Lack of nitric oxide synthase depresses ion transporting enzyme function in cardiac muscle

Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart.     

The effect of thyroxine on the calmodulin-dependent (Ca2+-Mg2+)ATPase activity and protein phosphorylation in rabbit fast skeletal muscle sarcolemma

Enzymatic properties and the protein pattern of sarcolemma fractions isolated from three groups of rabbits: euthyroid, hyperthyroid and hypothyroid, were studied. The amount of phosphorylated intermediate formed by the calmodulin-dependent (Ca2+-Mg2+)ATPase and the activity of this enzyme as well as that of (Na+-K+)ATPase were the highest in membranes isolated at the hyperthyroid state. On the other hand, sarcolemma obtained from the hypothyroid animals exhibited a decreased activity of (Na+-K+)ATPase, while the activity of calmodulin-dependent (Ca2+-Mg2+)ATPase was the same as in the preparations obtained from euthyroid animals. Thyroid hormones also changed the protein pattern of muscle sarcolemma. Membranes isolated from hyperthyroid animals lacked peptides of apparent molecular masses of 41 kDa and 53 kDa, while a peptide of the apparent molecular mass of 63 kDa was enriched in the preparation from hypothyroid animals. Thyroid hormones affected endogenous cAMP-dependent protein phosphorylation. The sarcolemma fraction obtained from hyperthyroid animals exhibited a decreased phosphorylation of peptides of apparent molecular masses of 30 kDa and 47 kDa, while the cAMP-independent phosphorylation of several other peptides was augmented. Moreover, sarcolemma preparations isolated from hyperthyroid animals showed higher activity of cAMP-independent protein kinase(s) and lower activity of cAMP-dependent protein kinase when compared to the euthyroid preparations. It is proposed that thyroxine increases the content of calmodulin-dependent (Ca2+-Mg2+)ATPase protein and affects the activity of cAMP-independent and cAMP-dependent protein kinases bound to sarcolemma.     

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.     

Nitric oxide protects cardiac sarcolemmal membrane enzyme function and ion active transport against ischemia-induced inactivation

Nitric oxide (NO.) generated from nitric oxide synthase (NOS) isoforms bound to cellular membranes may serve to modulate oxidative stresses in cardiac muscle and thereby regulate the function of key membrane-associated enzymes. Ischemia is known to inhibit the function of sarcolemmal enzymes, including the (Na+ + K+)-ATPase, but it is unknown whether concomitant injury to sarcolemma (SL)-associated NOS isoforms may contribute to this process by reducing the availability of locally generated NO. Here we report that nNOS, as well as eNOS (SL NOSs), are tightly associated with cardiac SL membranes in several different species. In isolated perfused rat hearts, global ischemia caused a time-dependent irreversible injury to cardiac SL NOSs and a disruption of SL NO. generation. Pretreatment with low concentrations of the NO. donor 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7) markedly protected both SL NOS and (Na+ + K+)-ATPase functions against ischemia-induced inactivation. Moreover, ischemia impaired SL Na+/K+ binding, and NOC-7 significantly prevented ischemic injury to the ion binding sites on (Na+ + K+)-ATPase. These novel findings indicate that NO. can protect cardiac SL NOSs and (Na+ + K+)-ATPase against ischemia-induced inactivation and suggest that locally generated NO. may serve to regulate SL Na+/K+ ion active transport in the heart.     

Carbohydrate-binding proteins in bovine kidney have consensus amino acid sequences of annexin family proteins.

Ca(2+)-dependent carbohydrate-binding proteins were purified from bovine kidney extracts. Upon SDS-polyacrylamide gel electrophoresis under nonreducing conditions, the purified fraction gave doublet protein bands corresponding to 33 kDa (p33) and 41 kDa (p41). Under reducing conditions, a single protein band (p33) was observed. p33 and p41 were submitted to proteolytic digestion with endoproteinase Lys-C, the peptides produced were separated by reversed-phase high performance liquid chromatography, and their amino acid sequences were determined by an automated gas-phase protein sequenator. Most of the resulting partial amino acid sequences of these proteins were strikingly homologous to annexin IV, an annexin family protein, i.e. Ca2+/phospholipid-binding proteins, especially in the consensus sequences. In the presence of Ca2+, both proteins bound to vesicles composed of phosphatidylserine and phosphatidylethanolamine, but not phosphatidylcholine. These results indicated that p33 and p41 are members of annexin family proteins.     

Modification of sarcolemmal Na+-K+-ATPase and Na+/Ca2+ exchanger expression in heart failure by blockade of renin-angiotensin system

The activities of both sarcolemmal (SL) Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchanger, which maintain the intracellular cation homeostasis, have been shown to be depressed in heart failure due to myocardial infarction (MI). Because the renin-angiotensin system (RAS) is activated in heart failure, this study tested the hypothesis that attenuation of cardiac SL changes in congestive heart failure (CHF) by angiotensin-converting enzyme (ACE) inhibitors is associated with prevention of alterations in gene expression for SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchanger. CHF in rats due to MI was induced by occluding the coronary artery, and 3 wk later the animals were treated with an ACE inhibitor, imidapril (1 mg.kg(-1).day(-1)), for 4 wk. Heart dysfunction and cardiac hypertrophy in the infarcted animals were associated with depressed SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities. Protein content and mRNA levels for Na(+)/Ca(2+) exchanger as well as Na(+)-K(+)-ATPase alpha(1)-, alpha(2)- and beta(1)-isoforms were depressed, whereas those for alpha(3)-isoform were increased in the failing heart. These changes in SL activities, protein content, and gene expression were attenuated by treating the infarcted animals with imidapril. The beneficial effects of imidapril treatment on heart function and cardiac hypertrophy as well as SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities in the infarcted animals were simulated by enalapril, an ACE inhibitor, and losartan, an angiotensin receptor antagonist. These results suggest that blockade of RAS in CHF improves SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities in the failing heart by preventing changes in gene expression for SL proteins.     

A Ca2+-dependent tryptic cleavage site and a protein kinase A phosphorylation site are present in the Ca2+ regulatory domain of scallop muscle Na+-Ca2+ exchanger

Digestion of scallop muscle membrane fractions with trypsin led to release of soluble polypeptides derived from the large cytoplasmic domain of a Na(+)-Ca(2+) exchanger. In the presence of 1 mm Ca(2+), the major product was a peptide of approximately 37 kDa, with an N terminus corresponding to residue 401 of the NCX1 exchanger. In the presence of 10 mm EGTA, approximately 16- and approximately 19-kDa peptides were the major products. Polyclonal rabbit IgG raised against the 37-kDa peptide also bound to the 16- and 19-kDa soluble tryptic peptides and to a 105-110-kDa polypeptide in the undigested membrane preparation. The 16-kDa fragment corresponded to the N-terminal part of the 37-kDa peptide. The conformation of the precursor polypeptide chain in the region of the C terminus of the 16-kDa tryptic peptide was thus altered by the binding of Ca(2+). Phosphorylation of the parent membranes with the catalytic subunit of protein kinase A and [gamma-(32)P]ATP led to incorporation of (32)P into the 16- and 37-kDa soluble fragments. A site may exist within the Ca(2+) regulatory domain of a scallop muscle Na(+)-Ca(2+) exchanger that mediates direct modulation of secondary Ca(2+) regulation by cAMP.     

Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

Although the Na(+)/H(+) exchanger (NHE) is considered to be involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the Na(+)/Ca(2+) exchanger, the exact mechanisms of its participation in Ca(2+) handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca(2+) movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca(2+)](i) in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca(2+)](i) and augmented the KCl-induced increase in [Ca(2+)](i) in a concentration-dependent manner. The MIA-induced increase in basal [Ca(2+)](i) was unaffected by extracellular Ca(2+), antagonists of the sarcolemmal (SL) L-type Ca(2+) channel, and inhibitors of the SL Na(+)/Ca(2+) exchanger, SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. However, the MIA-induced increase in basal [Ca(2+)](i) was attenuated by inhibitors of SL Na(+)-K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+) transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca(2+) concentration and attenuated by agents that inhibit SL L-type Ca(2+) channels, the SL Na(+)/Ca(2+) exchanger, SL Na(+)-K(+)-ATPase, and SR Ca(2+) release channels and the SR Ca(2+) pump. However, the effect of MIA on the KCl-induced increase in [Ca(2+)](i) remained unaffected by treatment with inhibitors of SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca(2+)](i), whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca(2+)](i) in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca(2+)](i) and that MIA-induced increases in basal [Ca(2+)](i), as well as augmentation of the KCl-induced increase in [Ca(2+)](i), in cardiomyocytes are regulated differentially.     

Ca2+/H+ exchange through plasma membrane in the system of transport of calcium and hydrogen ions

The survey is aimed to review the data from literature, concerning possible mechanisms of Ca2+ and H+ transport through the plasma membrane of a cells, and also possibility of existence of Ca2+/H(+)-exchange in the plasma membrane of the muscle cells. It is known that the modification of pHl (delta pH) also can influence the work of the contractile system of muscle cells, and the transition of Ca2+ through the plasma membrane of the cells. Thus, one can suppose a direct relation between Ca2+ and H+ transport, through Ca2+/H+ exchange, and indirect relation through connection with other systems of transport of both Ca2+ (Ca(2+)-ATPase, Na+/Ca2+ exchange), and H+ (Na+/H(+)-exchange, H(+)-ATPase). For example it is shown, that the activator (inhibitor) of the Na+/H(+)-exchange through the plasma membrane of muscle cells, influence the work of the retractive system. And as is known, Ca2+ takes main part in involvement in the system excitation--contraction, and, thus, influencing the work of the Na+/H(+)-exchange, it is possible to regulate transport of Ca2+ through the plasma membrane of a muscle cell. The problem about a possibility of existence of Ca2+/H+ exchange, or functioning of Ca2+/H(+)-exchanger, is still far from the solution. Therefore, in the given review the attempt is made to analyze available information about possible connection between Ca2+ and H+ transport through the plasma cell membrane.     

Calcium and magnesium binding to human centrin 3 and interaction with target peptides

There are four isoforms of centrin in mammals, with variable sequence, tissue expression, and functional properties. We have recently characterized a number of structural, ion, and target binding properties of human centrin isoform HsCen2. This paper reports a similar characterization of HsCen3, overexpressed in Escherichia coli and purified by phase-reversed chromatography. Equilibrium and dynamic binding studies revealed that HsCen3 has one mixed Ca(2+)/Mg(2+) binding site of high affinity (K(d) = 3 and 10 microM for Ca(2+) and Mg(2+), respectively) and two Ca(2+)-specific sites of low affinity (K(d) = 140 microM). The metal-free protein is fragmented by an unidentified protease into a polypeptide segment of 11 kDa, which was purified by HPLC, and identified by mass spectrometry as the segment of residues 21-112. Similarly, controlled trypsinolysis on Ca(2+)-bound HsCen3 yielded a mixture of segments of residues 1-124 and 1-125. The Ca(2+)/Mg(2+) site could be assigned to this segment and thus resides in the N-terminal half of HsCen3. Temperature denaturation experiments, circular dichroism, and utilization of fluorescence hydrophobic probes allowed us to propose that the metal-free protein has molten globule characteristics and that the dication-bound forms are compact with a polar surface for the Mg(2+) form and a hydrophobic exposed surface for the Ca(2+) form. Thus, HsCen3 could be classified as a Ca(2+) sensor protein. In addition, it is able to bind strongly to a model target peptide (melittin), as well as to peptides derived from the protein XPC and Kar1p, with a moderate Ca(2+) dependence.     

The N-terminal region of the plasma membrane Ca(2+) pump does not separate from the main catalytic fragments after proteolysis

The purified plasma membrane Ca(2+) pump (PMCA) was digested with trypsin, and the proteolytic products were identified by immunoblotting with monoclonal antibodies JA9 or 5F10 directed against the extreme N-terminal segment and the central portion of the molecule, respectively. After a short treatment with low concentrations of the protease, JA9 reacted predominantly with a peptide of 35 kDa whereas 5F10 detected a peptide of 90 kDa. The trypsin cut leading to the production of these fragments had no effect on the maximal activity of the enzyme. At higher concentrations of trypsin, JA9 detected a main fragment of 33 kDa and smaller fragments of 19 and 15 kDa. The persistence of fragments reacting with JA9 indicates that the N-terminal region containing its epitope (residues 51-75) was not easily accessible to the protease in the native PMCA. However, the reactivity with JA9 was rapidly lost during proteolysis of the denatured protein. The passage of the mixture of PMCA fragments through a calmodulin-Sepharose column resulted in the retention of the N-terminal 35 kDa fragment together with that of 90 kDa, despite the fact that only the latter binds calmodulin. The ethylenediaminetetraacetic acid (EDTA) eluate, which contained about equal amounts of both fragments, had a Ca(2+) ATPase activity similar to that of the intact enzyme. The tight association between the two peptides was evidenced by the fact that concentrations of polyoxyethylene 10 lauryl ether (C(12)E(10)), sodium dodecyl sulfate (SDS) high enough for inactivating the enzyme and dissociate the pump from calmodulin were unable of breaking the interaction between the 35 and 90 kDa fragments. Altogether, these results show that after digestion with trypsin, the N-terminal portion of the PMCA, including the extreme N-terminal segment, remains part of a fully functional catalytic complex.     

Leishmania amazonensis: PKC-like protein kinase modulates the (Na++K+)ATPase activity

The present study aimed to identify the presence of protein kinase C-like (PKC-like) in Leishmania amazonensis and to elucidate its possible role in the modulation of the (Na(+)+K(+))ATPase activity. Immunoblotting experiments using antibody against a consensus sequence (Ac 543-549) of rabbit protein kinase C (PKC) revealed the presence of a protein kinase of 80 kDa in L. amazonensis. Measurements of protein kinase activity showed the presence of both (Ca(2+)-dependent) and (Ca(2+)-independent) protein kinase activity in plasma membrane and cytosol. Phorbol ester (PMA) activation of the Ca(2+)-dependent protein kinase stimulated the (Na(+)+K(+))ATPase activity, while activation of the Ca(2+)-independent protein kinase was inhibitory. Both effects of protein kinase on the (Na(+)+K(+))ATPase of the plasma membrane were lower than that observed in intact cells. PMA induced the translocation of protein kinase from cytosol to plasma membrane, indicating that the maximal effect of protein kinase on the (Na(+)+K(+))ATPase activity depends on the synergistic action of protein kinases from both plasma membrane and cytosol. This is the first demonstration of a protein kinase activated by PMA in L. amazonensis and the first evidence for a possible role in the regulation of the (Na(+)+K(+))ATPase activity in this trypanosomatid. Modulation of the (Na(+)+K(+))ATPase by protein kinase in a trypanosomatid opens up new possibilities to understand the regulation of ion homeostasis in this parasite.     

Discovery of a novel potent Na+/Ca2+ exchanger inhibitor: design, synthesis and structure-activity relationships of 3,4-dihydro-2(1H)-quinazolinone derivatives

Design, synthesis and structure-activity relationships for 3,4-dihydro-2(1H)-quinazolinone derivatives with the inhibitory activities of the Na(+)/Ca(2+) exchanger are discussed. These studies based on lead compound 1a lead to the discovery of a structurally novel and highly potent inhibitor against the Na(+)/Ca(2+) exchanger 4f (SM-15811), which directly inhibited the Na(+)-dependent Ca(2+) influx via the Na(+)/Ca(2+) exchanger in cardiomyocytes with a high potency.     

Ca2+ binding alters the interdomain flexibility between the two cytoplasmic calcium-binding domains in the Na+/Ca2+ exchanger

The Na(+)/Ca(2+) exchanger (NCX) is a membrane protein, which catalyzes the counter transport of Na(+) and Ca(2+) ions across the plasma membrane, playing a key role in the maintenance of the intracellular Ca(2+) homeostasis in various cell types. NCX consists of a transmembrane part and a large intracellular loop. The activation of the NCX transport function requires the binding of Ca(2+) to two tandem C2 domains, CBD1 and CBD2, which are an integral part of the exchanger's intracellular loop. Although high-resolution structures of individual CBD1 and CBD2 are available, their interdomain structure and dynamics and the atomic level mechanism of allosteric Ca(2+)-regulation remains unknown. Here, we use solution NMR spectroscopy to study the interdomain dynamics of CBD12, a 32 kDa construct that contains both the CBD1 and CBD2 domains connected by a short linker. Analysis of NMR residual dipolar couplings shows that CBD12 assumes on average an elongated shape both in the absence and in the presence of Ca(2+). NMR (15)N relaxation data of the Apo state indicate that the two domains sample a wide range of relative arrangements on the nanosecond time scale. These arrangements comprise significantly non-linear interdomain orientations. Binding of Ca(2+) to CBD1 significantly restricts the interdomain flexibility, stabilizing a more rigid elongated conformation. These findings suggest a molecular mechanism for the role of CBD12 in the function of NCX.     

alpha-Lactalbumin: structure and function

Small milk protein alpha-lactalbumin (alpha-LA), a component of lactose synthase, is a simple model Ca(2+) binding protein, which does not belong to the EF-hand proteins, and a classical example of molten globule state. It has a strong Ca(2+) binding site, which binds Mg(2+), Mn(2+), Na(+), and K(+), and several distinct Zn(2+) binding sites. The binding of cations to the Ca(2+) site increases protein stability against action of heat and various denaturing agents, while the binding of Zn(2+) to the Ca(2+)-loaded protein decreases its stability. Functioning of alpha-LA requires its interactions with membranes, proteins, peptides and low molecular weight substrates and products. It was shown that these interactions are modulated by the binding of metal cations. Recently it was found that some folding variants of alpha-LA demonstrate bactericidal activity and some of them cause apoptosis of tumor cells.     

Interactions of the exchange inhibitory peptide with Na-Ca exchange in bovine cardiac sarcolemmal vesicles and ferret red cells.

The Na-Ca exchange inhibitory peptide (XIP), which corresponds to residues 251-270 of the Na-Ca exchange protein, specifically inhibits exchange activity (Li, Z., Nicoll, D. A, Collins, A., Hilgemann, D. W., Filoteo, A. G., Penniston, J. T., Weiss, J. N., Tomich, J. M., and Philipson, K. D. (1991) J. Biol. Chem. 266, 1014-1020). We have found that XIP decreased Na+i-dependent Ca2+ uptake to 46 and 20% of control in mixed and inside-out bovine sarcolemmal (SL) vesicles, respectively, and to 22% of control in ferret red cell vesicles. XIP inhibited uptake in bovine SL vesicles after proteolytic digestion. XIP also inhibited Na+o-dependent Ca2+ efflux in bovine SL vesicles but did not inhibit Ca2+ uptake in reconstituted proteoliposomes. Extracellular XIP did not inhibit Ca2+ uptake into intact ferret red cells. Inhibition of uptake in bovine SL vesicles was reduced as the ionic strength was increased. 125I-labeled XIP (1 microM) was cross-linked to proteins of bovine SL vesicles, ferret red cell vesicles, and intact ferret red cells. Labeling of bands at approximately 75, 120, and 220 kDa (in bovine SL vesicles) and bands at 55 and 85 kDa (in ferret red cell vesicles) was detected. No cross-linking was detected in intact ferret red cells. We conclude that XIP inhibition is insensitive to proteolytic digestion and is partially dependent on charge association and conformation of the exchanger. XIP binds to and interacts with the intracellular side of the Na-Ca exchanger.     

Overlapping effects of S3 stalk segment mutations on the affinity of Ca2+-ATPase (SERCA) for thapsigargin and cyclopiazonic acid

Chimeric exchanges and mutations were produced in the Ca(2+)-ATPase (SERCA) to match (in the majority of cases) corresponding sequences of the Na(+),K(+)-ATPase. The effects of these mutations on the concentration dependence of the specific Ca(2+)-ATPase inhibition by thapsigargin (TG) and cyclopiazonic acid (CPA) were then determined. Extensive chimeric mutations on the large cytosolic loop, on the S4 stalk segment, and on the M3 transmembrane segments produced little or no modification of the Ca(2+)-ATPase sensitivity to either inhibitor. On the other hand, the presence of a six amino acid Na(+), K(+)-ATPase sequence within the S3 stalk segment of the Ca(2+)-ATPase raised 60-fold the apparent K(i) for TG and 250-fold the apparent K(i) for CPA. More limited mutations within the same S3 segment, however, affected differently the concentration dependence of the Ca(2+)-ATPase inhibition by TG or CPA. Specifically, single mutation of Phe256 to Val increased 20-fold the apparent K(i) for TG, while having very little effect on the apparent K(i) for CPA. These findings indicate significant overlap of the TG and CPA binding domains within the S3 stalk segment of the Ca(2+)-ATPase, where the contribution of each protein residue is dependent on the structures of the two inhibitors. Saturating concentrations of either or both TG and CPA produce an identical reduction of the affinity of the ATPase for ATP, suggesting that only one inhibitor can bind at any time due to significant overlap of their binding domains. It is suggested that perturbations produced by binding of either inhibitor within the stalk segment interfere with the long-range functional linkage between ATP utilization in the ATPase cytosolic region and Ca(2+) binding in the membrane-bound region.     

Structural basis for Ca2+ -dependent formation of ALG-2/Alix peptide complex: Ca2+/EF3-driven arginine switch mechanism

ALG-2 belongs to the penta-EF-hand (PEF) protein family and interacts with various intracellular proteins, such as Alix and TSG101, that are involved in endosomal sorting and HIV budding. Through X-ray crystallography, we solved the structures of Ca(2+)-free and -bound forms of N-terminally truncated human ALG-2 (des3-20ALG-2), Zn(2+)-bound form of full-length ALG-2, and the structure of the complex between des3-23ALG-2 and the peptide corresponding to Alix799-814 in Zn(2+)-bound form. Binding of Ca(2+) to EF3 enables the side chain of Arg125, present in the loop connecting EF3 and EF4, to move enough to make a primary hydrophobic pocket accessible to the critical PPYP motif, which partially overlaps with the GPP motif for the binding of Cep55 (centrosome protein 55 kDa). Based on these results, together with the results of in vitro binding assay with mutant ALG-2 and Alix proteins, we propose a Ca(2+)/EF3-driven arginine switch mechanism for ALG-2 binding to Alix.     

Expression of Ca2+-ATPase and Na+/Ca2+-exchanger is upregulated during epithelial Ca2+ transport in hypodermal cells of the isopod Porcellio scaber

It is thought that a plasma membrane Ca(2+)-transport ATPase (PMCA) and a Na(+)/Ca(2+)-exchange (NCE) mechanism are involved in epithelial Ca(2+) transport (ECT) in a variety of crustacean epithelia. The sternal epithelium of the terrestrial isopod Porcellio scaber was used as a model for the analysis of Ca(2+)-extrusion mechanisms in the hypodermal epithelium. Using RT-PCR, we amplified a cDNA fragment of 1173 bp that encodes a protein sequence possessing 72% identity to the PMCA from Drosophila melanogaster and a cDNA fragment of 791 bp encoding a protein sequence with 50% identity to the NCE from Loligo opalescens. Semiquantitative RT-PCR revealed that the expression of both mRNAs increases from the non-Ca(2+)-transporting condition to the stages of CaCO(3) deposit formation and degradation. During Ca(2+)-transporting stages, the expression of PMCA and NCE was larger in the anterior sternal epithelium (ASE) than in the posterior sternal epithelium (PSE). The results demonstrate for the first time the expression of a PMCA and a NCE in the hypodermal epithelium of a crustacean and indicate a contribution of these transport mechanisms in ECT.     

Phosphorylated intermediates of (Ca2+ + Mg2+)-ATPase and alkaline phosphatase in renal plasma membranes

Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [gamma-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as (Na+ + K+)-ATPase and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius Mr of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 microM) in a similar way as the (Ca2+ + Mg2+)-ATPase from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte (Ca2+ + Mg2+)-ATPase. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal (Na+ + K+)-ATPase. In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.