Superparamagnetic iron oxide nanoparticles (SPIONs) modulate hERG ion channel activity.

Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used in various biomedical applications, such as diagnostic agents in magnetic resonance imaging (MRI), for drug delivery vehicles and in hyperthermia treatment of tumors. Although the potential benefits of SPIONs are considerable, there is a distinct need to identify any potential cellular damage associated with their use. Since human ether à go-go-related gene (hERG) channel, a protein involved in the repolarization phase of cardiac action potential, is considered one of the main targets in the drug discovery process, we decided to evaluate the effects of SPIONs on hERG channel activity and to determine whether the oxidation state, the dimensions and the coating of nanoparticles (NPs) can influence the interaction with hERG channel. Using patch clamp recordings, we found that SPIONs inhibit hERG current and this effect depends on the coating of NPs. In particular, SPIONs with covalent coating aminopropylphosphonic acid (APPA) have a milder effect on hERG activity. We observed that the time-course of hERG channel modulation by SPIONs is biphasic, with a transient increase (∼20% of the amplitude) occurring within the first 1-3 min of perfusion of NPs, followed by a slower inhibition. Moreover, in the presence of SPIONs, deactivation kinetics accelerated and the activation and inactivation I-V curves were right-shifted, similarly to the effect described for the binding of other divalent metal ions (e.g. Cd2+ and Zn2+). Finally, our data show that a bigger size and the complete oxidation of SPIONs can significantly decrease hERG channel inhibition. Taken together, these results support the view that Fe2+ ions released from magnetite NPs may represent a cardiac risk factor, since they alter hERG gating and these alterations could compromise the cardiac action potential.

Drug Interactions With the Ca2+-ATPase From Sarco(Endo)Plasmic Reticulum (SERCA).

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is an intracellular membrane transporter that utilizes the free energy provided by ATP hydrolysis for active transport of Ca2+ ions from the cytoplasm to the lumen of sarco(endo)plasmic reticulum. SERCA plays a fundamental role for cell calcium homeostasis and signaling in muscle cells and also in cells of other tissues. Because of its prominent role in many physiological processes, SERCA dysfunction is associated to diseases displaying various degrees of severity. SERCA transport activity can be inhibited by a variety of compounds with different chemical structures. Specific SERCA inhibitors were identified which have been instrumental in studies of the SERCA catalytic and transport mechanism. It has been proposed that SERCA inhibition may represent a novel therapeutic strategy to cure certain diseases by targeting SERCA activity in pathogens, parasites and cancer cells. Recently, novel small molecules have been developed that are able to stimulate SERCA activity. Such SERCA activators may also offer an innovative and promising therapeutic approach to treat diseases, such as heart failure, diabetes and metabolic disorders. In the present review the effects of pharmacologically relevant compounds on SERCA transport activity are presented. In particular, we will discuss the interaction of SERCA with specific inhibitors and activators that are potential therapeutic agents for different diseases.

Conformational memory in the association of the transmembrane protein phospholamban with the sarcoplasmic reticulum calcium pump SERCA.

The sarcoplasmic reticulum Ca2+-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.

Hofmeister effect of anions on calcium translocation by sarcoplasmic reticulum Ca(2+)-ATPase.

The occurrence of Hofmeister (specific ion) effects in various membrane-related physiological processes is well documented. For example the effect of anions on the transport activity of the ion pump Na(+), K(+)-ATPase has been investigated. Here we report on specific anion effects on the ATP-dependent Ca(2+) translocation by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA). Current measurements following ATP concentration jumps on SERCA-containing vesicles adsorbed on solid supported membranes were carried out in the presence of different potassium salts. We found that monovalent anions strongly interfere with ATP-induced Ca(2+) translocation by SERCA, according to their increasing chaotropicity in the Hofmeister series. On the contrary, a significant increase in Ca(2+) translocation was observed in the presence of sulphate. We suggest that the anions can affect the conformational transition between the phosphorylated intermediates E1P and E2P of the SERCA cycle. In particular, the stabilization of the E1P conformation by chaotropic anions seems to be related to their adsorption at the enzyme/water and/or at the membrane/water interface, while the more kosmotropic species affect SERCA conformation and functionality by modifying the hydration layers of the enzyme.

A sulfur-based transport pathway in Cu+-ATPases.

Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. PIB -type Cu(+)-ATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu(+) across cellular membranes. Crystal structures of a copper-free Cu(+)-ATPase are available, but the mechanism of Cu(+) recognition, binding, and translocation remains elusive. Through X-ray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solid-supported membranes using wild-type and mutant forms of the Legionella pneumophila Cu(+)-ATPase (LpCopA), we identify a sulfur-lined metal transport pathway. Structural analysis indicates that Cu(+) is bound at a high-affinity transmembrane-binding site in a trigonal-planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Met residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport. Additionally, the studies indicate essential roles of other conserved intramembranous polar residues in facilitating copper binding to the high-affinity site and subsequent release through the exit pathway.

Lipoic-based TRPA1/TRPV1 antagonist to treat orofacial pain.

Inflammation of the trigeminal nerve is considered one of the most painful conditions known to humankind. The diagnosis is often difficult; moreover, safe and effective pharmacological treatments are lacking. A new molecule, ADM_12, formed by a lipoic and omotaurine residues covalently linked, is here reported. In vitro and in vivo tests showed that ADM_12 is a very attractive original compound presenting (i) a remarkable safety profile; (ii) a high binding constant versus TRPA1; (iii) an intriguing behavior versus TRPV1; and (iv) the ability to significantly and persistently reduce mechanical facial allodynia in rats. Noteworthy, by testing ADM_12, we shed light on the unprecedented involvement of TRPA1 and TRPV1 channels in orofacial pain.

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).

Enhanced adsorption of Ca-ATPase containing vesicles on a negatively charged solid-supported-membrane for the investigation of membrane transporters.

A convenient model system for a biological membrane is a solid-supported membrane (SSM), which consists of a gold-supported alkanethiol|phospholipid bilayer. In combination with a concentration jump method, SSMs have been used for the investigation of several membrane transporters. Vesicles incorporating sarcoplasmic reticulum Ca-ATPase (SERCA) were adsorbed on a negatively charged SSM (octadecanethiol|phosphatidylserine bilayer). The current signal generated by the adsorbed vesicles following an ATP concentration jump was compared to that produced by SERCA-containing vesicles adsorbed on a conventional SSM (octadecanethiol|phosphatidylcholine bilayer). A significantly higher current amplitude was recorded on the serine-based SSM. The adsorption of SERCA-incorporating vesicles on the SSM was then characterized by surface plasmon resonance (SPR). The SPR measurements clearly indicate that in the presence of Ca(2+) and Mg(2+), the amount of adsorbed vesicles on the serine-based SSM is about twice that obtained using the conventional SSM, thereby demonstrating that the higher current amplitude recorded on the negatively charged SSM is correlated with a greater quantity of adsorbed vesicles. The enhanced adsorption of membrane vesicles on the PS-based SSM may be useful to study membrane preparations with a low concentration of transport protein generating small current signals, as in the case of various recombinantly expressed proteins.

Parthenolide inhibits nociception and neurogenic vasodilatation in the trigeminovascular system by targeting the TRPA1 channel.

Although feverfew has been used for centuries to treat pain and headaches and is recommended for migraine treatment, the mechanism for its protective action remains unknown. Migraine is triggered by calcitonin gene-related peptide (CGRP) release from trigeminal neurons. Peptidergic sensory neurons express a series of transient receptor potential (TRP) channels, including the ankyrin 1 (TRPA1) channel. Recent findings have identified agents either inhaled from the environment or produced endogenously that are known to trigger migraine or cluster headache attacks, such as TRPA1 simulants. A major constituent of feverfew, parthenolide, may interact with TRPA1 nucleophilic sites, suggesting that feverfew's antimigraine effect derives from its ability to target TRPA1. We found that parthenolide stimulates recombinant (transfected cells) or natively expressed (rat/mouse trigeminal neurons) TRPA1, where it, however, behaves as a partial agonist. Furthermore, in rodents, after initial stimulation, parthenolide desensitizes the TRPA1 channel and renders peptidergic TRPA1-expressing nerve terminals unresponsive to any stimulus. This effect of parthenolide abrogates nociceptive responses evoked by stimulation of peripheral trigeminal endings. TRPA1 targeting and neuronal desensitization by parthenolide inhibits CGRP release from trigeminal neurons and CGRP-mediated meningeal vasodilatation, evoked by either TRPA1 agonists or other unspecific stimuli. TRPA1 partial agonism, together with desensitization and nociceptor defunctionalization, ultimately resulting in inhibition of CGRP release within the trigeminovascular system, may contribute to the antimigraine effect of parthenolide.

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 TRPA1 antagonist reverts oxaliplatin-induced neuropathic pain.

Neuropathic pain (NeP) is generally considered an intractable problem, which becomes compelling in clinical practice when caused by highly effective chemotherapeutics, such as in the treatment of cancer with oxaliplatin (OXA) and related drugs. In the present work we describe a structurally new compound, ADM_09, which proved to effectively revert OXA-induced NeP in vivo in rats without eliciting the commonly observed negative side-effects. ADM_09 does not modify normal behavior in rats, does not show any toxicity toward astrocyte cell cultures, nor any significant cardiotoxicity. Patch-clamp recordings demonstrated that ADM_09 is an effective antagonist of the nociceptive sensor channel TRPA1, which persistently blocks mouse as well as human variants of TRPA1. A dual-binding mode of action has been proposed for ADM_09, in which a synergic combination of calcium-mediated binding of the carnosine residue and disulphide-bridge-forming of the lipoic acid residue accounts for the observed persistent blocking activity toward the TRPA1 channel.

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.

Structure-function relation of phospholamban: modulation of channel activity as a potential regulator of SERCA activity.

Phospholamban (PLN) is a small integral membrane protein, which binds and inhibits in a yet unknown fashion the Ca(2+)-ATPase (SERCA) in the sarcoplasmic reticulum. When reconstituted in planar lipid bilayers PLN exhibits ion channel activity with a low unitary conductance. From the effect of non-electrolyte polymers on this unitary conductance we estimate a narrow pore with a diameter of ca. 2.2 Å for this channel. This value is similar to that reported for the central pore in the structure of the PLN pentamer. Hence the PLN pentamer, which is in equilibrium with the monomer, is the most likely channel forming structure. Reconstituted PLN mutants, which either stabilize (K27A and R9C) or destabilize (I47A) the PLN pentamer and also phosphorylated PLN still generate the same unitary conductance of the wt/non-phosphorylated PLN. However the open probability of the phosphorylated PLN and of the R9C mutant is significantly lower than that of the respective wt/non-phosphorylated control. In the context of data on PLN/SERCA interaction and on Ca(2+) accumulation in the sarcoplasmic reticulum the present results are consistent with the view that PLN channel activity could participate in the balancing of charge during Ca(2+) uptake. A reduced total conductance of the K(+) transporting PLN by phosphorylation or by the R9C mutation may stimulate Ca(2+) uptake in the same way as an inhibition of K(+) channels in the SR membrane. The R9C-PLN mutation, a putative cause of dilated cardiomyopathy, might hence affect SERCA activity also via its inherent low open probability.

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).

Phospholamban generates cation selective ion channels.

Phosholamban (PLN) is involved in the contractility of cardiac muscles by regulating the intracellular calcium concentration (Ca(2+)(cyt)) of cardiac myocytes. This occurs via a modulation of the sarco-/endoplasmic CaATPase (SERCA). In spite of high-resolution structures the molecular mode of PLN action is yet not known. In the present paper we readdress the question whether PLN proteins can generate ion channel activity. Reconstitution of PLN in planar lipid bilayers reveals single channel fluctuations, which are characterized by two conductance levels, long open/closed dwell times, moderate selectivity between monovalent cations and no perceivable Ca(2+) permeability. The PLN generated channel activity could be inhibited by a PLN antibody (abPLN) implying that the channel activity is indeed due to the inherent channel function of the PLN protein.

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.