Heterologous expression of Na(+)-K(+)-ATPase in insect cells: intracellular distribution of pump subunits.

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 alpha- and beta-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, [(3)H]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-sensitive (86)Rb(+) 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 alpha-subunit or beta-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the alpha-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the beta-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the alpha-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the alpha-beta heterodimer within the endoplasmic reticulum apparently does not require a Na(+) pump-specific chaperone.

Conformational coupling: the moving parts of an ion pump.

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.

Expression of an active Na,K-ATPase with an alpha-subunit lacking all twenty-three native cysteine residues.

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.

Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged Dicopper(I) cluster.

Copper binding to the human copper chaperone for superoxide dismutase (hCCS) has been investigated by X-ray absorption spectroscopy. Stoichiometry measurements on the dialyzed, as-isolated protein indicated that up to 3.5 Cu ions bound per hCCS molecule. Reduction with either sodium dithionite or dithiothreitol decreased the copper binding ratio to 2 coppers per hCCS monomer. Analysis of the as-isolated EXAFS data indicated coordination of Cu by a mixture of S and N backscatterers, suggestive of heterogeneous binding of copper between Cu-cysteine binding sites of domain I or III and copper-histidine SOD1-like metal binding sites of domain II. The best fit was obtained with 1.6 Cu-S (cysteine) at 2.24 A (2sigma(2) = 0.011 A(2)) and 1.1 N (histidine) at 1.98 A (2sigma(2) = 0.005 A(2)). A peak of variable intensity in the Fourier transform (FT) of the as-isolated protein at 2.7 A was suggestive of the presence of a heavy atom scatterer such as Cu. Analysis of the dithionite- and DTT-reduced derivatives indicated that copper was lost from the histidine coordinating sites, resulting in a S-only environment with copper coordinated to three S backscatterers at 2. 26 A. The heavy atom scatterer peak was now prominent in the FT and could be well fit by a Cu-Cu interaction at 2.72 A. The data were best interpreted by a dinuclear mu(2)()-bridged cluster with doubly bridging cysteine ligands similar to the cluster proposed to exist in the cytochrome c oxidase chaperone COX17. Analysis of primary sequence and X-ray structural information on yeast CCS strongly suggests that this cluster bridges between domains I and III in hCCS. A mechanism for copper translocation is briefly discussed.

Site-directed chemical labeling of extracellular loops in a membrane protein. The topology of the Na,K-ATPase alpha-subunit.

We have mapped the membrane topology of the renal Na,K-ATPase alpha-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 (Cys(911) and Cys(964)) in the wild-type alpha-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 alpha-subunit mutants were co-expressed with the beta-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 alpha-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 Met(973) is apparently exposed, Val(969) and Leu(976) 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 alpha-subunit. This strategy utilized only functional Na,K-ATPase mutants to establish the membrane topology of the entire alpha-subunit, in contrast to most previously applied methods.

Cys(577) is a conformationally mobile residue in the ATP-binding domain of the Na,K-ATPase alpha-subunit.

2-[4'-Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mM ATP or low K(+) (<1 mM); the protective effect K(+) is reversed at higher concentrations. This biphasic effect was also observed with K(+) congeners. In contrast, Na(+) ions did not protect. MIANS inactivation disrupted high affinity ATP binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography. ATP clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of approximately 5 kDa. N-terminal amino acid sequencing identified the peptide (V(545)LGFCH...). This hydrophobic peptide contains only two Cys residues in all sodium pump alpha-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with ATP binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys(577). The cation-dependent change in reactivity of Cys(577) implies structural alterations in the ATP-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-ATPase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the ATP hydrolysis domain.

Stabilization of the H,K-ATPase M5M6 membrane hairpin by K+ ions. Mechanistic significance for p2-type atpases.

The integral membrane protein, the gastric H,K-ATPase, is an alpha-beta heterodimer, with 10 putative transmembrane segments in the alpha-subunit and one such segment in the beta-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.

Structural changes associated with the coupling of ATP hydrolysis and cation transport by the Na pump.

Most of the residues associated with cation coordination seem to reside within transmembrane segments of the alpha-subunit of the Na,K-ATPase, whereas amino acids which appear to be involved in the coordination of ATP are found in the major cytoplasmic loop between transmembrane segments M4 and M5 (see Lingrel & Kuntzweiler, 1994; Lutsenko & Kaplan, 1995). The coupling of the two functions of cation transport and ATP hydrolysis involved in the active transport of Na and K ions must involve interactions between these two structural units. This paper summarizes recent experimental results and conclusions of studies on the renal Na,K-ATPase which have employed controlled proteolysis in the presence of physiological ligands, chemical modification with a range of reagents and a variety of functional assays. The data provide evidence for movements between specific transmembrane segments associated with cation-binding conformations and coupled changes which take place in the ATP binding domain. The binding of different cations in the cation-binding domain is sensed in the ATP binding domain and manifested as a change in reactivity. This occurs at amino acid residues which are widely spaced in primary structure. It is apparent that structural changes are transmitted through much of the ATP-binding domain as a consequence of the occupancy of the cation-binding domain. We also provide evidence that both the number and identity of cations bound are also sensed in the ATP-binding domain.

The M4M5 cytoplasmic loop of the Na,K-ATPase, overexpressed in Escherichia coli, binds nucleoside triphosphates with the same selectivity as the intact native protein.

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.

N-terminal domains of human copper-transporting adenosine triphosphatases (the Wilson's and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal-binding repeat.

N-terminal domains of the Wilson's and Menkes disease proteins (N-WND and N-MNK) were overexpressed in a soluble form in Escherichia coli as fusions with maltose-binding protein, purified, and their metal-binding properties were characterized. Both N-MNK and N-WND bind copper specifically as indicated by the results of metal-chelate chromatography, direct copper-binding measurements, and chemical modification of Cys residues in the presence of different heavy metals. When E. coli cells are grown in the presence of copper, N-MNK and N-WND bind copper in vivo with stoichiometry of 5-6 nmol of copper/nmol of protein. Copper released from the copper-N-MNK and copper-N-WND complexes reacts with the Cu(I)-selective chelator bicinchoninic acid in the absence of reducing agents. This suggests that in proteins, it is bound in reduced Cu(I) form, in agreement with the spectroscopic properties of the copper-bound domains. Copper bound to the domains in vivo or in vitro specifically protects the N-MNK and N-WND against labeling with the cysteine-directed probe; this indicates that Cys residues in the repetitive motifs GMTCXXCXXXIE are involved in coordination of copper. Direct involvement of the N-terminal domains in the binding of copper suggests their important role in copper-dependent functions of human copper-transporting adenosine triphosphatases (Wilson's and Menkes disease proteins).

Chemical modification with dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate reveals the distance between K480 and K501 in the ATP-binding domain of the Na,K-ATPase.

Dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate (H2DIDS) inactivates the renal Na,K-ATPase in an ATP- and K-preventable fashion; inactivation results in the covalent incorporation of a single [3H2]DIDS molecule into the Na pump alpha-subunit. K+ protection is observed at low concentrations (< 2 mM) 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 3H2DIDS-inactivated enzyme, a single radiolabeled peptide is seen in 16.5% Tricine gels. N-terminal amino acid sequencing revealed two sequences 470IVEIPFNSTNxYQLS and 495HLLVMxGAPER, the unidentified residues were K480 and K501, respectively. These data provide suggestive evidence of cross-linking by H2DIDS between the two lysines. CNBr digestion of 3H2DIDS-labeled alpha-subunit produced a single radioactive band of the predicted 15-kDa mass for cross-linking between K480 an K501 produced by cleavage at known methione residues. The 15-kDa band combined two N-terminal sequences 464RDRYAKIVEI and 501xGAPERILDR which include K480 and K501. Thus K480 and K501 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.

Identification of two conformationally sensitive cysteine residues at the extracellular surface of the Na,K-ATPase alpha-subunit.

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.

Photolabile amiloride derivatives as cation site probes of the Na,K-ATPase.

Treatment of purified canine renal Na,K-ATPase with a range of photoactivatable amiloride derivatives results in inhibition of ATPase activity prior to illumination. Inhibition by amiloride derivatives substituted on a guanidium N could not be prevented by the presence of either K or Na; however, these cations could protect the enzyme against inhibition by derivatives substituted on the 5-position of the pyrazine ring. In the case of 5-(N-ethyl-[2'-methoxy-4'-nitrobenzyl])amiloride (NENMBA), the presence of monovalent cations (Na, K, and Rb) protected the enzyme effectively against inhibition, with concentrations in the millimolar range. ATP did not prevent inhibition; furthermore, native and NENMBA-treated enzyme exhibited normal levels of high affinity [3H]ADP (and hence ATP) binding. The rate of inhibition increased with increasing concentrations of NENMBA. Extensive washing of NENMBA-inhibited enzyme did not restore ATPase activity, showing that NENMBA has an extremely slow off-rate for dissociation from its inhibitory site. Partially inhibited enzyme could be rapidly pelleted and resuspended in NENMBA-free buffer and inhibition was observed to continue, albeit at a somewhat diminished rate, suggesting that NENMBA gains access to its inhibitory site after partitioning into the lipid phase rather than directly from the aqueous solution. Photolysis of NENMBA-inhibited enzyme resulted in covalent incorporation of the reagent into the alpha-subunit of the Na,K-ATPase, as observed by separation of labeled protein on a Laemmli gel and Western analysis using a polyclonal amiloride antibody. Almost all of the covalent labeling could be prevented by the presence of Rb in the incubation and labeling medium. These results suggest that NENMBA inhibits the Na, K-ATPase by disruption of the cation transport domain rather than the catalytic domain of the enzyme and that it promises to be a useful tool for cation site localization.

Laser photolysis of caged calcium: rates of calcium release by nitrophenyl-EGTA and DM-nitrophen.

Nitrophenyl-EGTA and DM-nitrophen are Ca2+ cages that release Ca2+ when cleaved upon illumination with near-ultraviolet light. Laser photolysis of nitrophenyl-EGTA produced transient intermediates that decayed biexponentially with rates of 500,000 s-1 and 100,000 s-1 in the presence of saturating Ca2+ and 290,000 s-1 and 68,000 s-1 in the absence of Ca2+ at pH 7.2 and 25 degrees C. Laser photolysis of nitrophenyl-EGTA in the presence of Ca2+ and the Ca2+ indicator Ca-orange-5N produced a monotonic increase in the indicator fluorescence, which had a rate of 68,000 s-1 at pH 7.2 and 25 degrees C. Irradiation of DM-nitrophen produced similar results with somewhat slower kinetics. The transient intermediates decayed with rates of 80,000 s-1 and 11,000 s-1 in the presence of Ca2+ and 59,000 s-1 and 3,600 s-1 in the absence of Ca2+ at pH 7.2 and 25 degrees C. The rate of increase in Ca(2+)-indicator fluorescence produced upon photolysis of the DM-nitrophen: Ca2+ complex was 38,000 s-1 at pH 7.2 and 25 degrees C. In contrast, pulses in Ca2+ concentration were generated when the chelator concentrations were more than the total Ca2+ concentration. Photoreleased Ca2+ concentration stabilized under these circumstances to a steady state within 1-2 ms.