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• W2007708493 abstract "The β-subunits of Na,K-ATPase and H,K-ATPase have important functions in maturation and plasma membrane targeting of the catalytic α-subunit but also modulate the transport activity of the holoenzymes. In this study, we show that tryptophan replacement of two highly conserved tyrosines in the transmembrane domain of both Na,K- and gastric H,K-ATPase β-subunits resulted in considerable shifts of the voltage-dependent E1P/E2P distributions toward the E1P state as inferred from presteady-state current and voltage clamp fluorometric measurements of tetramethylrhodamine-6-maleimide-labeled ATPases. The shifts in conformational equilibria were accompanied by significant decreases in the apparent affinities for extracellular K+ that were moderate for the Na,K-ATPase β-(Y39W,Y43W) mutation but much more pronounced for the corresponding H,K-ATPase β-(Y44W,Y48W) variant. Moreover in the Na,K-ATPase β-(Y39W,Y43W) mutant, the apparent rate constant for reverse binding of extracellular Na+ and the subsequent E2P-E1P conversion, as determined from transient current kinetics, was significantly accelerated, resulting in enhanced Na+ competition for extracellular K+ binding especially at extremely negative potentials. Analogously the reverse binding of extracellular protons and subsequent E2P-E1P conversion was accelerated by the H,K-ATPase β-(Y44W,Y48W) mutation, and H+ secretion was strongly impaired. Remarkably tryptophan replacements of residues in the M7 segment of Na,K- and H,K-ATPase α-subunits, which are at interacting distance to the β-tyrosines, resulted in similar E1 shifts, indicating their participation in stabilization of E2. Thus, interactions between selected residues within the transmembrane regions of α- and β-subunits of P2C-type ATPases exert an E2-stabilizing effect, which is of particular importance for efficient H+ pumping by H,K-ATPase under in vivo conditions. The β-subunits of Na,K-ATPase and H,K-ATPase have important functions in maturation and plasma membrane targeting of the catalytic α-subunit but also modulate the transport activity of the holoenzymes. In this study, we show that tryptophan replacement of two highly conserved tyrosines in the transmembrane domain of both Na,K- and gastric H,K-ATPase β-subunits resulted in considerable shifts of the voltage-dependent E1P/E2P distributions toward the E1P state as inferred from presteady-state current and voltage clamp fluorometric measurements of tetramethylrhodamine-6-maleimide-labeled ATPases. The shifts in conformational equilibria were accompanied by significant decreases in the apparent affinities for extracellular K+ that were moderate for the Na,K-ATPase β-(Y39W,Y43W) mutation but much more pronounced for the corresponding H,K-ATPase β-(Y44W,Y48W) variant. Moreover in the Na,K-ATPase β-(Y39W,Y43W) mutant, the apparent rate constant for reverse binding of extracellular Na+ and the subsequent E2P-E1P conversion, as determined from transient current kinetics, was significantly accelerated, resulting in enhanced Na+ competition for extracellular K+ binding especially at extremely negative potentials. Analogously the reverse binding of extracellular protons and subsequent E2P-E1P conversion was accelerated by the H,K-ATPase β-(Y44W,Y48W) mutation, and H+ secretion was strongly impaired. Remarkably tryptophan replacements of residues in the M7 segment of Na,K- and H,K-ATPase α-subunits, which are at interacting distance to the β-tyrosines, resulted in similar E1 shifts, indicating their participation in stabilization of E2. Thus, interactions between selected residues within the transmembrane regions of α- and β-subunits of P2C-type ATPases exert an E2-stabilizing effect, which is of particular importance for efficient H+ pumping by H,K-ATPase under in vivo conditions. The ubiquitous sodium pump and the closely related gastric proton pump are members of the P-type ATPase family that comprises more than 200 identified members (1Palmgren M.G. Axelsen K.B. Biochim. Biophys. Acta.. 1998; 1365: 37-45Google Scholar, 2Axelsen K.B. Palmgren M.G. J. Mol. Evol.. 1998; 46: 84-101Google Scholar). Apart from the highly homologous Na,K-ATPase α-isoforms, the catalytic subunits of Na,K-ATPase and H,K-ATPase share the highest sequence identity in the whole family (of about 62% between Na,K-ATPase α1-subunit and gastric H,K-ATPase α-subunit (3Shull G.E. Lingrel J.B. J. Biol. Chem.. 1986; 261: 16788-16791Google Scholar)). Besides the bacterial Kdp-ATPase, which is composed of four essential subunits (4Altendorf K. Gassel M. Puppe W. Mollenkamp T. Zeeck A. Boddien C. Fendler K. Bamberg E. Drose S. Acta Physiol. Scand. Suppl.. 1998; 643: 137-146Google Scholar), Na,K- and H,K-ATPases are the only P-type ATPase family members that require an additional β-subunit for folding, membrane insertion, and plasma membrane delivery of the catalytically active α-subunits (5Gottardi C.J. Caplan M.J. J. Biol. Chem.. 1993; 268: 14342-14347Google Scholar, 6McDonough A.A. Geering K. Farley R.A. FASEB J.. 1990; 4: 1598-1605Google Scholar, 7Geering K. FEBS Lett.. 1991; 285: 189-193Google Scholar). With only 20-30% overall sequence identity the three Na,K-ATPase β-subunit isoforms and the H,K-ATPase β-subunits from difference species are much less conserved than the α-subunits. However, all known β-subunits share the same basic overall structure: a short intracellular N-terminal domain followed by a single transmembrane span and a large C-terminal ectodomain, which contains highly conserved glycosylation sites and disulfide bridge-forming cysteine residues. Because a unique feature of the oligomeric members of the P-type ATPase family is the ability to transport K+ ions, it was speculated (8Geering K. J. Bioenerg. Biomembr.. 2001; 33: 425-438Google Scholar) that some structural particularities in the α-subunit that are required for potassium transport may be detrimental for proper folding and membrane integration and that the negative influence on these critical processes is overcome by the association with accessory subunits. In addition to this chaperone-like function, the β-subunit is essential for enzyme activity (9Noguchi S. Mishina M. Kawamura M. Numa S. FEBS Lett.. 1987; 225: 27-32Google Scholar) and influences the transport properties of mature sodium and proton pumps. The existence of various tissue-specific Na,K-ATPase β-subunit isoforms, which result in holoenzymes with different cation affinities (10Jaisser F. Jaunin P. Geering K. Rossier B.C. Horisberger J.D. J. Gen. Physiol.. 1994; 103: 605-623Google Scholar, 11Crambert G. Hasler U. Beggah A.T. Yu C. Modyanov N.N. Horisberger J.D. Lelievre L. Geering K. J. Biol. Chem.. 2000; 275: 1976-1986Google Scholar), and the fact that Na,K-ATPase α-subunits co-expressed with H,K-ATPase β-subunits form active pumps (12Horisberger J.D. Jaunin P. Reuben M.A. Lasater L.S. Chow D.C. Forte J.G. Sachs G. Rossier B.C. Geering K. J. Biol. Chem.. 1991; 266: 19131-19134Google Scholar), albeit with altered ion affinities (10Jaisser F. Jaunin P. Geering K. Rossier B.C. Horisberger J.D. J. Gen. Physiol.. 1994; 103: 605-623Google Scholar, 13Eakle K.A. Kim K.S. Kabalin M.A. Farley R.A. Proc. Natl. Acad. Sci. U. S. A.. 1992; 89: 2834-2838Google Scholar), strongly suggest a modulatory function of the β-subunit for ion translocation. Results from several studies indicate that it is mainly the C-terminal extracellular domain of the β-subunit that modulates cation transport. For example, reduction of the disulfide bonds in the ectodomain of the β-subunit impairs the function of purified Na,K- or H,K-ATPase. Because this was prevented in the presence of cations (14Kawamura M. Ohmizo K. Morohashi M. Nagano K. Biochim. Biophys. Acta.. 1985; 821: 115-120Google Scholar, 15Lutsenko S. Kaplan J.H. Biochemistry.. 1993; 32: 6737-6743Google Scholar, 16Chow D.C. Browning C.M. Forte J.G. Am. J. Physiol.. 1992; 263: C39-C46Google Scholar), a potential role of the β-subunit in K+ occlusion has been suggested. Functional analysis of chimeras between Na,K- and H,K-ATPase β-subunits confirmed that mostly α-β ectodomain interactions are responsible for the observed effects of the β-subunit on cation affinity and occlusion of the sodium pump (17Jaunin P. Jaisser F. Beggah A.T. Takeyasu K. Mangeat P. Rossier B.C. Horisberger J.D. Geering K. J. Cell Biol.. 1993; 123: 1751-1759Google Scholar, 18Eakle K.A. Kabalin M.A. Wang S.G. Farley R.A. J. Biol. Chem.. 1994; 269: 6550-6557Google Scholar, 19Hasler U. Wang X. Crambert G. Beguin P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem.. 1998; 273: 30826-30835Google Scholar). This is also in line with the electron density of the first 10-15 residues of the ectodomain, which was tentatively traced in the recent x-ray structure of the pig Na,K-ATPase holoenzyme (20Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sorensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature.. 2007; 450: 1043-1049Google Scholar) and clearly indicates that the extracellular M5/M6 and M7/M8 loops of the α-subunit are covered by a “lid” formed by the ectodomain of the β-subunit as suggested previously (15Lutsenko S. Kaplan J.H. Biochemistry.. 1993; 32: 6737-6743Google Scholar). Yet not only ectodomain modifications have been shown to alter the transport properties. N-terminal truncation of the cytoplasmic domain of the β-subunit of the Na,K-ATPase resulted in changes of the apparent K+ (19Hasler U. Wang X. Crambert G. Beguin P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem.. 1998; 273: 30826-30835Google Scholar, 21Geering K. Beggah A. Good P. Girardet S. Roy S. Schaer D. Jaunin P. J. Cell Biol.. 1996; 133: 1193-1204Google Scholar) and Na+ affinities (19Hasler U. Wang X. Crambert G. Beguin P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem.. 1998; 273: 30826-30835Google Scholar) and affected the conformational equilibrium (22Abriel H. Hasler U. Geering K. Horisberger J.D. Biochim. Biophys. Acta.. 1999; 1418: 85-96Google Scholar). Likewise an inhibitory antibody, which recognizes an epitope within the first 36 N-terminal amino acids of the β-subunit of H,K-ATPase (23Chow D.C. Forte J.G. Am. J. Physiol.. 1993; 265: C1562-C1570Google Scholar), altered the K+ affinity. However, cytoplasmic interactions are probably not directly responsible for the functional effects of β-subunits on cation binding of the sodium pump because, in contrast to a complete truncation, deletions or multiple mutational alterations of the N terminus did not affect the K+ activation of Na,K-ATPase expressed in Xenopus oocytes (19Hasler U. Wang X. Crambert G. Beguin P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem.. 1998; 273: 30826-30835Google Scholar). Furthermore results from a glycosylation mapping assay indicated a repositioning of the transmembrane segment as a consequence of the N-terminal truncation (24Hasler U. Greasley P.J. von Heijne G. Geering K. J. Biol. Chem.. 2000; 275: 29011-29022Google Scholar). This in turn may also impair the conformation of the ectodomain, whose significance for cation occlusion has already been out-lined above. Even the observed repositioning of the transmembrane domain (TMD) 2The abbreviations used are: TMD, transmembrane domain; TMRM, tetramethylrhodamine-6-maleimide; VCF, voltage clamp fluorometry; wt, wild type; MES, 2-(N-morpholino)ethanesulfonic acid; TMACl, tetramethylammonium chloride; MOPS, 3-(N-morpholino)propanesulfonic acid. itself could be responsible for the reported K+ effects in the N-terminally truncated β-variant because mutations in the TMD have also been shown to modify cation transport: tryptophan scanning mutagenesis in the TMD of the Na,K-ATPase β-subunit revealed that the replacement of two tyrosines by tryptophan has distinct and additive consequences for the cation affinities of the holoenzyme (25Hasler U. Crambert G. Horisberger J.D. Geering K. J. Biol. Chem.. 2001; 276: 16356-16364Google Scholar). Interestingly these tyrosines are highly conserved; they are actually present in all known β-subunits (represented by red capital letters in the β-TMD alignment shown in Fig. 1C). The apparent K½ for extracellular K activation of pump currents in Xenopus oocytes was significantly increased for a simultaneous tryptophan replacement of the two tyrosines in various β-subunit isoforms. Of note, this was accompanied by an increase of the apparent affinity for intracellular sodium and a reduced sensitivity toward the E2-specific inhibitor vanadate. Therefore, it was concluded that the affinity changes might occur secondarily to a conformational, i.e. E2-destabilizing, effect of the β-subunit mutations (25Hasler U. Crambert G. Horisberger J.D. Geering K. J. Biol. Chem.. 2001; 276: 16356-16364Google Scholar). However, direct evidence for a shift in the E1P/E2P distribution of the enzyme has not been provided yet. Given the high propensity of native gastric H,K-ATPase to occur in the E2 state (26Helmich-de Jong M.L. van Emst-de Vries S.E. De Pont J.J. Schuurmans Stekhoven F.M. Bonting S.L. Biochim. Biophys. Acta.. 1985; 821: 377-383Google Scholar), which has been speculated to be of primary importance for efficient H+ delivery to the luminal fluid against a 106-fold H+ gradient in vivo, it is of high interest to identify possible interaction sites on the β- and ultimately also on the transmembrane domains of the α-subunit that are crucial for this unique E2-specific structural stabilization. In the current study we set out to identify molecular determinants of E2-specific intersubunit interactions and utilized the technique of voltage clamp fluorometry (VCF) to directly determine the distribution between E1 and E2 states of wild type and mutated Na,K- and H,K-ATPase enzymes (27Geibel S. Kaplan J.H. Bamberg E. Friedrich T. Proc. Natl. Acad. Sci. U. S. A.. 2003; 100: 964-969Google Scholar, 28Dempski R.E. Friedrich T. Bamberg E. J. Gen. Physiol.. 2005; 125: 505-520Google Scholar). Because this method can be applied to the electrogenic Na,K-ATPase as well as to the electroneutrally operating gastric H,K-ATPase (29Geibel S. Zimmermann D. Zifarelli G. Becker A. Koenderink J.B. Hu Y.K. Kaplan J.H. Friedrich T. Bamberg E. Ann. N. Y. Acad. Sci.. 2003; 986: 31-38Google Scholar, 30Dürr K.L. Tavraz N.N. Zimmermann D. Bamberg E. Friedrich T. Biochemistry.. 2008; 47: 4288-4297Google Scholar), whether mutation of the conserved tyrosines causes similar conformational shifts in both enzymes can be investigated. Evidence is provided that the observed conformational effects are of a more general significance for ion translocation by oligomeric P-type ATPases. Furthermore we compared for both enzymes the effects of these β-subunit mutations on apparent cation affinities, which occur in conjunction with shifts in the distribution of E1P/E2P conformational states. Moreover we studied the effect of mutations of selected residues in the TMD7 of the Na,K- and H,K-ATPase α-subunit that are at interacting distance to the two β-subunit tyrosines (Fig. 1B). This strategy provided novel insights about the molecular details of interactions between β- and α-subunit transmembrane domains of oligomeric P2C-type ATPases that are responsible for stabilization of the E2 conformational state. Molecular Biology—The cDNAs of the sheep Na,K-ATPase β1-subunit, rat H,K-ATPase β-subunit, rat gastric H,K-ATPase α-subunit, and a modified form of the sheep Na,K-ATPase α1-subunit without extracellularly exposed cysteine residues (containing mutations C911S and C964A (31Hu Y.K. Kaplan J.H. J. Biol. Chem.. 2000; 275: 19185-19191Google Scholar)) and with reduced ouabain sensitivity in the millimolar range (achieved by the mutations Q111R and N122D (32Price E.M. Lingrel J.B. Biochemistry.. 1988; 27: 8400-8408Google Scholar)) were subcloned into vector pTLN (33Lorenz C. Pusch M. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A.. 1996; 92: 13362-13366Google Scholar) as described previously (27Geibel S. Kaplan J.H. Bamberg E. Friedrich T. Proc. Natl. Acad. Sci. U. S. A.. 2003; 100: 964-969Google Scholar, 28Dempski R.E. Friedrich T. Bamberg E. J. Gen. Physiol.. 2005; 125: 505-520Google Scholar). The reduced ouabain sensitivity of the latter construct allows selective inhibition of the endogenous Xenopus Na,K-ATPase and was therefore used for all co-expression studies with mutated β1-constructs or as template for site-directed mutagenesis of the Na,K-ATPase α-subunit. Furthermore to exclude any background signals in voltage clamp fluorometric studies that could possibly arise from Na,K-ATPase enzymes assembled from heterologously expressed α-subunits and endogenous β-subunits we utilized the β1-subunit sequence variant S62C for mutagenesis. The introduced cysteine is close to the transmembrane/extracellular interface and has been shown to give rise to voltage-dependent fluorescence changes upon site-directed fluorescence labeling with tetramethylrhodamine-6-maleimide (TMRM) without impairing enzyme function (28Dempski R.E. Friedrich T. Bamberg E. J. Gen. Physiol.. 2005; 125: 505-520Google Scholar). To enable voltage clamp fluorometry on gastric H,K-ATPase, we co-expressed the wild type (or mutated) H,K-ATPase β-subunits with a modified H,K-ATPase α-subunit with a single cysteine replacement of a serine in the M5/M6 extracellular loop (S806C) that is homologous to the N790C mutation in the M5/M6 loop of the Na,K-ATPase α-subunit (28Dempski R.E. Friedrich T. Bamberg E. J. Gen. Physiol.. 2005; 125: 505-520Google Scholar) and thus also suited for environmentally sensitive TMRM labeling (29Geibel S. Zimmermann D. Zifarelli G. Becker A. Koenderink J.B. Hu Y.K. Kaplan J.H. Friedrich T. Bamberg E. Ann. N. Y. Acad. Sci.. 2003; 986: 31-38Google Scholar). Rubidium uptake measurements confirmed that the S806C mutation did not affect the transport properties of H,K-ATPase (30Dürr K.L. Tavraz N.N. Zimmermann D. Bamberg E. Friedrich T. Biochemistry.. 2008; 47: 4288-4297Google Scholar). This construct was therefore used as a template for site-directed mutagenesis to introduce mutations in TMD7 of the H,K-ATPase α-subunit. All mutations were done using the QuikChange multi site-directed mutagenesis kit (Stratagene) and verified by DNA sequencing. Oocyte Preparation and cRNA Injection—Xenopus oocytes were obtained by collagenase treatment after partial ovariectomy from Xenopus laevis females. cRNAs were prepared using the SP6 mMessage mMachine kit (Ambion, Austin, Texas). A 50-nl aliquot containing 20-25 ng of Na,K-ATPase and 1.5-2.5 ng of Na,K-ATPase β1-subunit cRNA (or 20-25 ng of H,K-ATPase α-subunit cRNA and 5 ng of H,K-ATPase β-subunit) were injected into each cell. After injection, oocytes were kept in ORI buffer (110 mm NaCl, 5 mm KCl, 2 mm CaCl2, 5 mm HEPES, pH 7.4) containing 50 mg/liter gentamycin at 18 °C for 3-5 and 2 days for the Na,K-ATPase and H,K-ATPase, respectively. Isolation of Plasma Membranes from X. laevis Oocytes—The isolation of plasma membranes was carried out using positively charged silica beads as described by Kamsteeg and Deen (35Kamsteeg E.J. Deen P.M. Am. J. Physiol.. 2000; 279: F778-F784Google Scholar), who reported a 25- or 450-fold higher yield with this technique compared with standard isolation or biotinylation procedures. Moreover because binding of these beads is carried out on intact oocytes, a high purity of the plasma membrane fraction is achieved with only minor contaminations by internal membranes (36Kamsteeg E.J. Deen P.M. Biochem. Biophys. Res. Commun.. 2001; 282: 683-690Google Scholar). After removal of their follicular cell layer, 8-12 oocytes were rotated in 1% colloidal silica (Ludox Cl, Sigma-Aldrich) in MES-buffered saline for silica (MBSS; 20 mm MES, 80 mm NaCl, pH 6.0) for 30 min at 4 °C. After washing two times in MBSS, the oocytes were rotated at 4 °C in 0.1% polyacrylic acid (Sigma-Aldrich) in MBSS for 30 min. This blocking agent is added to coat unbound beads and solvent-exposed surfaces of beads that were already bound to oocytes. Afterward the oocytes were washed two times in modified Barth's solution (MBS; 0.33 mm Ca(NO3)2, 0.41 mm CaCl2, 88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 0.82 mm MgSO4, 10 mm HEPES, pH 7.5). Subsequently oocytes were homogenized in 1.5 ml of buffer HbA (20 mm Tris, 5 mm MgCl2, 5 mm NaH2PO4, 1 mm EDTA, 80 mm sucrose, pH 7.4) containing protease inhibitor (Complete; Roche Applied Science) and centrifuged for 30 s at 10 × g at 4 °C after which 1.3 ml of the sample supernatant was removed (to be saved for subsequent preparation of total membranes; see below), and 1 ml of HbA was added to the silica beads. This centrifugation and exchange of HbA was repeated three times, but centrifugation changed from twice at 10 × g to once at 20 × g to once at 40 × g. After the last centrifugation step, HbA was removed, and plasma membranes were spun down for 30 min at 16,000 × g at 4 °C and resuspended in Laemmli buffer (37Laemmli U.K. Nature.. 1970; 227: 680-685Google Scholar) (4 μl/oocyte). Preparation of Total Membranes from X. laevis Oocytes—To remove yolk platelets, the supernatant from homogenized oocytes in HbA containing protease inhibitor (see above) was centrifuged once for 3 min at 2,000 × g at 4 °C, and the pellet was discarded. Total membranes in the supernatant were spun down for 30 min at 16,000 × g at 4 °C and resuspended in 4 μl/oocyte Laemmli buffer (37Laemmli U.K. Nature.. 1970; 227: 680-685Google Scholar). Immunoblotting—Protein samples equivalent to two oocytes were separated on 10% SDS-polyacrylamide gels and blotted on nitrocellulose membranes (Roth). The α- and β-subunits of the rat gastric H,K-ATPase were detected with the polyclonal anti-H,Kα-antibody HK12.18 (5Gottardi C.J. Caplan M.J. J. Biol. Chem.. 1993; 268: 14342-14347Google Scholar) (Merck) and the monoclonal anti-H,Kβ-antibody 2G11 (23Chow D.C. Forte J.G. Am. J. Physiol.. 1993; 265: C1562-C1570Google Scholar) (Acris Antibodies), respectively. Subsequently blots were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Dako), and proteins were visualized by using an enhanced chemiluminescence kit (Roche Applied Science). Rb+ Uptake Assay Using Atomic Absorption Spectrometry—Two days after injection, noninjected control oocytes and H,K-ATPase-expressing oocytes were preincubated for 15 min in a Rb+- and K+-free solution (90 mm TMACl or NaCl, 20 mm tetraethylammonium chloride, 5 mm BaCl2, 5 mm NiCl2, 10 mm HEPES, pH 7.4) containing 100 μm ouabain to ensure inhibition of the endogenous Na,K-ATPase and then incubated for 15 min under temperature control in Rb+ flux buffer at 21 °C (5 mm RbCl, 85 mm TMACl or NaCl, 20 mm tetraethylammonium chloride, 5 mm BaCl2, 5 mm NiCl2, 10 mm MES, pH 5.5, 100 μm ouabain). After three washing steps in Rb+-free washing buffer (90 mm TMACl or NaCl, 20 mm tetraethylammonium chloride, 5 mm BaCl2, 5 mm NiCl2, 10 mm MES, pH 5.5) and one wash in water, each individual oocyte was homogenized in 1 ml of Milli-Q® water (Millipore, Billerica, MA). To determine the apparent constant K½ for half-maximal activation of the H,K-ATPase by rubidium, the sum of [TMACl or NaCl] plus [RbCl] in the Rb+ flux buffer was kept constant at 90 mm, e.g. 1 mm RbCl + 89 mm TMACl (or NaCl). After subtraction of the mean of Rb+ uptake into control oocytes of the same batch at a given RbCl concentration, the data were fitted to a Michaelis-Menten type function. V=Vmax·[S]K0.5+[S](Eq. 1) Oocyte homogenates were analyzed by atomic absorption spectroscopy using an AAnalyst800™ spectrometer (Perkin Elmer Life Sciences). From oocyte homogenates (typically 1 ml) samples of 20 μl were automatically transferred into a transversely heated graphite furnace and subjected to a temperature protocol according to the manufacturer's procedures (conditions are available on request), and absorption was measured at 780 nm using a rubidium hollow cathode lamp (Photron, Melbourne, Australia). After Zeeman background correction, Rb+ contents were calculated by comparison with standard calibration curves (measured between 0 and 50 μg/liter Rb+). The detection limit (characteristic mass) of Rb+ is ∼10 pg. Oocyte Pretreatment, Fluorescence Labeling, and Experimental Solutions—Prior to functional studies on Na,K-ATPase-expressing oocytes, cells were first incubated for 45 min in Na+ loading buffer (110 mm NaCl, 2.5 mm sodium citrate, 5 mm MOPS, 5 mm Tris, pH 7.4), and then for 15 min in postloading buffer (100 mm NaCl, 1 mm CaCl2, 5 mm BaCl2, 5 mm NiCl2, 5 mm MOPS/Tris, pH 7.4 (38Rakowski R.F. J. Gen. Physiol.. 1993; 101: 117-144Google Scholar)) to elevate the intracellular Na+ concentration. For voltage clamp fluorometry, site-specific labeling was achieved by incubating oocytes in postloading buffer containing 5 μm TMRM (Molecular Probes; stock solution, 5 mm in DMSO) for 5 min at room temperature in the dark followed by extensive washes in dye-free postloading buffer. Measurements under high extracellular Na+/K+-free conditions were carried out in Na+ test solution (100 mm NaCl, 5 mm BaCl2, 5 mm NiCl2, 5 mm MOPS/Tris, pH 7.4, 10 μm ouabain). The following solution was used for VCF measurements on H,K-ATPase: 90 mm TMACl or NaCl, 20 mm tetraethylammonium chloride, 5 mm BaCl2, 5 mm NiCl2, 10 mm MES, pH 5.5. Stationary currents of the Na,K-ATPase were measured upon a solution exchange from 0 to 10 mm K+ (10 mm KCl, 90 mm NaCl, 5 mm BaCl2, 5 mm NiCl2, 5 mm MOPS/Tris, pH 7.4, 10 μm ouabain). To determine the apparent constant K½ for half-maximal activation of the Na,K-ATPase by K+, the sum of [NaCl] plus [KCl] in the Na+ test solution was kept constant at 100 mm, e.g. 1 mm KCl + 99 mm NaCl. After rundown correction of stationary currents (if necessary) at a given K+ concentration, the data were fitted to a Michaelis-Menten type function (Equation 1). The sheep Na,K-ATPase could be inhibited by 10 mm ouabain, and the rat gastric H,K-ATPase could be inhibited by 10 μm SCH28080 (Sigma-Aldrich) or 30 μm omeprazole (Biotrend, Zürich, Switzerland). Voltage Clamp Fluorometry—An oocyte perfusion chamber was mounted in an Axioskop 2FS epifluorescence microscope (Carl Zeiss, Göttingen, Germany) equipped with a 40× water immersion objective (numerical aperture = 0.8). Currents were measured using a two-electrode voltage clamp amplifier (Turbotec 05, npi electronic GmbH, Tamm, Germany). Fluorescence was excited with a 100-watt tungsten lamp using filters 535DF50 (excitation), 565EFLP (emission), and 570DRLP (dichroic; all from Omega Optical, Brattleboro, VT). Fluorescence was measured with a PIN-022A photodiode (United Detector Technologies, Hawthorne, CA) mounted to the microscope camera port. Photocurrents were amplified by a low noise current amplifier (DLPCA-200, FEMTO, Berlin, Germany). Fluorescence and currents were recorded simultaneously using a Digidata 1322A interface and subsequently analyzed with Clampex 9.2 and Clampfit 9.2 software (Molecular Devices, Sunnyvale, CA). Apparent K½ values for extracellular K+ were obtained by titration experiments as described previously (28Dempski R.E. Friedrich T. Bamberg E. J. Gen. Physiol.. 2005; 125: 505-520Google Scholar). Analysis of Transient Currents of the Na,K-ATPase—Presteady-state currents under high extracellular Na+/K+-free conditions were obtained by subtracting the current responses to voltage steps from -40 mV to values between +60 and -180 mV (20-mV increments) in the presence of 10 mm ouabain (inhibiting the endogenously as well as the heterologously expressed pump) from currents measured in the presence of 10 μm ouabain (inhibiting only the endogenous Na,K-ATPase). The resulting difference currents were fitted monoexponentially, disregarding the first 3-5 ms after the voltage step to exclude capacitive artifacts. The translocated charge Q was calculated from the integral of the fitted transient currents, and the resulting Q-V curves were approximated by a Boltzmann-type function, Q(V)=Qmax+Qmax−Qmin(1+exp(Zq⋅FR⋅T(V−V0.5)))(Eq. 2) where Qmin and Qmax are the saturating values of translocated charge, V0.5 is the midpoint potential, zq is the fraction of charge displaced through the entire transmembrane field, F is the Faraday constant, R is the molar gas constant, T is the temperature (in K), and V is the transmembrane potential. All experiments were performed at 22-24 °C. Extracellular pH Measurements—A qualitative assay for the acidification of the extracellular medium mediated by H+ secretion of Xenopus oocytes expressing gastric H,K-ATPase was carried out according to Jaisser et al. (39Jaisser F. Horisberger J.D. Geering K. Rossier B.C. J. Cell Biol.. 1993; 123: 1421-1429Google Scholar). Two days after injection, oocytes were incubated for 5 min in a weakly buffered solution (70 mm TMACl, 20 mm RbCl, 5 mm BaCl2, 5 mm NiCl2, 500 μm MOPS, adjusted to pH 7.4 with tetramethylammonium hydroxide) containing the pH indicator phenol red (200 μg/ml). In some experiments, SCH28080 (100 μm) was added to the solution. Oocytes were placed individually in a small drop (0.5-1 μl) of the same solution under mineral oil. Every 2 min a color picture was taken at room temperature. E1P/E2P Conformational Distribution and Kinetics of the E1P/E2P Transition for Na,K-ATPase Wild Type and β-(Y39W,Y43W) Mutant Enzymes—To determine whether the double mutation Y39W/Y43W in the Na,K-ATPase β1-subunit causes the proposed shift of the conformational equilibrium of the enzyme toward E1 (25Hasler U. Crambert G. Horisberger J.D. Geering K. J. Biol. Chem.. 2001; 276: 16356-16364Google Scholar), the two tryptophans were introduced into the reporter construct β1-S62C and co-expressed with the α1-subunit in Xenopus oocytes. Upon labeling with the environmentally sensitive dye TMRM, we applied voltage jumps under extracellularly high Na+, K+-free conditions. Under these conditions, the sodium pump carries out Na+/Na+ exchange where the enzyme shuttles exclusively between E1P/E2P states of the catalytic cycle. The ratio E1P/E2Pis incr" @default.
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• W2007708493 title "Functional Significance of E2 State Stabilization by Specific α/β-Subunit Interactions of Na,K- and H,K-ATPase" @default.
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