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Objective: Copper (Cu) is essential micronutrient, and its dysregulation is implicated in aortic aneurysm (AA) development. The Cu exporter ATP7A (copper-transporting P-type ATPase/Menkes ATPase) delivers Cu via the Cu chaperone Atox1 (antioxidant 1) to secretory Cu enzymes, such as lysyl oxidase, and excludes excess Cu. Lysyl oxidase is shown to protect against AA formation. However, the role and mechanism of ATP7A in AA pathogenesis remain unknown. Approach and Results: Here, we show that Cu chelator markedly inhibited Ang II (angiotensin II)–induced abdominal AA (AAA) in which ATP7A expression was markedly downregulated. Transgenic ATP7A overexpression prevented Ang II–induced AAA formation. Conversely, Cu transport dysfunctional ATP7A mut/+ /ApoE −/− mice exhibited robust AAA formation and dissection, excess aortic Cu accumulation as assessed by X-ray fluorescence microscopy, and reduced lysyl oxidase activity. In contrast, AAA formation was not observed in Atox1 −/− /ApoE −/− mice, suggesting that decreased lysyl oxidase activity, which depends on both ATP7A and Atox1, was not sufficient to develop AAA. Bone marrow transplantation suggested importance of ATP7A in vascular cells, not bone marrow cells, in AAA development. MicroRNA (miR) array identified miR-125b as a highly upregulated miR in AAA from ATP7A mut/+ /ApoE −/− mice. Furthermore, miR-125b target genes (histone methyltransferase Suv39h1 and the NF-κB negative regulator TNFAIP3 [tumor necrosis factor alpha induced protein 3]) were downregulated, which resulted in increased proinflammatory cytokine expression, aortic macrophage recruitment, MMP (matrix metalloproteinase)-2/9 activity, elastin fragmentation, and vascular smooth muscle cell loss in ATP7A mut/+ /ApoE −/− mice and reversed by locked nucleic acid-anti-miR-125b infusion. Conclusions: ATP7A downregulation/dysfunction promotes AAA formation via upregulating miR-125b, which augments proinflammatory signaling in a Cu-dependent manner. Thus, ATP7A is a potential therapeutic target for inflammatory vascular disease.

Background: VEGFR2 (KDR/Flk1) signaling in endothelial cells (ECs) plays a central role in angiogenesis. Copper (Cu) is essential micronutrient and has been implicated in angiogenesis. The P-type ATPase transporter ATP7A is key regulator of Cu homeostasis but its role in VEGFR2 signaling in ECs and post-ischemic neovascularization is entirely unknown. Results: Here we show that ATP7A expression was dramatically increased in the angiogenic ECs in mice hindlimb ischemia model. EC-specific ATP7A deficient mice or Cu transporter-dysfunctional ATP7A mut mice showed significant decrease in blood flow recovery and CD31+ capillary density (angiogenesis) in ischemic tissues compared to their control mice. In cultured human ECs, ATP7A knockdown with siRNA significantly inhibited VEGF-induced migration (67%), capillary network formation on Matrigel (46%). Immunofluorescence, co-immunoprecipitation, and proximity ligation assays showed that VEGF stimulated ATP7A translocation from the trans-Golgi network to the plasma membrane where it bound to VEGFR2. Surprisingly, loss of ATP7A promoted VEGF-induced VEGFR2 protein degradation (56%) and inhibited VEGFR2 signaling via enhancing VEGFR2 ubiquitination (2.0-fold) in a Cu-independent manner. This was associated with reduced cell surface VEGFR2 expression, increased VEGFR2 binding to selective autophagic cargo/adaptor p62/SQSTM1 (60%) and LC3-GFP+autophagic puncta (34%) and autolysosome formation (transmission electron microscopy). Inhibition of autophagy by bafilomycin A1 or chloroquine prevented enhanced VEGFR2 degradation in ATP7A-depleted ECs. Furthermore, overexpression of p62, but not p62 lacking ubiquitin binding domain, or autophagy inducer rapamycin treatment increased VEGFR2 degradation (26% and 87%, respectively) and p62 overexpression inhibited ECs migration (29%). Enhanced autophagy flux due to ATP7A dysfunction in vivo was confirmed by employing autophagy reporter CAG-ATP7A mut -RFP-EGFP-LC3 transgenic mice. Conclusion: Our study uncovers the unexpected and novel function of ATP7A to limit autophagic degradation of VEGFR2, thereby promoting VEGFR2 signaling and angiogenesis, which restores neovascularization in ischemic disease.

The central importance of the role of the sodium pump in almost all eukaryotic cells is by now well appreciated. In cells of the central nervous system the sodium pump is responsible for maintaining the low intracellular [Na] following the inward Na currents of action potentials. In cells of the kidney and gastrointestinal system and in some (nucleated) red blood cells the sodium pump is required to maintain an inwardly directed Na gradient, the energy of which is used to accumulate solutes, and in many cells the sodium pump in intimately involved in the regulation and control of cell volume. The structure, function, and mechanism of the sodium pump has been the subject of several recent reviews [1–6]. In the present chapter the focus will be on the sodium pump of human red blood cells. The substantial and important literature on other systems that has contributed to our understanding of the sodium pump will only be drawn on to understand the red blood cell phenomena.

Vascular endothelial growth factor receptor type 2 (VEGFR2, also known as KDR and FLK1) signalling in endothelial cells (ECs) is essential for developmental and reparative angiogenesis. Reactive oxygen species and copper (Cu) are also involved in these processes. However, their inter-relationship is poorly understood. Evidence of the role of the endothelial Cu importer CTR1 (also known as SLC31A1) in VEGFR2 signalling and angiogenesis in vivo is lacking. Here, we show that CTR1 functions as a redox sensor to promote angiogenesis in ECs. CTR1-depleted ECs showed reduced VEGF-induced VEGFR2 signalling and angiogenic responses. Mechanistically, CTR1 was rapidly sulfenylated at Cys189 at its cytosolic C terminus after stimulation with VEGF, which induced CTR1-VEGFR2 disulfide bond formation and their co-internalization to early endosomes, driving sustained VEGFR2 signalling. In vivo, EC-specific Ctr1-deficient mice or CRISPR-Cas9-generated redox-dead Ctr1(C187A)-knockin mutant mice had impaired developmental and reparative angiogenesis. Thus, oxidation of CTR1 at Cys189 promotes VEGFR2 internalization and signalling to enhance angiogenesis. Our study uncovers an important mechanism for sensing reactive oxygen species through CTR1 to drive neovascularization.

VEGFR2 (KDR/Flk1) signaling in endothelial cells (ECs) plays a central role in angiogenesis. The P-type ATPase transporter ATP7A regulates copper homeostasis, and its role in VEGFR2 signaling and angiogenesis is entirely unknown. Here, we describe the unexpected crosstalk between the Copper transporter ATP7A, autophagy, and VEGFR2 degradation. The functional significance of this Copper transporter was demonstrated by the finding that inducible EC-specific ATP7A deficient mice or ATP7A-dysfunctional ATP7Amut mice showed impaired post-ischemic neovascularization. In ECs, loss of ATP7A inhibited VEGF-induced VEGFR2 signaling and angiogenic responses, in part by promoting ligand-induced VEGFR2 protein degradation. Mechanistically, VEGF stimulated ATP7A translocation from the trans-Golgi network to the plasma membrane where it bound to VEGFR2, which prevented autophagy-mediated lysosomal VEGFR2 degradation by inhibiting autophagic cargo/adapter p62/SQSTM1 binding to ubiquitinated VEGFR2. Enhanced autophagy flux due to ATP7A dysfunction in vivo was confirmed by autophagy reporter CAG-ATP7Amut -RFP-EGFP-LC3 transgenic mice. In summary, our study uncovers a novel function of ATP7A to limit autophagy-mediated degradation of VEGFR2, thereby promoting VEGFR2 signaling and angiogenesis, which restores perfusion recovery and neovascularization. Thus, endothelial ATP7A is identified as a potential therapeutic target for treatment of ischemic cardiovascular diseases., The role of endothelial copper transporter ATP7A in vascular function and angiogenesis remains largely unexplored. Here the authors show that ATP7A promotes VEGFR2 signaling and angiogenesis by limiting autophagy-mediated degradation of VEGFR2, which enhances reparative neovascularization.

The Na,K-ATPase α2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, whereas α1 has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α2 in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α2 is preferentially expressed with β2 in T-tubules of cardiac myocytes, forming α2β2 heterodimers. We have expressed human α1β1, α2β1, α2β2, and α2β3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2β2 and α2β3 differ significantly from both α2β1 and α1β1 in having a higher K0.5K+ and lower K0.5Na+ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1P-E2P conformational equilibrium toward E1P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α2β2 and α2β3 over α1β1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K+ affinity of α2β2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2β2 for cardiac muscle contractility. The high sensitivity of α2β2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2β2-selective digoxin derivatives for reducing cardiotoxicity.

The photolysis quantum yield, Qp, of 1-(2-nitrophenyl)ethyl phosphate (caged Pi) measured in the near-UV (342 nm peak with 60 nm half-bandwidth) is 0.53 and is based on results reported in 1978 (Biochemistry, 17, 1929-1935). This article amplifies methodology for determining that Qp in view of different recent estimates. Some general principles together with other examples relating to measurement of Qp values are discussed together with their relevance to biological research.

Copper (Cu) has emerged as an important modifier of body lipid metabolism. However, how Cu contributes to the physiology of fat cells remains largely unknown. We found that adipocytes require Cu to establish a balance between main metabolic fuels. Differentiating adipocytes increase their Cu uptake along with the ATP7A-dependent transport of Cu into the secretory pathway to activate a highly up-regulated amino-oxidase copper-containing 3 (AOC3)/semicarbazide-sensitive amine oxidase (SSAO); in vivo, the activity of SSAO depends on the organism's Cu status. Activated SSAO oppositely regulates uptake of glucose and long-chain fatty acids and remodels the cellular proteome to coordinate changes in fuel availability and related downstream processes, such as glycolysis, de novo lipogenesis, and sphingomyelin/ceramide synthesis. The loss of SSAO-dependent regulation due to Cu deficiency, limited Cu transport to the secretory pathway, or SSAO inactivation shifts metabolism towards lipid-dependent pathways and results in adipocyte hypertrophy and fat accumulation. The results establish a role for Cu homeostasis in adipocyte metabolism and identify SSAO as a regulator of energy utilization processes in adipocytes.

Copper (Cu) is an essential micronutrient but excess Cu is potentially toxic. Its important propensity to cycle between two oxidation states accounts for its frequent presence as a cofactor in many physiological processes through Cu-containing enzymes, including mitochondrial energy production (via cytochrome c-oxidase), protection against oxidative stress (via superoxide dismutase), and extracellular matrix stability (via lysyl oxidase). Since free Cu is potentially toxic, the bioavailability of intracellular Cu is tightly controlled by Cu transporters and Cu chaperones. Recent evidence reveals that these Cu transport systems play an essential role in the physiological responses of cardiovascular cells, including cell growth, migration, angiogenesis and wound repair. In response to growth factors, cytokines, and hypoxia, their expression, subcellular localization, and function are tightly regulated. Cu transport systems and their regulators have also been linked to various cardiovascular pathophysiologies such as hypertension, inflammation, atherosclerosis, diabetes, cardiac hypertrophy, and cardiomyopathy. A greater appreciation of the central importance of Cu transporters and Cu chaperones in cell signaling and gene expression in cardiovascular biology offers the possibility of identifying new therapeutic targets for cardiovascular disease.

This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na(+)/Ca(2+) exchange (NCX) and Na(+)/K(+)-ATPase (NKA). While the relevance of Ca(2+) homeostasis in cardiac function has been extensively investigated, the role of Na(+) regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na(+) content have multiple effects on the heart by influencing intracellular Ca(2+) and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na(+) homeostasis. Among the proteins that accomplish this task are the Na(+)/Ca(2+) exchanger (NCX) and the Na(+)/K(+) pump (NKA). By transporting three Na(+) ions into the cytoplasm in exchange for one Ca(2+) moved out, NCX is one of the main Na(+) influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na(+) ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na(+) and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na(+) homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)/K(+) pump and the controversies that still persist in the field.

Restoration of the functional potency of pancreatic islets either through enhanced proliferation (hyperplasia) or increase in size (hypertrophy) of beta cells is a major objective for intervention in diabetes. We have obtained experimental evidence that global knock-out of a small, single-span regulatory subunit of Na,K-ATPase, FXYD2, alters glucose control. Adult Fxyd2−/− mice showed significantly lower blood glucose levels, no signs of peripheral insulin resistance, and improved glucose tolerance compared with their littermate controls. Strikingly, there was a substantial hyperplasia in pancreatic beta cells from the Fxyd2−/− mice compared with the wild type littermates, compatible with an observed increase in the level of circulating insulin. No changes were seen in the exocrine compartment of the pancreas, and the mice had only a mild, well-adapted renal phenotype. Morphometric analysis revealed an increase in beta cell mass in KO compared with WT mice. This appears to explain a phenotype of hyperinsulinemia. By RT-PCR, Western blot, and immunocytochemistry we showed the FXYD2b splice variant in pancreatic beta cells from wild type mice. Phosphorylation of Akt kinase was significantly higher under basal conditions in freshly isolated islets from Fxyd2−/− mice compared with their WT littermates. Inducible expression of FXYD2 in INS 832/13 cells produced a reduction in the phosphorylation level of Akt, and phosphorylation was restored in parallel with degradation of FXYD2. Thus we suggest that in pancreatic beta cells FXYD2 plays a role in Akt signaling pathways associated with cell growth and proliferation. Background: Reduction in functional beta cells in pancreas is the major obstacle in diabetes. Results: Mice deficient in FXYD2 subunit of Na,K-ATPase possess a metabolic phenotype of low blood glucose along with hyperplastic pancreatic islets and hyperinsulinemia. Conclusion: The phenotype observed in Fxyd2−/− mice results from an increase in beta cell mass. Significance: FXYD2 may be a novel target for development of cell-based interventions in diabetes.

Cu is an essential micronutrient, and its role in an array of critical physiological processes is receiving increasing attention. Among these are wound healing, angiogenesis, protection against reactive oxygen species, neurotransmitter synthesis, modulation of normal cell and tumor growth, and many others. Free Cu is absent inside cells, and a network of proteins has evolved to deliver this essential, but potentially toxic, metal ion to its intracellular target sites following uptake. Although the total body content is low (∼100 mg), dysfunction of proteins involved in Cu homeostasis results in several well-characterized human disease states. The initial step in cellular Cu handling is its transport across the plasma membrane, a subject of study for only about the last 25 years. This review focuses on the initial step in Cu homeostasis, the properties of the major protein, hCTR1, that mediates Cu uptake, and the status of our understanding of this highly specialized transport system. Although a high-resolution structure of the protein is still lacking, an array of biochemical and biophysical studies have provided a picture of how hCTR1 mediates Cu(I) transport and how Cu is delivered to the proteins in the intracellular milieu. Recent studies provide evidence that the transporter also plays a key protective role in the regulation of cellular Cu via regulatory endocytosis, lowering its surface expression, in response to elevated Cu loads.

Cu ion (Cu) entry into human cells is mediated by CTR1 (also known as SLC31A1), the high-affinity Cu transporter. When extracellular Cu is raised, the cell is protected against excess accumulation by rapid internalization of the transporter. When Cu is lowered, the transporter returns to the membrane. We show in HEK293 cells overexpressing CTR1 that expression of either the C-terminal domain of AP180 (also known as SNAP91), a clathrin-coat assembly protein that sequesters clathrin, or a dominant-negative mutant of dynamin, decreases Cu-induced endocytosis of CTR1, as does a dynamin inhibitor and clathrin knockdown using siRNA. Utilizing imaging, siRNA techniques and a new high-throughput assay for endocytosis employing CLIP-tag methodology, we show that internalized CTR1 accumulates in early sorting endosomes and recycling compartments (containing Rab5 and EEA1), but not in late endosomes or lysosomal pathways. Using live cell fluorescence, we find that upon extracellular Cu removal CTR1 recycles to the cell surface through the slower-recycling Rab11-mediated pathway. These processes enable cells to dynamically alter transporter levels at the plasma membrane and acutely modulate entry as a safeguard against excess cellular Cu.

To catalyze ion transport, the Na,K-ATPase must contain one α and one β subunit. When expressed by transfection in various expression systems, each of the four α subunit isoforms can assemble with each of the three β subunit isoforms and form an active enzyme, suggesting the absence of selective α-β isoform assembly. However, it is unknown whether in vivo conditions the α-β assembly is random or isoform-specific. The α(2)-β(2) complex was selectively immunoprecipitated by both anti-α(2) and anti-β(2) antibodies from extracts of mouse brain, which contains cells co-expressing multiple Na,K-ATPase isoforms. Neither α(1)-β(2) nor α(2)-β(1) complexes were detected in the immunoprecipitates. Furthermore, in MDCK cells co-expressing α(1), β(1), and β(2) isoforms, a greater fraction of the β(2) subunits was unassembled with α(1) as compared with that of the β(1) subunits, indicating preferential association of the α(1) isoform with the β(1) isoform. In addition, the α(1)-β(2) complex was less resistant to various detergents than the α(1)-β(1) complex isolated from MDCK cells or the α(2)-β(2) complex isolated from mouse brain. Therefore, the diversity of the α-β Na,K-ATPase heterodimers in vivo is determined not only by cell-specific co-expression of particular isoforms, but also by selective association of the α and β subunit isoforms.

Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ∼90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60–70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in dietary copper acquisition.

Copper is an essential co-factor in many important physiological processes, but at elevated levels it is toxic to cells. Thus at both the organism and cellular level mechanisms have evolved to finely tune copper homeostasis. The protein responsible for copper entry from the circulation in most human cells is hCTR1, a small protein (190 amino acid residues) that functions as a trimer in the plasma membrane. In the present work we employ cell surface biotinylation and isotopic copper uptake studies of overexpressed hCTR1 in HEK293 cells to examine the acute (minutes) response of hCTR1 to changes in extracellular copper. We show that within 10 min of exposure to copper at 2.5 microM or higher, plasma membrane hCTR1 levels are reduced (by approximately 40%), with a concomitant reduction in copper uptake rates. We are unable to detect any degradation of internalized hCTR1 in the presence of cycloheximide after up to 2 h of exposure to 0-100 microM copper. Using a reversible biotinylation assay, we quantified internalized hCTR1, which increased upon the addition of copper and corresponded to the hCTR1 lost from the surface. In addition, when extracellular copper is then removed, internalized hCTR1 is promptly (within 30 min) recycled to the plasma membrane. We have shown that in the absence of added extracellular copper, there is a small but detectable amount of internalized hCTR1 that is increased in the presence of copper. Similar studies on endogenous hCTR1 show a cell-specific response to elevated extracellular copper. Copper-dependent internalization and recycling of hCTR1 provides an acute and reversible mechanism for the regulation of cellular copper entry.

The human copper transporter hCTR1 is a homotrimer composed of a plasma membrane protein of 190 amino acids that contains three transmembrane segments. The extracellular 65-amino acid amino terminus of hCTR1 contains both N-linked (at Asn(15)) and O-linked (at Thr(27)) sites of glycosylation. If O-glycosylation at Thr(27) is prevented, hCTR1 is efficiently cleaved, removing approximately 30 amino acids from the amino terminus. We have now investigated (i) the site of this cleavage, determining which peptide bonds are cleaved, (ii) the mechanism by which glycosylation prevents cleavage, and (iii) where in the cell the proteolytic cleavage takes place. Cleavage occurs in the sequence Ala-Ser-His-Ser-His (residues 29-33), which does not contain previously recognized protease cleavage sites. Using a series of hCTR1 mutants, we show that cleavage occurs preferentially between residues Ala(29)-Ser(30)-His(31). We also show that the O-linked polysaccharide at Thr(27) blocks proteolysis due to its proximity to the cleavage site. Moving the cleavage site away from the Thr(27) polysaccharide by insertion of as few as 5 amino acids allows cleavage to occur in the presence of glycosylation. Imaging studies using immunofluorescence in fixed cells and a functional green fluorescent protein-tagged hCTR1 transporter in live cells showed that the cleaved peptide accumulates in punctate structures in the cytoplasm. These puncta overlap compartments were stained by Rab9, indicating that hCTR1 cleavage occurs in a late endosomal compartment prior to delivery of the transporter to the plasma membrane.

The Na,K-ATPase is an alphabeta heterodimer responsible for maintaining fluid and electrolyte homeostasis in mammalian cells. We engineered Madin-Darby canine kidney cell lines expressing alpha(1)FLAG, beta(1)FLAG, or beta(2)MYC subunits via a tetracycline-regulated promoter and a line expressing both stable beta(1)MYC and tetracycline-regulated beta(1)FLAG to examine regulatory mechanisms of sodium pump subunit expression. When overexpression of exogenous beta(1)FLAG increased total beta subunit levels by200% without changes in alpha subunit abundance, endogenous beta(1) subunit (beta(1)E) abundance decreased. beta(1)E down-regulation did not occur during beta(2)MYC overexpression, indicating isoform specificity of the repression mechanism. Measurements of RNA stability and content indicated that decreased beta subunit expression was not accompanied by any change in mRNA levels. In addition, the degradation rate of beta subunits was not altered by beta(1)FLAG overexpression. Cells stably expressing beta(1)MYC, when induced to express beta(1)FLAG subunits, showed reduced beta(1)MYC and beta(1)E subunit abundance, indicating that these effects occur via the coding sequences of the down-regulated polypeptides. In a similar way, Madin-Darby canine kidney cells overexpressing exogenous alpha(1)FLAG subunits exhibited a reduction of endogenous alpha(1) subunits (alpha(1)E) with no change in alpha mRNA levels or beta subunits. The reduction in alpha(1)E compensated for alpha(1)FLAG subunit expression, resulting in unchanged total alpha subunit abundance. Thus, regulation of alpha subunit expression maintained its native level, whereas beta subunit was not as tightly regulated and its abundance could increase substantially over native levels. These effects also occurred in human embryonic kidney cells. These data are the first indication that cellular sodium pump subunit abundance is modulated by translational repression. This mechanism represents a novel, potentially important mechanism for regulation of Na,K-ATPase expression.

Human Cu-ATPases ATP7A and ATP7B maintain copper homeostasis through regulated trafficking between intracellular compartments. Inactivation of these transporters causes Menkes disease and Wilson disease, respectively. In Menkes disease, copper accumulates in kidneys and causes tubular damage, indicating that the renal ATP7B does not compensate for the loss of ATP7A function. We show that this is likely due to a kidney-specific regulation of ATP7B. Unlike ATP7A (or hepatic ATP7B) which traffics from the TGN to export copper, renal ATP7B does not traffic and therefore is unlikely to mediate copper export. The lack of ATP7B trafficking is not on account of the loss of a kinase-mediated phosphorylation or simultaneous presence of ATP7A in renal cells. Rather, the renal ATP7B appears 2-3 kDa smaller than hepatic ATP7B. Recombinant ATP7B expressed in renal cells is similar to hepatic protein in size and trafficking. The analysis of ATP7B mRNA revealed a complex behavior of exon 1 upon amplification, suggesting that it could be inefficiently translated. Recombinant ATP7B lacking exon 1 traffics differently in renal and hepatic cells, but does not fully recapitulate the endogenous phenotype. We discuss factors that may contribute to cell-specific behavior of ATP7B and propose a role for renal ATP7B in intracellular copper storage.

Kidneys regulate their copper content more effectively than many other organs in diseases of copper deficiency or excess. We demonstrate that two copper-transporting ATPases, ATP7A and ATP7B, contribute to this regulation. ATP7A is expressed, to a variable degree, throughout the kidney and shows age-dependent intracellular localization. In 2-wk-old mice, ATP7A is located in the vicinity of the basolateral membrane, whereas in 20-wk-old mice, ATP7A is predominantly in intracellular vesicles. Acute elevation of serum copper, via intraperitoneal injection, results in the in vivo redistribution of ATP7A from intracellular compartments toward the basolateral membrane, illustrating a role for ATP7A in renal response to changes in copper load. Renal copper homeostasis also requires functional ATP7B, which is coexpressed with ATP7A in renal cells of proximal and distal origin. The kidneys of Atp7b−/−mice, an animal model of Wilson disease, show metabolic alterations manifested by the appearance of highly fluorescent deposits; however, in marked contrast to the liver, renal copper is not significantly elevated. The lack of notable copper accumulation in the Atp7b−/−kidney is likely due to the compensatory export of copper by ATP7A. This interpretation is supported by the predominant localization of ATP7A at the basolateral membrane of Atp7b−/−cortical tubules. Our results suggest that both Cu-ATPases regulate renal copper, with ATP7A playing a major role in exporting copper via basolateral membranes and protecting renal tissue against copper overload.

In epithelial cells, the sodium pump, in coordination with several other ion transporting proteins and channels, acts to regulate directional water and ion flux across the epithelial barrier. This function is dependant on the polarized localization of the sodium pump to a single plasma membrane domain. In most epithelial cell types the sodium pump is found in an exclusively basolateral position. Despite the clear importance of maintaining a polarized distribution of the sodium pump, surprisingly little is known about the specific mechanisms responsible for the targeting and trafficking of the sodium pump to the basolateral surface. We briefly discuss our current understanding of factors which may act to regulate the cellular distribution of the sodium pump, including the potential role of the sodium pump beta-subunit. Several previous, studies have suggested that the expression of the beta2 isoform (instead of beta1) may cause the apical localization of the sodium pump. This appeared to be confirmed by Wilson et al. Am J Pathol, 156: 253-268, 2000 who found that MDCK cells stably transfected with the beta2 subunit express the sodium pump at the apical surface. However, careful examination by Laughery et al.,Am J Physiol, 292: F1718-F1725, 2007, showed that the apical targeting of the pump was caused by the presence of butyrate in the cell growth media and was not due to the presence of the beta2 isoform. These findings are discussed below, along with potential explanations as to how butyrate may influence the polarity of the sodium pump in epithelial cells.

Copper is essential for human growth and survival. Enterocytes mediate the absorption of dietary copper from the intestinal lumen into blood as well as utilizing copper for their biosynthetic needs. Currently, the pathways for copper entry into enterocytes remain poorly understood. We demonstrate that the basolateral copper uptake into intestinal cells greatly exceeds the apical uptake. The basolateral but not apical transport is mediated by the high affinity copper transporter hCTR1. This unanticipated conclusion is supported by cell surface biotinylation and confocal microscopy of endogenous hCTR1 in Caco2 cells as well as copper influx measurements that show saturable high affinity uptake at the basolateral but not the apical membrane. Basolateral localization of hCTR1 and polarized copper uptake are also conserved in T84 cells, models for intestinal crypt cells. The lateral localization of hCTR1 seen in intestinal cell lines is recapitulated in immunohistochemical staining of mouse intestinal sections. Biochemical and functional assays reveal the basolateral localization of hCTR1 also in renal Madin-Darby canine kidney cells and opossum kidney cells. Overexpression of hCTR1 in Madin-Darby canine kidney cells results in both apical and basolateral delivery of the overexpressed protein and greatly enhanced copper uptake at both cell surfaces. We propose a model of intestinal copper uptake in which basolateral hCTR1 plays a key role in the physiologically important delivery of copper from blood to intracellular proteins, whereas its role in the initial apical uptake of dietary copper is indirect.

In this brief review we summarize what is known about the role of hCTR1 in mediating the entry of copper into human cells. There is a body of information that clearly identifies this protein as being a major source (though not the only source) of copper entry into human cells, and thus a crucial element of copper homeostasis. However, much remains that is poorly understood and key aspects of the physiological roles of hCTR1 and its regulation are only superficially appreciated. The particular characteristics of a transport process that in vivo involves the binding, transmembrane transport and release of a substrate that is not present in a free form in the intracellular or extracellular compartments poses particular challenges that are not encountered in the transport of more familiar physiologically important metal cations. Thus much of what we have learned about the more commonly encountered transported ions provides an inadequate model for studies of copper homeostasis. In this article we review progress made and identify the major questions that need to be resolved before an adequate description is attained of how copper entry into human cells is mediated and regulated by hCTR1.

Cellular copper uptake is a prerequisite for the biosynthesis of many copper-dependent enzymes; disruption of copper uptake results in embryonic lethality. In humans, copper is transported into cells by hCTR1, a membrane protein, composed of 190 amino acids with only three trans-membrane segments. To provide insight into the mechanism of this unusual transporter, we characterized the functional properties of various hCTR1 mutants stably expressed in Sf9 cells. Most single amino acid substitutions involving charged and potential copper-coordinating residues have some influence on the Vmax and Km values for copper uptake but do not greatly alter hCTR1-mediated copper transport. However, there were two notable exceptions. Replacement of Tyr156 with Ala greatly reduced the maximal transport rate without effect on the Km value for copper. Also, replacement of His139 in the second trans-membrane segment with Arg caused a dramatic increase in the rate of copper uptake and a large increase in the Km value for copper. This effect was not seen with an Ala replacement, pointing to the role of a positive charge in modulating copper exit from the pathway. Truncated mutants demonstrated that the deletion of a large portion of the N-terminal domain only slightly decreased the apparent Km value for copper and decreased the rate of transport. Similar effects were observed with the removal of the last 11 C-terminal residues. The results suggested that the N and C termini, although nonessential for transport, may have an important role in facilitating the delivery of copper to and retrieving copper from, respectively, the translocation pathway. A model of how hCTR1 mediates copper entry into cells was proposed that included a rate-limiting site in the pore close to the intracellular exit.

Cysteine-to-serine mutants of a maltose binding protein fusion with the human copper chaperone for superoxide dismutase (hCCS) were studied with respect to (i) their ability to transfer Cu to E,Zn superoxide dismutase (SOD) and (ii) their Zn and Cu binding and X-ray absorption spectroscopic (XAS) properties. Previous work has established that Cu(I) binds to four cysteine residues, two of which, C22 and C25, reside within an Atox1-like N-terminal domain (DI) and two of which, C244 and C246, reside in a short unstructured polypeptide chain at the C-terminus (DIII). The wild-type (WT) protein shows an extended X-ray absorption fine structure (EXAFS) spectrum characteristic of cluster formation, but it is not known how such a cluster is formed. Cys to Ser mutagenesis was used to investigate the Cu binding in more detail. Single Cys to Ser mutations, as represented by C22S and C244S, did little to affect the metal binding ratios of hCCS. Both mutants still showed approximately 2 Cu(I) ions and 1 Zn ion per protein. The double mutants C22/24S and C244/246S, on the other hand, showed Cu binding stoichiometries close to 1:1. The Zn-EXAFS of WT CCS showed a 3-4 histidine ligand environment that is consistent with Zn binding in the SOD-like domain II of CCS. The Zn environment remained unchanged between wild type and all of the mutant CCS proteins. Single Cys to Ser mutations displayed lower activity than WT protein, although close to full activity could be rescued by increasing the CCS:SOD ratios to 8:1 in the assay mixture. The structure of the Cu centers of the single mutants as revealed by EXAFS was also similar to that of WT protein, with clear indications of a Cu cluster. On the other hand, the double mutants showed a greater degree of perturbation. The DI C22/25S mutant was 70% active and formed a cluster with a more intense Cu-Cu interaction. The DIII C244/246S mutant retained only a fraction (16%) of activity and did not form a cluster. The results suggest the formation of a DIII-DIII cluster within a dimeric or tetrameric protein and further suggest that this cluster may be an important element of the copper transfer machinery.

The reaction mechanism of the Na,K-ATPase is thought to involve a number of ligand-induced conformational changes. The specific amino acid residues responsible for binding many of the important ligands have been identified; however, details of the specific conformational changes produced by ligand binding are largely undescribed. The experiments described in this paper begin to identify interactions between domains of the Na,K-ATPase alpha-subunit that depend on the presence of particular ligands. The major cytoplasmic loop (between TM4 and TM5), which we have previously shown contains the ATP-binding domain, was overexpressed in bacteria either with a His(6) tag or as a fusion protein with glutathione S-transferase. We have observed that these polypeptides associate in the presence of MgATP. Incubation with [gamma-(32)P]ATP under conditions that result in phosphorylation of the full-length Na,K-ATPase did not result in (32)P incorporation into either the His(6) tag or glutathione S-transferase fusion proteins. The MgATP-induced association was strongly inhibited by prior modification of the fusion proteins with fluorescein isothiocyanate or by simultaneous incubation with 10 microm eosin, indicating that the effect of MgATP is due to interactions within the nucleotide-binding domain. These data are consistent with Na,K-ATPase associating within cells via interactions in the nucleotide-binding domains. Although any functional significance of these associations for ion transport remains unresolved, they may play a role in cell function and in modulating interactions between the Na,K-ATPase and other proteins.

We have expressed hCTR1, the human copper transporter, in Sf9 cells using a baculovirus-mediated expression system, and we observed greatly enhanced copper uptake. Western blots showed that the protein is delivered to the plasma membrane, where it mediates saturable copper uptake with a K(m) of approximately 3.5 microm. We also expressed functional transporters where the N-linked glycosylation sites were substituted, and we provided evidence for the extracellular location of the amino terminus. Accessibility of amino-terminal FLAG epitope to antibody prior to permeabilization and of carboxyl-terminal FLAG only after permeabilization confirmed the extracellular location of the amino terminus and established the intracellular location of the carboxyl terminus. Tryptic digestion of hCTR1 occurred within the cytoplasmic loop and generated a 10-Da carboxyl-terminal peptide; cleavage was prevented by the presence of copper. hCTR1 mutants where Cys-161 and Cys-189, the two native cysteines, were replaced with serines also mediated copper uptake, indicating that neither cysteine residue was essential for transport. However, the mutants provided evidence that these residues may stabilize hCTR1 oligomerization. Western blots of hCTR1 in Sf9 cells showed expression levels 100-fold higher than in mammalian (HepG2) cells. The high level of functional expression and the low level of endogenous copper uptake will enable future structure-function analysis of this important protein.

Copper-transporting ATPase ATP7B is essential for normal distribution of copper in human cells. Mutations in the ATP7B gene lead to copper accumulation in a number of tissues and to a severe multisystem disorder, known as Wilson's disease. Primary sequence analysis suggests that the copper-transporting ATPase ATP7B or the Wilson's disease protein (WNDP) belongs to the large family of cation-transporting P-type ATPases, however, the detailed characterization of its enzymatic properties has been lacking. Here, we developed a baculovirus-mediated expression system for WNDP, which permits direct and quantitative analysis of catalytic properties of this protein. Using this system, we provide experimental evidence that WNDP has functional properties characteristic of a P-type ATPase. It forms a phosphorylated intermediate, which is sensitive to hydroxylamine, basic pH, and treatments with ATP or ADP. ATP stimulates phosphorylation with an apparent K(m) of 0.95 +/- 0.25 microm; ADP promotes dephosphorylation with an apparent K(m) of 3.2 +/- 0.7 microm. Replacement of Asp(1027) with Ala in a conserved sequence motif DKTG abolishes phosphorylation in agreement with the proposed role of this residue as an acceptor of phosphate during the catalytic cycle. Catalytic phosphorylation of WNDP is inhibited by the copper chelator bathocuproine; copper reactivates the bathocuproine-treated WNDP in a specific and cooperative fashion confirming that copper is required for formation of the acylphosphate intermediate. These studies establish the key catalytic properties of the ATP7B copper-transporting ATPase and provide a foundation for quantitative analysis of its function in normal and diseased cells.

Renal tissue hypoxia is a major cause of acute renal failure. The goal of this study was to elucidate the mechanism(s) underlying role of the Na,K-ATPase in the adaptive response of renal cells to a hypoxic stress. Kidney microsome membranes were isolated from mice exposed to 7% O2 (hypoxia) or to room air for three days, proteins were extracted by a non-ionic detergent, immunoprecipitated using anti-Na,K-ATPase antibodies, and analyzed by nLC-MS/MS and Western blot analyses. Hypoxia did not change the total amount of the Na,K-ATPase, as was assessed by comparing the Mascot scores and confirmed by a Western blot analysis. In contrast, hypoxia significantly decreased the amount of the Na,K-ATPase-co-immunoprecipitated ER chaperones and enzymes presumably involved in the maturation of the Na,K-ATPase, including BiP, Hsp40, calnexin, PDI, glucosidase, mannosidase and DnaJ. Consistent with these results, a significant retention of the Na,K-ATPase was observed in cultured epithelial cells exposed to hypoxia. T...

Copper is an essential element for human growth and development. Abnormal copper metabolism is associated with severe and potentially lethal disorders. Previous studies of copper misbalance in the ...

The Na+-K+-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na+-K+-ATPase activity. We expressed sheep kidney Na+-K+-ATPase α- and β-subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na+-K+-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8–2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na+pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [3H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na+-K+-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive86Rb+uptake in whole cells as a means to specifically evaluate Na+-K+-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the α-subunit or β-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the α-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the β-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the α-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the α-β heterodimer within the endoplasmic reticulum apparently does not require a Na+pump-specific chaperone.

The Na,K-ATPase carries out the coupled functions of ATP hydrolysis and cation transport. These functions are performed by two distinct regions of the protein. ATP binding and hydrolysis is mediated by the large central cytoplasmic loop of about 430 amino-acids. Transmembrane cation transport is accomplished via coordination of the Na and K ions by side-chains of the amino-acids of several of the transmembrane segments. The way in which these two protein domains interact lies at the heart of the molecular mechanism of active transport, or ion pumping. We summarize evidence obtained from protein chemistry studies of the purified renal Na,K-ATPase and from bacterially expressed polypeptides which characterize these separate functions and point to various movements which may occur as the protein transits through its reaction cycle. We then describe recent work using heterologous expression of renal Na,K-ATPase in baculovirus-infected insect cells which provides a suitable system to characterize such protein motions and which can be employed to test specific models arising from recently acquired high resolution structural information on related ion pumps.

Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu) plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX), a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2) also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC). In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α) with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness.

We have constructed a mutant Na,K-ATPase alpha1-subunit with all native cysteine residues replaced. Using the baculovirus system, this cysteine-less alpha1-subunit and wild-type beta1-subunit were expressed in High Five cells. After 3 days of infection, cells were fractionated, and endoplasmic reticulum, Golgi apparatus, and plasma membranes were isolated. The molecular activity of the cysteine-less mutant in the plasma membranes was close to the wild-type protein (8223 min(-)(1) versus 6655 min(-)(1)). Cation and ATP activation of Na,K-ATPase activities revealed that replacing all 23 cysteines resulted in only a 50% reduction of K(m) for Na(+), a 2-fold increase in K(m) for K(+), and no changes in K(m) for ATP. The distribution of alpha-subunits among the membranes showed a high percentage of cysteine-less protein in the endoplasmic reticulum and Golgi apparatus compared with the wild-type protein. Furthermore, the cellular stability of the alphabeta assembly appeared reduced in the cysteine-less mutant. Cells harvested after more than 3 days of infection showed extensive degradation of the cysteine-less alpha-subunit, which is not observed with the wild-type enzyme. Thus the Na,K-ATPase contains no cysteine residues that are critical for function, but the folding and/or assembly pathway of this enzyme is affected by total cysteine substitution.

We have mapped the membrane topology of the renal Na,K-ATPase α-subunit by using a combination of introduced cysteine mutants and surface labeling with a membrane impermeable Cys-directed reagent, N-biotinylaminoethyl methanethiosulfonate. To begin our investigation, two cysteine residues (Cys911 and Cys964) in the wild-type α-subunit were substituted to create a background mutant devoid of exposed cysteines (Lutsenko, S., Daoud, S., and Kaplan, J. H. (1997) J. Biol. Chem. 272, 5249–5255). Into this background construct were then introduced single cysteines in each of the five putative extracellular loops (P118C, T309C, L793C, L876C, and M973C) and the resulting α-subunit mutants were co-expressed with the β-subunit in baculovirus-infected insect cells. All of our expressed Na,K-ATPase mutants were functionally active. Their ATPase, phosphorylation, and ouabain binding activities were measured, and the turnover of the phosphoenzyme intermediate was close to the wild-type enzyme, suggesting that they are folded properly in the infected cells. Incubation of the insect cells with the cysteine-selective reagent revealed essentially no labeling of the α-subunit of the background construct and labeling of all five mutants with single cysteine residues in putative extracellular loops. Two additional mutants, V969C and L976C, were created to further define the M9M10 loop. The lack of labeling for these two mutants showed that although Met973is apparently exposed, Val969 and Leu976 are not, demonstrating that this method may also be utilized to define membrane aqueous boundaries of membrane proteins. Our labeling studies are consistent with a specific 10-transmembrane segment model of the Na,K-ATPase α-subunit. This strategy utilized only functional Na,K-ATPase mutants to establish the membrane topology of the entire α-subunit, in contrast to most previously applied methods.

The integral membrane protein, the gastric H,K-ATPase, is an α-β heterodimer, with 10 putative transmembrane segments in the α-subunit and one such segment in the β-subunit. All transmembrane segments remain within the membrane domain following trypsinization of the intact gastric H,K-ATPase in the presence of K+ ions, identified as M1M2, M3M4, M5M6, and M7, M8, M9, and M10. Removal of K+ ions from this digested preparation results in the selective loss of the M5M6 hairpin from the membrane. The release of the M5M6 fragment is directed to the extracellular phase as evidenced by the accumulation of the released M5M6 hairpin inside the sealed inside out vesicles. The stabilization of the M5M6 hairpin in the membrane phase by the transported cation as well as loss to the aqueous phase in the absence of the transported cation has been previously observed for another P2-type ATPase, the Na,K-ATPase (Lutsenko, S., Anderko, R., and Kaplan, J. H. (1995)Proc. Natl. Acad. Sci. U. S. A. 92, 7936–7940). Thus, the effects of the counter-transported cation on retention of the M5M6 segment in the membrane as compared with the other membrane pairs may be a general feature of P2-ATPase ion pumps, reflecting a flexibility of this region that relates to the mechanism of transport.

Escherichia coli was used to overexpress the large cytoplasmic loop of the rat Na,K-ATPase. A 1260-base DNA segment encoding Lys354-Lys774 of the rat alpha1-subunit was constructed via polymerase chain reaction. The polymerase chain reaction product was successfully subcloned into the expression vector pET-28 (Novagen), which produces an N-terminal 6-histidine-tagged fusion protein. The pET-28 vector containing rat alpha-loop, i.e. pAN, was used to transform calcium-competent E. coli BL21(DE3) cells, and positive clones were selected by kanamycin resistance. Bacterial cultures were grown, and protein synthesis was induced with isopropyl beta-D-thiogalactoside. Cells were harvested and lysed, revealing production of the His-tagged fusion protein ( approximately 46 kDa). The fusion protein was affinity-purified from other soluble cellular proteins via a Ni-NTA column, which routinely yielded approximately 20 mg of soluble His6-alpha-loop/L cell culture. The His6-alpha-loop retained significant native structure, as evidenced by the ability of ATP and ADP (but not AMP, CTP, GTP, or UTP) to protect against chemical modification by either fluorescein isothiocyanate or maleimidylanilinonapthalene sulfonic acid. More specifically, circular dichroism spectroscopy was used to estimate the secondary structure of the His6 loop, revealing an ordered folding composed of 23% alpha-helix, 23% antiparallel beta-sheet, 4% parallel beta-sheet, 19% beta-turn, and 32% random coil. The 6-histidine loop bound the fluorescent ATP analog trinitrophenyl-ATP with high affinity, as determined by measuring the fluorescence changes associated with binding. Affinities for ATP ( approximately 350 microM) and ADP ( approximately 550 microM) were determined by their ability to compete with and displace 2',3'-O-[2,4,6,-trinitrophenyl]-ATP. These nucleotide affinities are similar to those observed for the E2 conformation of the intact Na,K-ATPase.

Dihydro-4,4′-diisothiocyanostilbene-2,2′-disulfonate (H 2 DIDS) inactivates the renal Na,K-ATPase in an ATP- and K-preventable fashion; inactivation results in the covalent incorporation of a single [ 3 H 2 ]DIDS molecule into the Na pump α-subunit. K + protection is observed at low concentrations ( m ) and reversed at higher concentrations. The biphasic effect is also seen with Rb + , to a lesser extent by Cs + , and not at all by Na + or choline. After extensive tryptic digestion of 3 H 2 DIDS-inactivated enzyme, a single radiolabeled peptide is seen in 16.5% Tricine gels. N-terminal amino acid sequencing revealed two sequences 470 IVEIPFNSTNxYQLS and 495 HLLVMxGAPER, the unidentified residues were K 480 and K 501 , respectively. These data provide suggestive evidence of cross-linking by H 2 DIDS between the two lysines. CNBr digestion of 3 H 2 DIDS-labeled α-subunit produced a single radioactive band of the predicted 15-kDa mass for cross-linking between K 480 an K 501 produced by cleavage at known methione residues. The 15-kDa band combined two N-terminal sequences 464 RDRYAKIVEI and 501 xGAPERILDR which include K 480 and K 501 . Thus K 480 and K 501 are within approximately 14 A of each other in the Na-bound form of the enzyme and information about the occupancy of the cation binding domain is transmitted to the ATP binding loop of the Na,K-ATPase.

Na,K-ATPase in right-side-out oriented vesicles was stabilized in different conformations, and the location of intramembrane Cys residues of the alpha-subunit was assessed with membrane-permeable and membrane-impermeable Cys-directed reagents. In the presence of Mg2+ and Pi, Cys964 was the most accessible for both membrane-impermeable 4-acetamido-4'-maleimidylstilbene-2, 2'disulfonic acid (or stilbene disulfonate maleimide, SDSM) and membrane-permeable 7-diethylamino-3-(4'-maleimidyl)-4-methylcoumarin (CPM). In the presence of K+, Cys964 was modified only by hydrophobic CPM, indicating that the environment around Cys964 was different in these two conformations. Cys964 seems to mark the extracellular border of transmembrane segment M9. Cys911 in transmembrane segment M8 showed similar behavior; however, it was not so readily modified. Complete modification of Cys964 and Cys911 causes only partial (about 50%) inactivation of both ATPase activity and Rb+ (or K+) occlusion, indicating that the effect on cation occlusion is indirect and not within the occlusion cavity. The ATP binding capacity remains unaltered by the modifications. Treatment of the K+-stabilized post-tryptic preparation of purified Na, K-ATPase revealed labeling of several cysteines by CPM, none of which were labeled with SDSM. Removal of K+ ions from the preparation, which we have previously shown is accompanied by release of the M5M6 hairpin to the supernatant (), causes changes in the organization of the C-terminal 21-kDa fragment. In particular Cys983 in M10 became labeled by both CPM and SDSM, pointing to a tight association between the C terminus and the M5M6 hairpin of the alpha-subunit.

Cisplatin (cDDP) is an anticancer drug used in a number of malignancies, including testicular, ovarian, cervical, bladder, lung, head, and neck cancers. Its use is limited by the development of resistance, often rationalized via effects on cellular uptake. It has been claimed that human copper transporter 1 (hCTR1), the human high-affinity copper transporter, is the major entry pathway for cDDP and related drugs via a mechanism that mimics copper. This is an unexpected property of hCTR1, a highly selective copper (I) transporter. We compared the uptake rates of copper with cDDP (and several analogs) into human embryonic kidney 293 cells overexpressing wild-type or mutant hCTR1, mouse embryonic fibroblasts that do or do not express CTR1, and human ovarian tumor cells that are sensitive or resistant to cDDP. We have also compared the effects of extracellular copper, which causes regulatory endocytosis of hCTR1, to those of cDDP. We confirm the correlation between higher hCTR1 levels and higher platinum drug uptake in tumor cells sensitive to the drug. However, we show that hCTR1 is not the major entry route of platinum drugs, and that the copper transporter is not internalized in response to extracellular drug. Our data suggest the major entry pathway for platinum drugs is not saturable at relevant concentrations and not protein-mediated. Clinical trials have been initiated that depend upon regulating membrane levels of hCTR1. If reduced drug uptake is a major factor in resistance, hCTR1 is unlikely to be a productive target in attempts to enhance efficacy, although the proteins involved in copper homeostasis may play a role.