selectivity sequence Ca²⁺≥Sr²⁺>Mg²⁺>Ba²⁺. Sulfhydryl modification of K1237C, W1531C or D1532C with methanethiosulfonate derivatives that introduce a positively charged ammonium group, large trimethylammonium moiety, or a negatively charged sulfonate group within the pore was ineffective in restoring Na⁺ selectivity to these channels. Selectivity of D1532C mutants could be largely restored by increasing extracellular pH suggesting altering the ionized state at this position influences selectivity. These data suggest that K1237 in domain III and W1531, D1532, and G1533 in domain IV play a critical role in determining the ionic selectivity of the Na⁺ channel.     

Calcium uptake by excised maize roots and interactions with alkali cations

Ca²⁺ uptake was studied in short-term experiments using 5-day-old excised maize roots. This tissue readily absorbs Ca²⁺, and inhibition by dinitrophenol and low temperature shows that the process is metabolically mediated. The uptake of Ca²⁺, like that of other cations, is influenced by the counter ion, the pH and concentration of the ambient solution, and the presence of other cations. The rate of uptake from various salts decreases in the following order: NO₃⁻ > Cl⁻ = Br⁻ > SO₄²⁻. K⁺ and H⁺ greatly interfere with Ca²⁺ absorption, while Li⁺ and Na⁺ have only slight effects.     

Adenosine triphosphatase from soybean callus and root cells

The ATPase activity of a membrane fraction from soybean (Glycine max L.) root and callus cells, presumed to be enriched in plasma membrane, has been characterized with respect to ion stimulation, pH requirement, and nucleotide specificity. The enzyme from both sources was activated by divalent cations (Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺ > Sr²⁺) and further stimulated by monovalent salts. Preparations from root cells were stimulated by monovalent ions according to the sequence: K⁺ > Rb⁺ > Choline⁺ > Na⁺ > Li⁺ > NH₄⁺ > Cs⁺ > tris⁺. Membrane preparations from callus cells showed similar stimulatory patterns except for a slight preference for Na⁺ over K⁺. No synergism between K⁺ and Na⁺ was found with preparations from either cell source.     

Selectivity of the Ca2+-activated and Light-dependent K+ Channels for Monovalent Cations

The ionic selectivity of the Ca²⁺-activated K⁺ channel of Aplysia neurons and of the light-dependent K⁺ channel of Pecten photoreceptors to metal and organic cations was studied. The selectivity sequence determined from reversal potential measurements is T1⁺ K⁺ > Rb⁺ > NH⁺₄ > Cs⁺ > Na⁺, Li⁺ and is identical to the sequence determined previously for voltage-dependent K⁺ channels in a variety of tissues. Our results suggest that some physical aspect of the K⁺ channel is conserved in phyllogenetically different tissues and cells.     

Cation amelioration of aluminum toxicity in wheat

Aluminum is a major constituent of most soils and limits crop productivity in many regions. Amelioration is of theoretical as well as practical interest because understanding amelioration may contribute to an understanding of the mechanisms of toxicity. In the experiments reported here 2-day-old wheat (Triticum aestivum L. cv Tyler) seedlings with 15-millimeter roots were transferred to solutions containing 0.4 millimolar CaCl₂ at pH 4.3 variously supplemented with AlCl₃ and additional amounts of a chloride salt. Root lengths, measured after 2 days in the test solutions, were a function of both Al activity and the cation activity of the added salt. Percent inhibition = 100 {Al³⁺}/({Al³⁺} + K_m + α{C}^β) where {Al³⁺} is the activity of Al³⁺ expressed in micromolar, {C} is the activity of the added cation expressed in millimolar, and K_m (= 1.2 micromolar) is the {Al³⁺} required for 50% inhibition in the absence of added salt. For Ca²⁺, Mg²⁺, and Na⁺ the values of α were 2.4, 1.6, and 0.011, respectively, and the values for β were 1.5, 1.5, and 1.8, respectively. With regard to relative ameliorative effectiveness, Ca²⁺ > Mg²⁺ ≈ Sr²⁺ K⁺ ≈ Na⁺. Other cations were tested, but La³⁺, Sc³⁺, Li⁺, Rb⁺, and Cs⁺ were toxic at potentially ameliorative levels. The salt amelioration is not solely attributable to reductions in {Al³⁺} caused by increases in ionic strength. Competition between the cation and Al for external binding sites may account for most of the amelioration.     

ATP-Induced Inhibition of Na+, K+, Cl− Cotransport in Madin-Darby Canine Kidney Cells: Lack of Involvement of Known Purinoceptor-Coupled Signaling Pathways

P_2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca²⁺] _i , activation of phospholipases C (PLC) and A₂ (PLA₂), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na⁺,K⁺,Cl⁻ cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier (∼50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca²⁺]_i that was abolished by a chelator of Ca²⁺ _i , BAPTA. However, neither BAPTA nor the Ca²⁺ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca²⁺-pump, thapsigargin, modified ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na⁺,K⁺,Cl⁻ cotransport activity. Inhibitors of PLA₂ (AACOCF₃), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na⁺ channels (amiloride), Cl⁻ channels (NPPB) and Na⁺/H⁺ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport and suggest the presence of a novel P₂-receptor-coupled signaling mechanism. Received: 29 July 1998/Revised: 19 October     

Ion conduction and selectivity in acid-sensing ion channel 1

The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na⁺ with Li⁺, K⁺, Rb⁺, or Cs⁺ altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with P_K/P_Na = 0.1 and G_K/G_Na = 0.7 and P_Rb/P_Na = 0.03 and G_Rb/G_Na = 0.3. Stimulation of Cl⁻ currents when Na⁺ was replaced with Ca²⁺, Sr²⁺, or Ba²⁺ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na⁺ in a hyperbolic manner, consistent with an apparent affinity (K_m) for Na⁺ conduction of 25 mM. Nitrogen-containing cations, including NH₄⁺, NH₃OH⁺, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na⁺ currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na⁺ (K_m = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule.     

Roles of Ca2+ and Protein Tyrosine Kinase in Insulin Action on Cell Volume via Na+ and K+ Channels and Na+/K+/2Cl− Cotransporter in Fetal Rat Alveolar Type II Pneumocyte

The aim of the present study was to investigate the roles of Ca²⁺ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca²⁺ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca²⁺ (<1 nm). This insulin action in a Ca²⁺-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na⁺/K⁺/2Cl⁻ cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na⁺ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca²⁺-free solution was completely blocked by quinine (1 mm, a blocker of Ca²⁺-activated K⁺ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm extracellular Ca²⁺, that the stimulatory action of insulin on an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter requires Ca²⁺, and that in a Ca²⁺-free solution insulin activates a quinine-sensitive K⁺ channel but not in the presence of 1 mm Ca²⁺. The insulin action on cell volume in a Ca²⁺-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca²⁺-ATPase which depletes the intracellular Ca²⁺ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca²⁺-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K⁺ channel is mediated through PTK activity in a cytosolic Ca²⁺-dependent manner. Lavendustin A, further, completely blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in a Ca²⁺-free solution, but only partially blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm Ca²⁺. This observation suggests that the activity of the Na⁺/K⁺/2Cl⁻ cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca²⁺-independent pathway and the other is a PTK-independent, Ca²⁺-dependent pathway. Further, we observed that removal of extracellular Ca²⁺ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na⁺ channel and the bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter, and that removal of extracellular Ca²⁺ abolished the activity of the quinine-sensitive K⁺ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca²⁺ results from diverse effects on the cotransporter and Na⁺ and K⁺ channels. Received: 2 September 1998/Revised: 30 November 1998     

Inhibition of the Na+/Ca2+ antiport of heart mitochondria by diethylpyrocarbonate

Diethylpyrocarbonate inhibits Na⁺/Ca²⁺ antiport activity in isolated heart mitochondria. The inhibition is time-dependent with maximum activity developed after 5 min at 25°C. The reaction of diethylpyrocarbonate with the mitochondrial membrane is biphasic with 25–30 nmol mg^–1 reacting rapidly and an additional 30 nmol mg^–1 taken up slowly over a 30-min incubation. Inhibition of mitochondrial Na⁺/Ca²⁺ antiport by diethylpyrocarbonate decreases theV _max of the reaction, and the inhibition cannot be reversed by washing the mitochondria or addition of excess histidine. The inhibition occurs at levels of inhibitor that have little or no effect on Ca²⁺ uptake, Na⁺/H⁺ antiport, or succinate respiration. A portion of the Na⁺-dependent efflux of Ca²⁺ is insensitive to diethylpyrocarbonate and this component is abolished by diltiazem. The mechanism by which diethylpyrocarbonate inactivates Na⁺/Ca²⁺ antiport is still uncertain, but may involve the modification of an unprotonated histidine residue in the transporter.     

Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter

Summary In rabbit ileum, Ca²⁺/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca²⁺/CaM stimulate linked Na⁺ and Cl^– absorption. The role of Ca²⁺/CaM-dependent phosphorylation in regulation of the brush-border Na⁺/H⁺ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na⁺/H⁺ antiporter and Ca²⁺/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca²⁺, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca²⁺/CaM and ATP inhibited Na⁺/H⁺ exchange by 45±13%. This effect was specific since Ca²⁺/CaM and ATP did not alter diffusive Na⁺ uptake, Na⁺-dependent glucose entry, or Na⁺ or glucose equilibrium volumes. The inhibition of the Na⁺/H⁺ exchanger by Ca²⁺/CaM/ATP was due to an effect on theV _max and not on theK _m for Na⁺. In the presence of CaM and ATP, Ca²⁺ caused a concentration-dependent inhibition of Na⁺ uptake, with an effect 50% of maximum occurring at 120nm. This Ca²⁺ concentration dependence was similar to the Ca²⁺ concentration dependence of Ca²⁺/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca²⁺/CaM/ATP-inhibition of Na⁺/H⁺ exchange was reversed by W₁₃, a Ca²⁺/CaM antagonist, but not by a hydrophobic control, W₁₂, or by H-7, a protein kinase C antagonist. we conclude that Ca²⁺, acting through CaM, regulates ileal brush-border Na⁺/H⁺ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.     

The Permeability of the Sodium Channel to Metal Cations in Myelinated Nerve

The relative permeability of sodium channels to eight metal cations is studied in myelinated nerve fibers. Ionic currents under voltage-clamp conditions are measured in Na-free solutions containing the test ion. Measured reversal potentials and the Goldman equation are used to calculate the permeability sequence: Na⁺ ≈ Li⁺ > Tl⁺ > K⁺. The ratio P_K/P_Na is 1/12. The permeabilities to Rb⁺, Cs⁺, Ca⁺⁺, and Mg⁺⁺ are too small to measure. The permeability ratios agree with observations on the squid giant axon and show that the reversal potential E_Na differs significantly from the Nernst potential for Na⁺ in normal axons. Opening and closing rates for sodium channels are relatively insensitive to the ionic composition of the bathing medium, implying that gating is a structural property of the channel rather than a result of the movement or accumulation of particular ions around the channel. A previously proposed pore model of the channel accommodates the permeant metal cations in a partly hydrated form. The observed sequence of permeabilities follows the order expected for binding to a high field strength anion in Eisenman's theory of ion exchange equilibria.     

Ionic Regulation for Cytokinin-dependent Betacyanin Synthesis in Amaranthus Seedlings

Potassium ions at low concentrations stimulate cytokinin-dependent betacyanin synthesis in Amaranthus tricolor seedlings more than other alkali metal ions when tested as the chloride salts. The sequence of relative stimulation is K⁺ > Rb⁺ > (Na⁺ = Li⁺). Calcium and Mg²⁺ ions are inhibitory at concentrations > 1 millimolar when tested as chlorides. Anions also have an effect on the degree of alkali metal stimulation in the order PO₄³⁻ > NO₃⁻ > Cl⁻. The high activity of phosphate may be partly due to its chelating effect on inhibitory Ca²⁺ ions, or to effects on K⁺ uptake. A mixture of Na⁺ and K⁺ in the presence of phosphate is more effective than either cation alone. This result may be due either to effects on tyrosine transport or on the potassium uptake system. Phytochrome-dependent betacyanin synthesis shows the same stimulation by Na⁺ plus K⁺. The effect of a number of inhibitors of transport systems on betacyanin accumulation is reported. The possible role of the ionic environment of cells in their metabolic regulation is discussed, particularly in relation to cytokinin action.     

Mitochondrial cation transport: A progress report

This report summarizes recent work in our laboratory aimed at understanding protein-mediated mitochondrial cation transport. We are studying three distinct cation cycles that contain porters catalyzing influx and efflux of cations between cytosol and mitochondrial matrix. Each of these cation cycles plays a major physiological role in the overall energy economy. The K⁺ cycle maintains the integrity of the vesicular structure and includes the K⁺/H⁺ antiporter, the K_ATP channel, and K⁺ leak driven by the high membrane potential. The Ca²⁺ cycle relays the signals calling for modulation of ATP production and includes the Ca²⁺ channel, the Na⁺/Ca²⁺ antiporter, and the Na⁺/H⁺ antiporter. The H⁺ cycle of brown adipose tissue mitochondria provides heat to hibernating and newborn mammals and consists of the uncoupling protein, which catalyzes regulated H⁺ influx.     

Plasma membrane potential of Lettré cells does not depend on cation gradients but on pumps

Summary The plasma membrane potential of Lettré cells has been determined with the optical indicator oxonol-V and found to be –57 mV at 37°C (range –20 to –80 mV depending on the physiological condition of the cells). Increasing extracellular K⁺ does not depolarize cells: even in the presence of 155mM K⁺ the potential is –41 mV; membrane potential is also insensitive to the chemical gradient of Na⁺,Mg²⁺, Ca²⁺ or Cl^–. Ouabain depolarizes the cells; H⁺ efflux from cells is stimulated by extracellular Na⁺. We propose that in Lettré cells the plasma membrane potential is generated by electrogenic cation pumps. The balancing fluxes of Na⁺ and K⁺ are mainly through electroneutral cation exchanges (Na⁺/K⁺ and Na⁺/H⁺) and the magnitude of the potential is limited by organic anion leaks. Such a mechanism may operate in other biological membranes also.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Calcium-Binding Properties of the Mitochondrial Channel-Forming Hydrophobic Component

A hydrophobic, low-molecular weight component extracted from mitochondria forms aCa²⁺-activated ion channel in black-lipid membranes (Mironova et al., 1997). At pH 8.3–8.5, thecomponent has a high-affinity binding site for Ca²⁺ with a K_d of 8 × 10^–6 M, while at pH7.5 this K_d was decreased to 9 × 10^–5 M. B_max for the Ca²⁺-binding site did not changesignificantly with pH. In the range studied, 0.2 ± 0.06 mmol Ca²⁺/g component were boundor one calcium ion to eight molecules of the component. The Ca²⁺ binding was stronglydecreased by 50–100 mM Na⁺, but not by K⁺. Treatment of mitochondria withCaCl₂ priorto ethanolic extraction resulted in a high level of Ca²⁺-binding capacity of the partially purifiedcomponent. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition,when added to the mitochondrial suspension, decreased the Ca²⁺-binding activity of thepurified extract severalfold. The calcium-binding capability of the partially purified componentcorrelates with its calcium-channel activity. This indicates that the channel-forming componentmight be involved in the permeability transition that stimulates its formation.     

Ouabain-induced perturbations in intracellular ionic homeostasis regulate death receptor-mediated apoptosis

Apoptosis is defined by specific morphological and biochemical characteristics including cell shrinkage (termed apoptotic volume decrease), a process that results from the regulation of ion channels and plasma membrane transporter activity. The Na⁺–K⁺-ATPase is the predominant pump that controls cell volume and plasma membrane potential in cells and alterations in its function have been suggested to be associated with apoptosis. We report here that the Na⁺–K⁺-ATPase inhibitor ouabain, potentiates apoptosis in the human lymphoma Jurkat cells exposed to Fas ligand (FasL) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not other apoptotic agents such as H₂O₂, thapsigargin or UV-C implicating a role for the Na⁺–K⁺-ATPase in death receptor-induced apoptosis. Interestingly, ouabain also potentiated perturbations in cell Ca²⁺ homeostasis only in conjunction with the apoptotic inducer FasL but not TRAIL. Ouabain did not affect alterations in the intracellular Ca²⁺ levels in response to H₂O₂, thapsigargin or UV-C. FasL-induced alterations in Ca²⁺ were not abolished in Ca²⁺-free medium but incubation of cells with BAPTA-AM inhibited both Ca²⁺ perturbations and the ouabain-induced potentiation of FasL-induced apoptosis. Our data suggest that the impairment of the Na⁺–K⁺-ATPase activity during apoptosis is linked to perturbations in cell Ca²⁺ homeostasis that modulate apoptosis induced by the activation of Fas by FasL.     

Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion

Summary In the presence of inhibitors for mitochondrial H⁺-ATPase, (Na⁺+K⁺)- and Ca²⁺-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane (ecto-ATPases). These ATPases accept several nucleotides, are stimulated by Ca²⁺ and Mg²⁺, and are inhibited by N,N-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brushborder and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg²⁺ and Mn²⁺, but not by Ca²⁺, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator, ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H⁺ secretion is electrogenic and requires either exit of a permeant anion (Cl^–) or entry of a cation, e.g., Na⁺ via electrogenic Na⁺/d-glucose and Na⁺/l-phenylalanine uptake. In the presence of Na⁺, ATP-driven H⁺ efflux is stimulated by blocking the Na⁺/H⁺ exchanger with amiloride. These data prove the coexistence of Na⁺-coupled substrate transporters, Na⁺/H⁺ exchanger, and an ATP-driven H⁺ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H⁺ pumps with (a part of) the DCCD- and NEM-sensitive ATPases at the cytosolic side of the brush-border membrane.     

Extracellular calcium depletion transiently elevates oxygen consumption in neurosecretory PC12 cells through activation of mitochondrial Na/Ca exchange

Fluctuating extracellular Ca²⁺ regulates many aspects of neuronal (patho)physiology including cell metabolism and respiration. Using fluorescence-based intracellular oxygen sensing technique, we demonstrate that depletion of extracellular Ca²⁺ from 1.8 to ≤ 0.6 mM by chelation with EGTA induces a marked spike in O₂ consumption in differentiated PC12 cells. This respiratory response is associated with the reduction in cytosolic and mitochondrial Ca²⁺, minor depolarization on the mitochondrial membrane, moderate depolarization of plasma membrane, and no changes in NAD(P)H and ATP. The response is linked to the influx of extracellular Na⁺ and the subsequent activation of mitochondrial Na⁺/Ca²⁺ and Na⁺/H⁺ exchange. The mitochondrial Na⁺/Ca²⁺ exchanger (_mNCX) activated by Na⁺ influx reduces Ca²⁺ and increases Na⁺ levels in the mitochondrial matrix. The excess of Na⁺ activates the mitochondrial Na⁺/H⁺ exchanger (NHE) increasing the outward pumping of protons, electron transport and O₂ consumption. Reduction in extracellular Na⁺ and inhibition of Na⁺ influx through the receptor operated calcium channels and plasmalemmal NHE reduce the respiratory response. Inhibition of the _mNCX, L-type voltage gated Ca²⁺ channels or the release of Ca²⁺ from the endoplasmic reticulum also reduces the respiratory spike, indicating that unimpaired intercompartmental Ca²⁺ exchange is critical for response development.