Antimony-Phosphomolybdate ATPase Assay.

Hydrolytic activity is an important functional parameter of enzymes like adenosinetriphosphatases (ATPases). It is measured to test enzyme functionality, but it also provides useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. Here, we describe a molybdenum-based protocol that makes use of potassium antimony (III) oxide tartrate and may be valuable in biochemical and biomedical investigations of ATPase enzymes as well as in high-throughput drug screening. This method has been successfully applied to native and recombinant ATPases.

Electrophysiological Measurements on Solid Supported Membranes.

The solid supported membrane (SSM) represents a convenient model system for a biological membrane with the advantage of being mechanically so stable that solutions can be rapidly exchanged at the surface. The SSM consists of a hybrid alkanethiol-phospholipid bilayer supported by a gold electrode. Proteoliposomes, membrane vesicles, or membrane fragments containing the transport protein of interest are adsorbed on the SSM surface and are subjected to a rapid substrate concentration jump. The substrate concentration jump activates the protein and the charge displacement concomitant with its transport activity is recorded as a current transient. Since this technique is well suited for the functional characterization of electrogenic membrane transporters, it is expected to become a promising platform technology for drug screening and development.

Anticancer ruthenium(III) complex KP1019 interferes with ATP-dependent Ca2+ translocation by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA).

Sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), a P-type ATPase that sustains Ca2+ transport and plays a major role in intracellular Ca2+ homeostasis, represents a therapeutic target for cancer therapy. Here, we investigated whether ruthenium-based anticancer drugs, namely KP1019 (indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)]), NAMI-A (imidazolium [trans-tetrachloro(1H-imidazole)(S-dimethylsulfoxide)ruthenate(III)]) and RAPTA-C ([Ru(η6-p-cymene)dichloro(1,3,5-triaza-7-phosphaadamantane)]), and cisplatin (cis-diammineplatinum(II) dichloride) might act as inhibitors of SERCA. Charge displacement by SERCA adsorbed on a solid-supported membrane was measured after ATP or Ca2+ concentration jumps. Our results show that KP1019, in contrast to the other metal compounds, is able to interfere with ATP-dependent translocation of Ca2+ ions. An IC50 value of 1 µM was determined for inhibition of calcium translocation by KP1019. Conversely, it appears that KP1019 does not significantly affect Ca2+ binding to the ATPase from the cytoplasmic side. Inhibition of SERCA at pharmacologically relevant concentrations may represent a crucial aspect in the overall pharmacological and toxicological profile of KP1019.

Translocation of platinum anticancer drugs by human copper ATPases ATP7A and ATP7B.

Cisplatin, carboplatin, and oxaliplatin are widely used anticancer drugs. Their efficacy is strongly reduced by development of cell resistance. Down-regulation of CTR1 and up-regulation of the Cu-ATPases, ATP7A and ATP7B, have been associated to augmented drug resistance. To gain information on translocation of Pt drugs by human Cu-ATPases, we performed electrical measurements on the COS-1 cell microsomal fraction, enriched with recombinant ATP7A, ATP7B, and selected mutants, and adsorbed on a solid supported membrane. The experimental results indicate that Pt drugs activate Cu-ATPases and undergo ATP-dependent translocation in a fashion similar to that of Cu. We then used NMR spectroscopy and ESI-MS to determine the binding mode of these drugs to the first N-terminal metal-binding domain of ATP7A (Mnk1).

Istaroxime stimulates SERCA2a and accelerates calcium cycling in heart failure by relieving phospholamban inhibition.

BACKGROUND AND PURPOSE: Calcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca(2) (+) cycling may represent a promising approach to heart failure therapy. Istaroxime is a new luso-inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na-K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity. EXPERIMENTAL APPROACH: To study the effect of istaroxime on SERCA2a-phospholamban (PLB) complex, we applied different methodologies in native dog healthy and failing heart preparations and heterologous canine SERCA2a/PLB co-expressed in Spodoptera frugiperda (Sf21) insect cells. KEY RESULTS: We showed that istaroxime enhances SERCA2a activity, Ca(2) (+) uptake and the Ca(2) (+) -dependent charge movements into dog healthy and failing cardiac SR vesicles. Although not directly demonstrated, the most probable explanation of these activities is the displacement of PLB from SERCA2a.E2 conformation, independently from cAMP/PKA. We propose that this displacement may favour the SERCA2a conformational transition from E2 to E1, thus resulting in the acceleration of Ca(2) (+) cycling. CONCLUSIONS AND IMPLICATIONS: Istaroxime represents the first example of a small molecule that exerts a luso-inotropic effect in the failing human heart through the stimulation of SERCA2a ATPase activity and the enhancement of Ca(2) (+) uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way.

A method to measure hydrolytic activity of adenosinetriphosphatases (ATPases).

The detection of small amounts (nanomoles) of inorganic phosphate has a great interest in biochemistry. In particular, phosphate detection is useful to evaluate the rate of hydrolysis of phosphatases, that are enzymes able to remove phosphate from their substrate by hydrolytic cleavage. The hydrolysis rate is correlated to enzyme activity, an extremely important functional parameter. Among phosphatases there are the cation transporting adenosinetriphosphatases (ATPases), that produce inorganic phosphate by cleavage of the γ-phosphate of ATP. These membrane transporters have many fundamental physiological roles and are emerging as potential drug targets. ATPase hydrolytic activity is measured to test enzyme functionality, but it also provides useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. We have optimized a molybdenum-based protocol that makes use of potassium antimony (III) oxide tartrate (originally employed for phosphate detection in environmental analysis) to allow its use with phosphatase enzymes. In particular, the method was successfully applied to native and recombinant ATPases to demonstrate its reliability, validity, sensitivity and versatility. Our method introduces significant improvements to well-established experimental assays, which are currently employed for ATPase activity measurements. Therefore, it may be valuable in biochemical and biomedical investigations of ATPase enzymes, in combination with more specific tests, as well as in high throughput drug screening.

Mimicking the intramolecular hydrogen bond: synthesis, biological evaluation, and molecular modeling of benzoxazines and quinazolines as potential antimalarial agents.

The intramolecular hydrogen bond formed between a protonated amine and a neighboring H-bond acceptor group in the side chain of amodiaquine and isoquine is thought to play an important role in their antimalarial activities. Here we describe isoquine-based compounds in which the intramolecular H-bond is mimicked by a methylene linker. The antimalarial activities of the resulting benzoxazines, their isosteric tetrahydroquinazoline derivatives, and febrifugine-based 1,3-quinazolin-4-ones were examined in vitro (against Plasmodium falciparum ) and in vivo (against Plasmodium berghei ). Compounds 6b,c caused modest inhibition of chloroquine transport via the parasite's "chloroquine resistance transporter" (PfCRT) in a Xenopus laevis oocyte expression system. In silico predictions and experimental evaluation of selected drug-like properties were also performed on compounds 6b,c. Compound 6c emerged from this work as the most promising analogue of the series; it possessed low toxicity and good antimalarial activity when administered orally to P. berghei -infected mice.

Distinctive features of catalytic and transport mechanisms in mammalian sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases.

Ca(2+) (sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA)) and Cu(+) (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca(2+), demonstrated by the addition of ATP and Ca(2+) to SERCA deprived of Ca(2+) (E2) as compared with ATP to Ca(2+)-activated enzyme (E1·2Ca(2+)). Activation by Ca(2+) is slower at low pH (2H(+)·E2 to E1·2Ca(2+)) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca(2+) translocation. A "H(+)-gated pathway," demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca(2+) release by H(+)/Ca(2+) exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu(+)/H(+) exchange. As opposed to SERCA after Ca(2+) chelation, ATP7A/B does not undergo reverse phosphorylation with P(i) after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms.

The Ca2+-ATPase (SERCA1) is inhibited by 4-aminoquinoline derivatives through interference with catalytic activation by Ca2+, whereas the ATPase E2 state remains functional.

Several clotrimazole (CLT) and 4-aminoquinoline derivatives were synthesized and found to exhibit in vitro antiplasmodial activity with IC(50) ranging from nm to µm values. We report here that some of these compounds produce inhibition of rabbit sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) with IC(50) values in the µm range. The highest affinity for the Ca(2+)-ATPase was observed with NF1442 (N-((3-chlorophenyl)(4-((4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl)phenyl)methyl)-7-chloro-4-aminoquinoline) and NF1058 (N-((3-chlorophenyl)(4-(pyrrolidin-1-ylmethyl)phenyl)methyl)-7-chloro-4-aminoquinoline),yielding IC(50) values of 1.3 and 8.0 µm as demonstrated by measurements of steady state ATPase activity as well as single cycle charge transfer. Characterization of sequential reactions comprising the ATPase catalytic and transport cycle then demonstrated that NF1058, and similarly CLT, interferes with the mechanism of Ca(2+) binding and Ca(2+)-dependent enzyme activation (E(2) to E(1)·Ca(2) transition) required for formation of phosphorylated intermediate by ATP utilization. On the other hand, Ca(2+) independent phosphoenzyme formation by utilization of P(i) (i.e. reverse of the hydrolytic reaction in the absence of Ca(2+)) was not inhibited by NF1058 or CLT. Comparative experiments showed that the high affinity inhibitor thapsigargin interferes not only with Ca(2+) binding and phosphoenzyme formation with ATP but also with phosphoenzyme formation by utilization of P(i) even though this reaction does not require Ca(2+). It is concluded that NF1058 and CLT inhibit SERCA by stabilization of an E(2) state that, as opposed to that obtained with thapsigargin, retains the functional ability to form E(2)-P by reacting with P(i).

ATP dependent charge movement in ATP7B Cu+-ATPase is demonstrated by pre-steady state electrical measurements.

ATP7B is a copper dependent P-type ATPase, required for copper homeostasis. Taking advantage of high yield heterologous expression of recombinant protein, we investigated charge transfer in ATP7B. We detected charge displacement within a single catalytic cycle upon ATP addition and formation of phosphoenzyme intermediate. We attribute this charge displacement to movement of bound copper within ATP7B. Based on specific mutations, we demonstrate that enzyme activation by copper requires occupancy of a site in the N-terminus extension which is not present in other transport ATPases, as well as of a transmembrane site corresponding to the cation binding site of other ATPases.

Confining the sodium pump in a phosphoenzyme form: the effect of lead(II) ions.

The effect of Pb(2+) ions on the Na(+),K(+)-ATPase was investigated in detail by means of steady-state fluorescence spectroscopy. Experiments were performed by using the electrochromic styryl dye RH421. It is shown that Pb(2+) ions can bind reversibly to the protein and do not affect the Na(+) and K(+) binding affinities in the E(1) and P-E(2) conformations of the enzyme. The pH titrations indicate that lead(II) favors binding of one H(+) to the P-E(2) conformation in the absence of K(+). A model scheme is proposed that accounts for the experimental results obtained for backdoor phosphorylation of the enzyme in the presence of Pb(2+) ions. Taken together, our results clearly indicate that Pb(2+) bound to the enzyme stabilizes an E(2)-type conformation. In particular, under conditions that promote enzyme phosphorylation, Pb(2+) ions are able to confine the Na(+),K(+)-ATPase into a phosphorylated E(2) state.

Inhibitory effect of Pb2+ on the transport cycle of the Na+,K+-ATPase.

The effect of Pb(2+) on the transport cycle of the Na(+),K(+)-ATPase was characterized in detail at a molecular level by combining electrical and biochemical measurements. Electrical measurements were performed by adsorbing purified membrane fragments containing Na(+),K(+)-ATPase on a solid-supported membrane. Upon adsorption, the Na(+),K(+)-ATPase was activated by carrying out concentration jumps of different activating substrates, for example, Na(+) and ATP. Charge movements following Na(+),K(+)-ATPase activation were measured in the presence of various Pb(2+) concentrations to investigate the effect of Pb(2+) on different ion translocating steps of the pump cycle. These charge measurements were then compared to biochemical measurements of ATPase activity in the presence of increasing Pb(2+) concentration. Our results indicate that Pb(2+) inhibits cycling of the enzyme, but it does not affect cytoplasmic Na(+) binding and release of Na(+) ions at the extracellular side at concentrations below 10 muM. To explain the inhibitory effect of Pb(2+) on the Na(+),K(+)-ATPase, we propose that Pb(2+) may interfere with the hydrolytic cleavage of the phosphorylated intermediate E(2)P, which occurs in the K(+)-related branch of the pump cycle.

High-yield heterologous expression of wild type and mutant Ca(2+) ATPase: Characterization of Ca(2+) binding sites by charge transfer.

High-yield heterologous SERCA1 (Ca(2+) ATPase) expression was obtained in COS-1 cells infected with recombinant adenovirus vector (rAdSERCA). Higher transcription and expression were obtained in the presence of a His(6) tag at the amino terminus, as compared with a His(6) tag at the carboxyl SERCA terminus, or no tag. The expressed protein was targeted extensively to intracellular membranes. Optimal yield of functional Ca(2+) ATPase corresponded to 10% of total protein, with phosphoenzyme levels, catalytic turnover and Ca(2+) transport identical with those of native SERCA1. This recombinant membrane-bound (detergent-free) enzyme was used for characterization of Ca(2+) binding at the two specific transmembrane sites (ATP-free) by measurements of net charge transfer upon Ca(2+) binding to the protein, yielding cooperative isotherms (K(1)=5.9+/-0.5x10(5) M(-1) and K(2)=5.7+/-0.3x10(6) M(-1)). Non-cooperative binding of only one Ca(2+), and loss of ATPase activation, were observed following E309 mutation at site II. On the other hand, as a consequence of the site II mutation, the affinity of site I for Ca(2+) was increased (K=4.4+/-0.2x10(6) M(-1)). This change was due to a pK(a) shift of site I acidic residues, and to contributions of oxygen functions from empty site II to Ca(2+) binding at site I. No charge movement was observed following E771Q mutation at site I, indicating no Ca(2+) binding to either site. Therefore, calcium occupancy of site I is required to trigger cooperative binding to site II and catalytic activation. In the presence of millimolar Mg(2+), the charge movement upon addition of Ca(2+) to WT ATPase was reduced by 50%, while it was reduced by 90% when Ca(2+) was added to the E309Q/A mutants, demonstrating that competitive Mg(2+) binding can occur at site I but not at site II.

Effect of clotrimazole on the pump cycle of the Na,K-ATPase.

The effect of the antimycotic drug clotrimazole (CLT) on the Na,K-ATPase was investigated using fluorescence and electrical measurements. The results obtained by steady-state fluorescence experiments with the electrochromic styryl dye RH421 were combined with those achieved by a pre-steady-state method based on fast solution exchange on a solid supported membrane that adsorbs the protein. Both techniques are suitable for monitoring the electrogenic steps of the pump cycle and are in general complementary, yielding distinct kinetic information. The experiments show clearly that CLT affects specific partial reactions of the pump cycle of the Na,K-ATPase with an affinity in the low micromolar range and in a reversible manner. All results can be consistently explained by proposing the CLT-promoted formation of an ion-occluded-CLT-bound conformational E(2) state, E(2)(CLT)(X(2)) that acts as a "dead-end" side track of the pump cycle, where X stands for H+ or K+. Na+ binding, enzyme phosphorylation, and Na+ transport were not affected by CLT, and at high CLT concentrations approximately (1/3) of the enzyme remained active in the physiological transport mode. The presence of Na+ and K+ destabilized the inactivated form of the Na,K-ATPase.

Charge transfer in P-type ATPases investigated on planar membranes.

Planar lipid bilayers, e.g., black lipid membranes (BLM) and solid supported membranes (SSM), have been employed to investigate charge movements during the reaction cycle of P-type ATPases. The BLM/SSM method allows a direct measurement of the electrical currents generated by the cation transporter following chemical activation by a substrate concentration jump. The electrical current transients provides information about the reaction mechanism of the enzyme. In particular, the BLM/SSM technique allows identification of electrogenic steps which in turn may be used to localize ion translocation during the reaction cycle of the pump. In addition, using the high time resolution of the technique, especially when rapid activation via caged ATP is employed, rate constants of electrogenic and electroneutral steps can be determined. In the present review, we will discuss the main results obtained by the BLM and SSM methods and how they have contributed to unravel the transport mechanism of P-type ATPases.

Effects of high-affinity inhibitors on partial reactions, charge movements, and conformational States of the Ca2+ transport ATPase (sarco-endoplasmic reticulum Ca2+ ATPase).

The inhibitory effects of thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone, and 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene on the Ca(2+) ATPase were characterized by comparative measurements of sequential reactions of the catalytic and transport cycle, including biochemical measurements and detection of charge movements within a single cycle. In addition, patterns of ATPase proteolytic digestion with proteinase K were derived to follow conformational changes through the cycle or after inhibitor binding. We find that thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone inhibit Ca(2+) binding and catalytic activation as demonstrated with isotopic tracers and lack of charge movement upon addition of Ca(2+) in the absence of ATP. It has been shown previously that binding of these inhibitors requires the E2 conformational state of the ATPase, obtained in the absence of Ca(2+). We demonstrate here that E2 state conformational features are in fact induced by these inhibitors on the ATPase even in the presence of Ca(2+). The resulting dead-end complex interferes with progress of the catalytic and transport cycle. Inhibition by 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene, on the other hand, is related to interference with a conformational transition of the phosphorylated intermediate (E1 approximately P . 2Ca(2+) to E2-P . 2Ca(2+) transition), as demonstrated by increased phosphoenzyme levels and absence of bound Ca(2+) translocation upon addition of ATP. This transition includes large movements of ATPase headpiece domains and transmembrane segments, produced through utilization of ATP-free energy as the "conformational work" of the pump. We conclude that the mechanism of high-affinity Ca(2+) ATPase inhibitors is based on global effects on protein conformation that interfere with ATPase cycling.

Electrogenic steps of the SR Ca-ATPase enzymatic cycle and the effect of curcumin.

Sarcoplasmic reticulum (SR) vesicles were adsorbed on an octadecanethiol/phosphatidylcholine mixed bilayer anchored to a gold electrode, and the Ca-ATPase contained in the vesicles was activated by ATP concentration jumps in the presence of calcium ions. The resulting capacitive current transients are compared with those calculated on the basis of the enzymatic cycle of the calcium pump. This comparison provides information on the kinetics of the E(2)-E(1) conformational change and on its pH dependence. The alteration in the current transients following ATP concentration jumps in the presence of curcumin is examined. In particular, curcumin decreases the rate of slippage of the Ca-ATPase, and at concentrations above 10 microM reduces calcium transport by this pump.

Pre-steady state electrogenic events of Ca2+/H+ exchange and transport by the Ca2+-ATPase.

Native or recombinant SERCA (sarco(endo)plasmic reticulum Ca(2+) ATPase) was adsorbed on a solid supported membrane and then activated with Ca(2+) and ATP concentration jumps through rapid solution exchange. The resulting electrogenic events were recorded as electrical currents flowing along the external circuit. Current transients were observed following Ca(2+) jumps in the absence of ATP and following ATP jumps in the presence of Ca(2+). The related charge movements are attributed to Ca(2+) reaching its binding sites in the ground state of the enzyme (E(1)) and to its vectorial release from the enzyme phosphorylated by ATP (E(2)P). The Ca(2+) concentration and pH dependence as well as the time frames of the observed current transients are consistent with equilibrium and pre-steady state biochemical measurements of sequential steps within a single enzymatic cycle. Numerical integration of the current transients recorded at various pH values reveal partial charge compensation by H(+) in exchange for Ca(2+) at acidic (but not at alkaline) pH. Most interestingly, charge movements induced by Ca(2+) and ATP vary over different pH ranges, as the protonation probability of residues involved in Ca(2+)/H(+) exchange is lower in the E(1) than in the E(2)P state. Our single cycle measurements demonstrate that this difference contributes directly to the reduction of Ca(2+) affinity produced by ATP utilization and results in the countertransport of two Ca(2+) and two H(+) within each ATPase cycle at pH 7.0. The effects of site-directed mutations indicate that Glu-771 and Asp-800, within the Ca(2+) binding domain, are involved in the observed Ca(2+)/H(+) exchange.

Clotrimazole inhibits the Ca2+-ATPase (SERCA) by interfering with Ca2+ binding and favoring the E2 conformation.

Clotrimazole (CLT) is an antimycotic imidazole derivative that is known to inhibit cytochrome P-450, ergosterol biosynthesis and proliferation of cells in culture, and to interfere with cellular Ca(2+) homeostasis. We found that CLT inhibits the Ca(2+)-ATPase of rabbit fast-twitch skeletal muscle (SERCA1), and we characterized in detail the effect of CLT on this calcium transport ATPase. We used biochemical methods for characterization of the ATPase and its partial reactions, and we also performed measurements of charge movements following adsorption of sarcoplasmic reticulum vesicles containing the ATPase onto a gold-supported biomimetic membrane. CLT inhibits Ca(2+)-ATPase and Ca(2+) transport with a K(I) of 35 mum. Ca(2+) binding in the absence of ATP and phosphoenzyme formation by the utilization of ATP in the presence of Ca(2+) are also inhibited within the same CLT concentration range. On the other hand, phosphoenzyme formation by utilization of P(i) in the absence of Ca(2+) is only minimally inhibited. It is concluded that CLT inhibits primarily Ca(2+) binding and, consequently, the Ca(2+)-dependent reactions of the SERCA cycle. It is suggested that CLT resides within the membrane-bound region of the transport ATPase, thereby interfering with binding and the conformational effects of the activating cation.

Time-resolved charge translocation by sarcoplasmic reticulum Ca-ATPase measured on a solid supported membrane.

Sarcoplasmic reticulum vesicles were adsorbed on an octadecanethiol/phosphatidylcholine mixed bilayer anchored to a gold electrode, and the Ca-ATPase contained in the vesicles was activated by ATP concentration jumps both in the absence and in the presence of K(+) ions and at different pH values. Ca(2+) concentration jumps in the absence of ATP were also carried out. The resulting capacitive current transients were analyzed together with the charge under the transients. The relaxation time constants of the current transients were interpreted on the basis of an equivalent circuit. The current transient after ATP concentration jumps and the charge after Ca(2+) concentration jumps in the absence of ATP exhibit almost the same dependence upon the Ca(2+) concentration, with a half-saturating value of approximately 1.5 microM. The pH dependence of the charge after Ca(2+) translocation demonstrates the occurrence of one H(+) per one Ca(2+) countertransport at pH 7 by direct charge-transfer measurements. The presence of K(+) decreases the magnitude of the current transients without altering their shape; this decrease is explained by K(+) binding to the cytoplasmic side of the pump in the E(1) conformation and being released to the same side during the E(1)-E(2) transition.