Entamoeba histolytica: localization and characterization of ca2+-dependent nucleotidases

An activity of Ca²⁺-dependent nucleotidase was detected in axenically-cultivated trophozoites of Entamoeba histolytica. The enzyme was concentrated by differential and sucrose density gradient centrifugation and catalyzed hydrolysis of nucleoside tri- and diphosphates and also thiamine pyrophosphate. Hydrolysis of nucleoside mono-phosphates was not affected by Ca²⁺. Among substrates tested, ATP was most active. Addition of Zn²⁺ or heat treatment almost abolished the enzyme activity. The enzyme exhibited almost the identical activity at acid and neutral pH. Among 6 bands isolated by polyacrylamide gel electrophoresis, 4 were stained with ATP, UTP, CTP and ADP, whereas the other 2 were stained only with ATP, UTP and CTP. The concentrated enzyme preparation, primarily composed of membrane fragments, also had activities of acid phosphatase, acid inorganic pyrophosphatase, 5'-nucleotidase and Mg²⁺-dependent ATPase. These observations suggest that E. histolytica has 2 Ca²⁺-dependent nucleotidases, i.e. one Ca²⁺-dependent ATPase and the other Ca²⁺-dependent nucleoside diphosphatase or an apyrase-like enzyme, and that these nucleotidases are at least partially associated with the plasma membrane or an organelle of lysosomal nature in this parasite.     

Localisation of thiamine pyrophosphatase within the cytoplasmic fine structure of trophozoites of Entamoeba histolytica and E. invadens

McCaul T.F. and Bird R.G. 1978. Localisation of thiamine pyrophosphatase within the cytoplasmic fine structure of trophozoites of Entamoeba histolytica and E. invadens. International Journal for Parasitology8: 501–506. The distribution of thiamine pyrophosphatase (TPPase) activity was studied in both formaldehyde and glutaraldehyde fixed trophozoites of Entamoeba histolytica and E. invadens. The activity was localised within certain vacuoles. No dense deposits for TPPase activity were seen in the small vesicles, elongated smooth-walled lacunae equated with endoplasmic reticulum, or the nucleus. The demonstration of small vesicles surrounding the larger vacuoles indicated that the Golgi-like vacuoles might be involved in the production of cell coat materials and primary lysosomes.     

Evidence for a plasma membrane calcium pump in bovine adrenal medulla but not adrenal cortex

Continuous sucrose density gradient subfractions from bovine adrenal medullary microsomes were found to accumulate ⁴⁵Ca²⁺ in the presence of ATP and ammonium oxalate mainly in subfractions of intermediate density. (Na⁺ + K⁺)-ATPase (plasma membrane marker) and Ca²⁺-ATPase activities were also concentrated in these intermediate subfractions but thiamine pyrophosphatase (Golgi apparatus marker) was not. NADH oxidase (endoplasmic reticulum marker) activity was distributed throughout all subfractions.⁴⁵Ca²⁺ accumulation in adrenal cortical microsomes was found to rise and fall in parallel with thiamine pyrophosphatase but not with (Na⁺ + K⁺)-ATPase or NADH oxidase activities.Accumulation of ⁴⁵Ca²⁺ in membrane vesicles in these experiments suggests the existence of a calcium transfer mechanism in plasma membranes of the adrenal medulla but not adrenal cortex.     

Entamoeba histolytica: Soluble and membrane-associated neutral sphingomyelinase-C and other unidentified esterase activity

Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5-96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg²⁺, Mn²⁺, or Co²⁺, and inhibited by Zn²⁺, Hg²⁺, Ca²⁺, and EDTA. SMase-C was active in the pH range of 3-10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence.     

Characterisation of a Ca2+-dependent ATP hydrolysis associated with the vacuolar membrane of wheat roots

Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca²⁺ than Mg²⁺. Here we characterise the Ca²⁺-dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca²⁺-dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co-migrates with the major peak of nitrate-inhibited Mg²⁺. Cl-ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca²⁺-dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca²⁺ on the plasma membrane K⁺, Mg²⁺-ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca²⁺-dependent activity is stimulated by free Ca²⁺ with a K_m of 8.1 μM in the absence of Mg²⁺ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca²⁺-dependent than Mg²⁺-dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion-dependent activities in vacuolar membrane-enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca²⁺ and Mg²⁺ The Ca²⁺-dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg²⁺-dependent activity functioned as a bafilomycin sensitive H⁺-pump in vacuolar membrane vesicles. Ca²⁺-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K⁺. Possible functions of Ca²⁺-dependent hydrolysis as a H⁺-pump or a Ca²⁺-pump was investigated using vacuolar membrane vesicles. No H⁺ or Ca²⁺ translocating activity was observed under conditions when the Ca²⁺-dependent ATP hydrolysis was active.     

Inorganic pyrophosphatase and nucleoside diphosphatase in the parasitic protozoon, Entamoeba histolytica.

Entamoeba histolytica contains two acid pyrophosphatases. One is an inorganic pyrophosphatase with a relatively high K_m ( ? 1 mM) and no cation requirement. The other is a nucleoside diphosphatase with a relatively low K_m ( ? 50 μM) and Ca²⁺ requirement. No Mg²⁺ dependent neutral or alkaline inorganic pyrophosphatase is present. The pyrophosphatases are localized in subcellular particles, display structure-linked latency and are tightly bound to membranes.     

Zn2+-Dependent Acid Inorganic Pyrophosphatase Activity as Related to Other Pyrophosphatases in Oryza sativa

Acid inorganic pyrophosphatase on the one hand, and Mg²⁺-dependent alkaline inorganic pyrophosphatase and Zn²⁺-dependent acid inorganic pyrophosphatase on the other hand showed opposite trends in their activities in rice (Oryza sativa L. cv. Ratna) seedlings grown in dark and sun. The opposite trends in their activities were also noted in rice seedlings grown from gamma-irradiated seeds and in detached rice leaves floated on water in dark. The ratios of Mg²⁺ dependent alkaline inorganic pyrophosphatase/acid inorganic pyrophosphatase and Zn²⁺-dependent acid inorganic pyrophosphatase/acid inorganic pyrophosphatase changed significantly in response to the above physical treatments, but the ratio of Mg²⁺ dependent alkaline inorganic pyrophosphatase/Zn²⁺ dependent acid inorganic pyrophosphatase remained relatively stable. The conclusion is that Zn²⁺-dependent acid inorganic pyrophosphatase activity is the same as that of Mg²⁺-dependent alkaline inorganic pyrophosphatase and is different from that of acid inorganic pyrophosphatase, which requires no metal ion for activity. The acid and alkaline inorganic pyrophosphatase activities are due to separate enzyme proteins.     

Alkaline and acid phosphatase in the intestines of rats infected with a virulent strain of Entamoeba histolytica and treated with emetine and Flagyl

The distribution patterns of alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2) in the intestine of rats inoculated intracaecally with a virulent strain of Entamoeba histolytica and treated with emetine hydrochloride and metronidazole (Flagyl) were studied. The caecum and the large intestine showed a highly significant increase in alkaline phosphatase activity after amoebic inoculation, and the enhanced activity was lowered by emetine and Flagyl treatment. There was no significant increase in acid phosphatase activity either in the caecum and the large intestine or in the small intestine (ileocaecal end). Intracaecal inoculation of bacterial associates alone from E. histolytica cultures did not produce any significant change in the level of these enzymes in the intestine.     

Channel Formation by Yeast F-ATP Synthase and the Role of Dimerization in the Mitochondrial Permeability Transition

Purified F-ATP synthase dimers of yeast mitochondria display Ca²⁺-dependent channel activity with properties resembling those of the permeability transition pore (PTP) of mammals. After treatment with the Ca²⁺ ionophore ETH129, which allows electrophoretic Ca²⁺ uptake, isolated yeast mitochondria undergo inner membrane permeabilization due to PTP opening. Yeast mutant strains ΔTIM11 and ΔATP20 (lacking the e and g F-ATP synthase subunits, respectively, which are necessary for dimer formation) display a striking resistance to PTP opening. These results show that the yeast PTP originates from F-ATP synthase and indicate that dimerization is required for pore formation in situ.     

Ca- and Mg-dependent association of phosphorylase kinase with human erythrocyte membranes

The interaction of rabbit muscle phosphorylase kinase (EC 2.7.1.38) with human erythrocyte membranes was investigated. It was found that at pH 7.0 the kinase binds to the inner face of the erythrocyte membrane (inside-out vesicles) and that this binding is Ca²⁺- and Mg²⁺-dependent. The sharpest increase in the binding reaction occurs at concentrations between 70 and 550 nM free Ca²⁺. Erythrocyte ghost or right-side out erythrocyte vesicles showed a significantly lower capacity to interact with phosphorylase kinase. Autophosphorylated phosphorylase kinase shows a similar Ca²⁺-dependent binding profile, while trypsin activation of the kinase and calmodulin decrease the original binding capacity by about 50%. Heparin (200 μg/ml) and high ionic strength (50 mM NaCl) almost completely blocks enzyme-membrane interaction; glycogen does not affect the interaction.     

Membrane-bound ATPase in chloroplasts of Euglena gracilis

Membrane-bound ATPase activities in chloroplasts of Euglena were examined. Ca²⁺- and Mg²⁺-dependent activities were relatively high in membrane preparations and could not be further activated by a number of procedures. The enzyme was found to be highly specific for purine nucleotides and was inhibited by the usual inhibitors of photophosphorylation. K_m values of Ca²⁺ and Mg²⁺ ATPase for ATP were 2.5 and 2.1 mM, respectively. Both activities were competitively inhibited by ADP and inorganic phosphate. A relationship was found between Ca²⁺- or Mg²⁺-dependent ATPase activities and chloroplast completeness. The possibilities that these activities result from one enzyme depending on Ca²⁺ or Mg²⁺ or from two different enzymes are discussed.     

Comparative study of Y<Superscript>3+</Superscript> effect on calcium-dependent processes in frog cardiac muscle and mitochondria of rat cardiomyocytes

Inotropic effects of yttrium acetate (Y³⁺) on contractions of myocardium preparations of the frog Rana ridibunda, as well as on respiration and the inner membrane potential (ΔΨ_mito) of isolated rat heart mitochondria were studied. 2 mM yttrium in Ringer solution was found to significantly reduce the amplitude of myocardium contractions, evoked by electric stimulation, and increase the half-relaxation time (n = 5). In experiments with Ca²⁺, Y³⁺ decreased the Ca²⁺-dependent basal respiration rate in rat heart mitochondria, energized by glutamate and malate, impeded the reduction in respiration of these mitochondria operating in state 3 after Chance or uncoupled by 2,4-dinitrophenol, and inhibited a Ca²⁺-induced reduction in their inner membrane potential. The data obtained are important for better understanding the mechanism underlying Y³⁺ effects on the myocardial Ca²⁺-dependent processes. Possible mechanisms of the negative inotropic effect of Y³⁺ on myocardium and its influence on the Ca²⁺-dependent processes in rat mitochondria are discussed.     

Protons and calcium modulate SV-type channels in the vacuolar-lysosomal compartment — channel interaction with calmodulin inhibitors

Slowly activating vacuolar (SV-type; Hedrich and Neher 1987, Nature 329: 833–835) ion channels provide the predominant membrane conductance of the vacuolar-lysosomal compartment of Vicia faba L. guard cells and sugar beet (Beta vulgaris L.) taproots. Applying the patch-clamp technique to isolated vacuoles of both tissues, the electrical and pharmacological properties of guard-cell SV-type currents were studied and compared to the sugar beet channel with regard to its modulation by cytoplasmic Ca²⁺ and pH. This outward rectifier of V. faba guard cells showed a half-maximum activation at 55–60 mV with an apparent gating charge equivalent of z ≈ 4. Studies on the single-channel and whole-vacuole level revealed an extremely high conductance of 280 pS for the guard-cell channels at a mean density of 0.37 μm^-2 compared to taproots (120–140 pS at about 0.16 channels per μm²). Guard-cell SV-type channels are weakly selective for cations over anions and lack saturation at KC1 concentrations of up to 1 M. Since in the absence of physiological K⁺ concentrations, Ca²⁺ is the major permeable ion, relative changes in the amounts of the two ions might control the permeation process. In spite of their different origins and physiological functions, in guard cells and beet taproot cells, cytoplasmic Ca²⁺ and protons, both considered as candidates for intracellular signalling in plants, modulate the voltage dependence of SV-type channels. While the two effectors do not alter the single-channel conductance, they strongly interact with the voltage sensor. The calmodulin (CaM) antagonists N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide hydrochloride (W-7), trifluoperazine (TFP) and calmidazolium hydrochloride (R 24571) effectively blocked the channel in an antagonist-specific manner. In agreement with the properties of a Ca²⁺-permeable channel, CaM could be involved in the modulation of the activation threshold of the SV-type channel. We therefore conclude that guard-cell SV-type channels, which might be responsible for the release of K⁺, Cl^- and to a smaller extent Ca²⁺ during stomatal closure, could serve as an intracellular sensor for changes in cytosolic calcium (calcium-CaM) and pH.     

Dependence of membrane potential on Ca2+ transport in cultured cytotrophoblasts of human immature placentas

The electrical membrane properties of cultured human cytotrophoblast were examined by means of a standard electrophysiological technique. The mean values of the membrane potential (E_m) and the membrane resistance in a physiological medium were around ?49 mV and 12 MΩ, respectively. The membrane potential was dependent, to a large extent, on the external Ca²⁺ concentration ([Ca²⁺]₀). Deprivation of external Ca²⁺ reduced membrane potential to about ?20 mV, and an increase in [Ca²⁺]₀ caused a hyperpolarization in a saturable manner. The Ca²⁺-dependency of membrane potential was affected remarkably by [K⁺]₀, but not by [Na⁺]₀ or [Cl^?]₀. The intracellular Ca²⁺ injection hyperpolarized the membrane in a Ca²⁺-free medium. A Ca²⁺ channel blocker, verapamil, completely abolished the Ca²⁺-dependent E_m. The Ca²⁺-dependent E_m was also suppressed by cooling or by the application of metabolic inhibitors. It is suggested that the Ca²⁺-dependent E_m in cultured human cytotrophoblast is caused by a Ca²⁺ influx which, in turn, increases the K⁺ conductance of the cell membrane, presumably due to stimulation of Ca²⁺-activated K⁺ channel.     

Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Modulation of L-type Ca²⁺ channels by tonic elevation of cytoplasmic Ca²⁺ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba²⁺ was used as charge carrier, and run down of Ca²⁺ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca²⁺ in intact cells by elevation of extracellular Ca²⁺ in the presence of the ionophore A23187 inhibited the activity of L-type Ca²⁺ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca²⁺ with fura-2 revealed a 50% inhibitory concentration (IC₅₀) of 260 nM and a Hill coefficient close to 4 for Ca²⁺- dependent inhibition. Ca²⁺-induced inhibition of Ca²⁺ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca²⁺-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca²⁺ at the cytoplasmic side of inside-out patches inhibited Ca²⁺ channels with an IC₅₀ of 2 μM and a Hill coefficient close to unity. Direct Ca²⁺-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca²⁺ concentration of 1 μM inhibited Ca²⁺ channel open probability and availability. Elevation of cytoplasmic Ca²⁺ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC₅₀ of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca²⁺ sensitivity of Ca²⁺ channels in intact cells. Our results suggest that L-type Ca²⁺ channels of smooth muscle are controlled by two Ca²⁺-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca²⁺ with a single membrane-associated site that may reside on the channel protein itself.     

A high affinity,calmodulin-responsive (Ca2+ + Mg2+)-ATPase in isolated bone cells

Although acute alterations in Ca²⁺ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca²⁺-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca²⁺-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca²⁺ of 280 nM, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg²⁺ could replace Ca²⁺ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg²⁺ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5′-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca²⁺-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca²⁺-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca²⁺ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function.     

Escherichia coli lacking the AcrAB multidrug efflux pump also lacks nonproteinaceous, PHB-polyphosphate Ca channels in the membrane

PHB(polyP) complexes bind calcium and form calcium channels in the cytoplasmic membrane in Escherichia coli and are likely to be important in Ca²⁺ homeostasis in this organism. E. coli N43, which lacks the AcrA component of a major multidrug resistance pump, was shown to be defective in calcium handling, with an inability to maintain submicromolar levels of free Ca²⁺ in the cytoplasm. Therefore, using an N-phenyl-1-napthylamine (NPN)-dependent fluorescence assay, we measured temperature-dependent phase transitions in the membranes of intact cells. These transitions specifically depend on the presence of PHB(Ca²⁺polyP) complexes. PHB(Ca²⁺polyP) channel complexes, particularly in stationary phase cultures, were detected in wild-type strains; however, in contrast, isogenic acrA⁻ strains had greatly reduced amounts of the complexes. This indicates that the AcrAB transporter may have a novel, hitherto undetected physiological role, either directly in the membrane assembly of the PHB complexes or the transport of a component of the membrane, which is essential for assembly of the complexes into the membrane. In other experiments, we showed that the particular defective calcium handling detected in N43 was not due to the absence of AcrA but to other unknown factors in this strain.     

Properties of enzyme activities involved in protein phosphorylatino-dephosphorylation of the thyroid plasma membranes

Bovine thyroid tissue exhibited cAMP-dependent and Ca²⁺-dependent protien kinase activities as well as a basal (cAMP- and Ca²⁺-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kiniase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent K_m values of cAMP and Ca²⁺ for the membrane-bound protein kinase were 5·10^?8 M and 8.3·10^?4M (in the presence of 1 mM EGTA), respectively. The apparent K_m values of Mg²⁺ were 7·10^?4 M (without cAMP and Ca²⁺, 5·10^?4 M (with cAMP) and 1.3·10^?3 M (with Ca²⁺), and those ATP were 3.5·10^?5 M (with or without cAMP) and 8.5·10^?5 M (with Ca²⁺). The Ca²⁺-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using ³²P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca²⁺, but was sitmulated by a rather broad range (5–25 mM) of Mg²⁺ and Mn²⁺. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca²⁺-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli.     

Cloning,Expression, and Characterization of the Squid Na+–Ca2+ Exchanger (NCX-SQ1)

We have cloned the squid neuronal Na⁺–Ca²⁺ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na⁺–Ca²⁺ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca²⁺ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na⁺-dependent ⁴⁵Ca²⁺ uptake; the uptake was inhibited by injection of Ca²⁺ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na⁺-dependent inactivation, secondary activation by cytoplasmic Ca²⁺, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATPγS, in the presence of F⁻ (0.2 mM) and vanadate (50 μM), and both effects reversed on application of a phosphatidylinositol-4′,5′-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATPγS. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4′,5′-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5–10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na⁺ and Ca²⁺ transport reactions. Outward current transients associated with Na⁺ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca²⁺ extrusion were much larger. For NCX-SQ1, charge movements of Ca²⁺ transport could be defined in voltage jump experiments with a low cytoplasmic Ca²⁺ (2 μM) in the presence of high extracellular Ca²⁺ (4 mM). The rates of charge movements showed “U”-shaped dependence on voltage, and the slopes of both charge–voltage and rate–voltage relations (1,600 s⁻¹ at 0 mV) indicated an apparent valency of −0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.