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• W2000341993 abstract "A lysine residue within the highly conserved center of the fifth transmembrane segment in PIIC-type ATPase α-subunits is uniquely found in H,K-ATPases instead of a serine in all Na,K-ATPase isoforms. Because previous studies suggested a prominent role of this residue in determining the electrogenicity of non-gastric H,K-ATPase and in pKa modulation of the proton-translocating residues in the gastric H,K-ATPases as well, we investigated its functional significance for ion transport by expressing several Lys-791 variants of the gastric H,K-ATPase in Xenopus oocytes. Although the mutant proteins were all detected at the cell surface, none of the investigated mutants displayed any measurable K+-induced stationary currents. In Rb+ uptake measurements, replacement of Lys-791 by Arg, Ala, Ser, and Glu substantially impaired transport activity and reduced the sensitivity toward the E2-specific inhibitor SCH28080. Furthermore, voltage clamp fluorometry using a reporter site in the TM5/TM6 loop for labeling with tetra-methylrhodamine-6-maleimide revealed markedly changed fluorescence signals. All four investigated mutants exhibited a strong shift toward the E1P state, in agreement with their reduced SCH28080 sensitivity, and an about 5–10-fold decreased forward rate constant of the E1P ↔ E2P conformational transition, thus explaining the E1P shift and the reduced Rb+ transport activity. When Glu-820 in TM6 adjacent to Lys-791 was replaced by non-charged or positively charged amino acids, severe effects on fluorescence signals and Rb+ transport were also observed, whereas substitution by aspartate was less disturbing. These results suggest that formation of an E2P-stabilizing interhelical salt bridge is essential to prevent futile proton exchange cycles of H+ pumping P-type ATPases. A lysine residue within the highly conserved center of the fifth transmembrane segment in PIIC-type ATPase α-subunits is uniquely found in H,K-ATPases instead of a serine in all Na,K-ATPase isoforms. Because previous studies suggested a prominent role of this residue in determining the electrogenicity of non-gastric H,K-ATPase and in pKa modulation of the proton-translocating residues in the gastric H,K-ATPases as well, we investigated its functional significance for ion transport by expressing several Lys-791 variants of the gastric H,K-ATPase in Xenopus oocytes. Although the mutant proteins were all detected at the cell surface, none of the investigated mutants displayed any measurable K+-induced stationary currents. In Rb+ uptake measurements, replacement of Lys-791 by Arg, Ala, Ser, and Glu substantially impaired transport activity and reduced the sensitivity toward the E2-specific inhibitor SCH28080. Furthermore, voltage clamp fluorometry using a reporter site in the TM5/TM6 loop for labeling with tetra-methylrhodamine-6-maleimide revealed markedly changed fluorescence signals. All four investigated mutants exhibited a strong shift toward the E1P state, in agreement with their reduced SCH28080 sensitivity, and an about 5–10-fold decreased forward rate constant of the E1P ↔ E2P conformational transition, thus explaining the E1P shift and the reduced Rb+ transport activity. When Glu-820 in TM6 adjacent to Lys-791 was replaced by non-charged or positively charged amino acids, severe effects on fluorescence signals and Rb+ transport were also observed, whereas substitution by aspartate was less disturbing. These results suggest that formation of an E2P-stabilizing interhelical salt bridge is essential to prevent futile proton exchange cycles of H+ pumping P-type ATPases. The ubiquitous Na,K-ATPase and the gastric H,K-ATPase belong to the PIIC subgroup of the extensive class of P-type ATPases, which use ATP hydrolysis for active transport of cations. The reversible phosphorylation of a highly conserved Asp residue, a hallmark of all P-type ATPases, is coupled to the transition between two principal conformational states (E1 and E2) and the corresponding phosphointermediates (E1P and E2P). Na,K- and H,K-ATPases share several similarities. The catalytic α-subunits are highly homologous (∼60% sequence identity), whereas the accessory β-subunits have lower sequence identity but nevertheless share similar basic structural features (1Geering K. J. Bioenerg. Biomembr. 2001; 33: 425-438Crossref PubMed Scopus (270) Google Scholar). Furthermore, Na,K- and H,K-ATPases are the only known K+-countertransporting P-type pumps in eukaryotes. Despite these common features, there are also important differences between the two enzymes. For example, the asymmetrical transport stoichiometry of Na+ and K+ exchange (3 versus 2) by all known Na,K-ATPase isoforms results in a net electrogenic transport (2Thomas R.C. J. Physiol. 1969; 201: 495-514Crossref PubMed Scopus (152) Google Scholar), whereas gastric and non-gastric H,K-ATPases operate strictly electroneutral with 2:2 (or 1:1) stoichiometry (3Sachs G. Chang H.H. Rabon E. Schackman R. Lewin M. Saccomani G. J. Biol. Chem. 1976; 251: 7690-7698Abstract Full Text PDF PubMed Google Scholar, 4Burnay M. Crambert G. Kharoubi-Hess S. Geering K. Horisberger J.D. Am. J. Physiol. Renal. Physiol. 2001; 281: F869-F874Crossref PubMed Google Scholar). Although the transport cycle of the H,K-ATPase is net electroneutral, evidence has been accumulated that several partial reactions of the cycle are electrogenic. Electrophysiological experiments using purified H,K-ATPase-containing membrane fragments on planar lipid bilayers (5van der Hijden H.T. Grell E. de Pont J.J. Bamberg E. J. Membr. Biol. 1990; 114: 245-256Crossref PubMed Scopus (24) Google Scholar, 6Stengelin M. Fendler K. Bamberg E. J. Membr. Biol. 1993; 132: 211-227Crossref PubMed Scopus (14) Google Scholar) have shown that the proton branch of the cycle, which involves the E1P-E2P conformational change investigated in the current study, includes an electrogenic step. As it is the case for the electrogenic Na,K-ATPase, the E1P-E2P distribution might be driven by the redistribution of cations within intra- or extracellularly oriented high-field access channels to the transport sites. The electrogenicity during the H+ branch of the cycle is most likely counterbalanced by another partial reaction of reversed electrogenicity in the K+ branch to bring about net electroneutrality (7Lorentzon P. Sachs G. Wallmark B. J. Biol. Chem. 1988; 263: 10705-10710Abstract Full Text PDF PubMed Google Scholar). Notably, mutagenesis studies have shown that Na,K-ATPase and H,K-ATPase extrude Na+ and H+ (or H3O+), generally utilizing the same conserved carboxylic acids of their respective cation binding sites in the transmembrane domains TM4 2The abbreviations used are: TMtransmembrane domainTMRMtetramethylrhodamine-6-maleimideVCFvoltage clamp fluorometry. to TM6 (see Fig. 1). This raises not only the important question of how the different stoichiometries are achieved on a molecular level but also why these carboxyls with an expected pK around 3–5 can release protons at a lumenal pH of ∼1 in the case of the gastric H,K-ATPase. Finally, a remarkable particularity of the gastric H,K-ATPase (and also some other P-type proton pumps like the plant/fungal H+-ATPase) is its inability to run backward and synthesize ATP (8Helmich-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: 377-383Crossref PubMed Scopus (25) Google Scholar, 9Nissen P. EMBO J. 2009; 28: 1535-1536Crossref PubMed Scopus (2) Google Scholar), whereas the reverse operation has readily been observed for Na,K-ATPase (10Garrahan P.J. Glynn I.M. Nature. 1966; 211: 1414-1415Crossref PubMed Scopus (26) Google Scholar, 11Garrahan P.J. Glynn I.M. J. Physiol. 1967; 192: 237-256Crossref PubMed Scopus (137) Google Scholar, 12Lew V.L. Glynn I.M. Ellory J.C. Nature. 1970; 225: 865-866Crossref PubMed Scopus (39) Google Scholar, 13Chmouliovsky M. Straub R.W. Pflügers Arch. 1974; 350: 309-320Crossref PubMed Scopus (5) Google Scholar, 14Taniguchi K. Post R.L. J. Biol. Chem. 1975; 250: 3010-3018Abstract Full Text PDF PubMed Google Scholar, 15De Weer P. Rakowski R.F. Nature. 1984; 309: 450-452Crossref PubMed Scopus (43) Google Scholar, 16Efthymiadis A. Schwarz W. Biochim. Biophys. Acta. 1991; 1068: 73-76Crossref PubMed Scopus (9) Google Scholar) and Ca2+-ATPases (17Knowles A.F. Racker E. J. Biol. Chem. 1975; 250: 1949-1951Abstract Full Text PDF PubMed Google Scholar, 18de Meis L. Tume R.K. Biochemistry. 1977; 16: 4455-4463Crossref PubMed Scopus (69) Google Scholar, 19Wüthrich A. Schatzmann H.J. Romero P. Experientia. 1979; 35: 1589-1590Crossref PubMed Scopus (14) Google Scholar, 20Chiesi M. Zurini M. Carafoli E. Biochemistry. 1984; 23: 2595-2600Crossref PubMed Scopus (38) Google Scholar) under sufficiently steep ion gradients and high ADP/ATP ratios in presence of Pi. transmembrane domain tetramethylrhodamine-6-maleimide voltage clamp fluorometry. Notably, all these unique H,K-ATPase properties have been linked to the presence of a lysine residue in the fifth transmembrane segment of the catalytic α-subunit (Lys-791 in gastric H,K-ATPase (shown in blue in Fig. 1 and in the sequence alignments in Fig. 9). This lysine in the otherwise highly conserved (K/S)NIPEIT sequence motif is replaced by an uncharged serine in Na,K-ATPases (see P-type ATPase alignments in Fig. 9). Remarkably, it is the only positively charged amino acid in the whole TM region of the H,K-ATPase α-subunit. Homology modeling of the cation binding pocket based on the SERCA (sarco(endo)plasmic reticulum calcium ATPase) structure in the E2-state together with mutagenesis studies have predicted an E2-conformation-specific salt bridge between the side chains of Lys-791 (in TM5) and Glu-820 (in TM6, highlighted in brown in FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6, FIGURE 7, FIGURE 8, FIGURE 9) of the cation binding pocket (21Koenderink J.B. Swarts H.G. Willems P.H. Krieger E. De Pont J.J. J. Biol. Chem. 2004; 279: 16417-16424Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Koenderink et al. (21Koenderink J.B. Swarts H.G. Willems P.H. Krieger E. De Pont J.J. J. Biol. Chem. 2004; 279: 16417-16424Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) concluded that this interhelical interaction could contribute to the inherent E2 preference of the gastric H,K-ATPase, which in turn could be relevant for preventing a backward running of the pump (see “Discussion”). Moreover, molecular dynamics simulations by Munson et al. (22Munson K. Garcia R. Sachs G. Biochemistry. 2005; 44: 5267-5284Crossref PubMed Scopus (82) Google Scholar) have shown that the positively charged side chain of Lys-791 probably reorients during the E1P ↔ E2P transition as a consequence of a change in the relative positions of the TM5 and TM6 helices. The reorientation may move the NH3+ group of the side chain closer to the plane of the ion binding site in E2P (Fig. 1), thereby lowering the effective pKa of the putative H3O+-coordinating carboxylates. This change in pKa could enable proton release at low lumenal pH (22Munson K. Garcia R. Sachs G. Biochemistry. 2005; 44: 5267-5284Crossref PubMed Scopus (82) Google Scholar, 23Shin J.M. Munson K. Vagin O. Sachs G. Pflügers Arch. 2009; 457: 609-622Crossref PubMed Scopus (179) Google Scholar).FIGURE 2Control measurements on TMRM-labeled oocytes expressing construct αS806C, demonstrating the specificity of the voltage-jump induced fluorescence changes. A–D, shown are fluorescence signals of TMRM-labeled oocytes in response to voltage jumps from a holding potential of −40 mV to values between −180 and +60 mV in 20-mV increments. A and B, shown are fluorescence signals of an individual oocyte expressing the αS806C construct together with the H,K-ATPase β-subunit before (A) and after (B) the addition of the specific inhibitor SCH28080 (100 μm). C and D, shown are voltage jump-induced fluorescence responses from two oocytes from the same batch, which were either injected with the cRNA of the αS806C construct alone (D) or co-injected with the H,K-ATPase β-subunit (C). E, shown is normalized cell fluorescence of TMRM-labeled oocytes, which were either uninjected or expressed the H,K-ATPase αS806C construct together with the wild-type β-subunit. Data were obtained from three batches of cells, and the fluorescence intensity was normalized to the mean fluorescence of uninjected oocytes in each batch. For the measurement of cellular fluorescence, all cells from each batch of oocytes were placed into the perfusion chamber of the experimental microscope and illuminated with constant excitation light intensity. A constant region of interest was chosen for all cells using a circular iris aperture that allowed measurement of the fluorescence of about 90% of the illuminated cell surface. a.u., arbitrary units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Rb+ uptake and cell surface expression of Xenopus oocytes expressing H,K-ATPase wild-type or Lys-791 variants. A, H,K-ATPase-mediated Rb+ uptake at 5 mm RbCl in the absence (hatched bars) or presence (black bars) of 10 μm SCH28080 is shown. Results from uninjected control oocytes, oocytes injected with the reference construct HKαS806C/βwt, or constructs with the indicated Lys-791 point mutations are shown. Rb+ uptake was measured on individual cells by atomic absorption spectrometry (see “Experimental Procedures”). Data are the means ± S.E. from two individual experiments with 15–20 oocytes, normalized to Rb+ uptake of the wild-type construct HKαS806C/βwt (corresponding to 20.2 and 38.9 pmol/oocyte/min, respectively). B, shown is Western blot analysis of plasma membrane (PP, upper panel) and total membrane fractions (TP, lower panel) isolated from H,K-ATPase-expressing oocytes. Detection used anti-H,Kα antibody HK12.18. One representative Western blot is shown. C, densitometric analysis is shown of Western blot bands from corresponding total membrane or plasma membrane fraction preparations as shown in B, normalized to the band intensity of the TP fraction of the WT protein. Data are expressed as the means ± S.D. from four to five experiments using oocytes from different Xenopus females. a.u., arbitrary units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Voltage dependence of the E1P/E2P distribution and kinetics of E1P ↔ E2P transitions of H,K-ATPase Lys-791 mutants studied by voltage clamp fluorometry. A–G, shown are fluorescence responses of site-specific labeled gastric H,K-ATPase under K+-free conditions (90 mm NaCl, pH 5.5) upon voltage jumps from a holding potential of −40 mV to voltages between −180 mV and +60 mV (A and F insets = voltage protocols). Recordings originated from single oocytes coexpressing the wild-type β-subunit with either the reference construct HKαS806C (A) or the indicated Lys-791 variant constructs (B–G). H, shown are reciprocal time constants from monoexponential fits to voltage jump-induced fluorescence changes under K+-free conditions for H,K-ATPase α(S806C,K791S)/βwt (pink diamonds), α(S806C,K791A)/βwt (red circles), α(S806C,K791E)/βwt (blue squares), or α(S806C,K791R)/βwt (green squares) in comparison to αS806C/βwt (black squares). Data are the means ± S.E. from 5–17 oocytes. I–L, voltage dependence of fluorescence amplitudes (1 − ΔF/F) under K+-free conditions for the indicated Lys-791 mutants, each in comparison to αS806C/βwt (same colors as in H). Data are the means ± S.E. of 5–13 oocytes. A curve resulting from a fit of a Boltzmann function is superimposed together with the corresponding wild-type curve (dotted lines in J–L). The fluorescence amplitudes 1 − ΔF/F were normalized to saturation values from the fits. Midpoint potentials V0.5 and slope factors zq derived from the fits are also shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Calculated voltage dependence of the forward and reverse rate constants of the E1P ↔ E2P conformational transition for wild-type H,K-ATPase and several Lys-791 variants. Shown is calculated voltage dependence of the forward (kf) and reverse (kb) rate constant of the E1P-E2P conformational transition in comparison to the experimentally obtained reciprocal time constants (ktot) from voltage clamp fluorometric measurements (using the H,K-ATPase αS806C/βWT as a background) for the wild-type (A) and mutants K791S (B), K791A (C), K791E (D), and K791R (E). Values were calculated using a simple two-state kinetic model; see supplemental Appendix A for details.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Cell surface expression and Rb+ uptake of Xenopus oocytes expressing H,K-ATPase wild-type or several Glu-820 variants. A, shown is a Western blot analysis of plasma membrane (PP, upper panel) and total membrane fractions (TP, lower panel) isolated from H,K-ATPase-expressing oocytes. Detection used anti-H,Kα antibody HK12.18. One representative Western blot of at least three from different oocyte batches is shown. B, shown is a densitometric analysis of Western blot bands from corresponding total membrane or plasma membrane fraction preparations as shown in A, normalized to the band intensity of the TP fraction of the WT protein. Data are expressed as the means ± S.D. from 2–5 experiments. a.u., arbitrary units. C, H,K-ATPase-mediated Rb+ uptake at 5 mm RbCl in the absence (hatched bars) or presence of 100 μm SCH28080 (black bars) is shown. D, H,K-ATPase-mediated Rb+ uptake at 5 mm RbCl and pHex = 7.4 (dark gray bars), pHex = 5.5 (light gray bars), or at pHex = 7.4 in the presence of 40 mm butyrate, which causes a slight intracellular acidification (by ∼ 0.5 pH units, hatched dark gray bars), is shown. Results from uninjected control oocytes or oocytes injected with the reference construct HKαS806C/βwt or HKαS806C/E820X (X = Ala, Gln, Asp) are shown. Rb+ uptake was measured on individual cells by atomic absorption spectrometry (see “Experimental Procedures”). Data are the means ± S.E. from four individual experiments with 15–20 oocytes, normalized to Rb+ uptake of the wild-type construct HKαS806C/βwt (corresponding to 23.9, 22.5, 21.2, and 24.9 pmol/oocyte/min, respectively). Inset, shown is inhibition of Rb+ uptake by sodium orthovanadate for oocytes expressing either the wild-type or the E820Q mutant proton pump at 5 mm RbCl and pH 7.4 in presence of 40 mm butyrate (black bars, see “Experimental Procedures” for details). One representative experiment is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Voltage dependence of the E1P/E2P distribution and kinetics of E1P/E2P transitions of H,K-ATPase mutants E820D and E820K. A and B, shown are fluorescence responses of site-specifically labeled gastric H,K-ATPase under K+-free conditions (90 mm NaCl, pH 5.5) upon voltage jumps from a holding potential of −40 mV to voltages between −180 and +60 mV (same voltage protocols as in Fig. 4, A and F). Recordings originated from a representative oocyte coexpressing the wild-type HKβ subunit with HKαS806C,E820D in A or HKαS806C,E820K in B, respectively. C and D, shown is voltage dependence of fluorescence amplitudes (1 − ΔF/F) under K+-free conditions for mutants HKαS806C,E820D (open red circle in C) and HKαS806C,E820K (open blue circle in D) compared with the reference construct HKαS806 (open square). Data are the means ± S.E. of 9–14 oocytes. A curve resulting from a fit of a Boltzmann function is superimposed. The fluorescence amplitudes 1 − ΔF/F were normalized to saturation values from the fits. Midpoint potentials V0.5 and slope factors zq derived from the fits are also shown. E and F, shown are reciprocal time constants from monoexponential fits to voltage jump-induced fluorescence changes under K+-free conditions for the mutants HKαS806C,E820D ((open red circle in E) and HKαS806C,E820K (open blue circle in F), each in comparison to αS806C (filled square). Data are means ± S.E. from 7–9 oocytes. G and H, shown is the calculated voltage dependence of the forward (kf) and reverse (kb) rate constant of the E1P-E2P conformational transition in comparison to the experimentally obtained reciprocal time constants (ktot) from voltage clamp fluorometric measurements for the mutants E820D (G) and E820K (H). Values were calculated using a simple two-state kinetic model; see supplemental Appendix A for details.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 8Cell surface expression and Rb+ uptake activity of the charge-inverting double mutation E820K/K791E compared with the wild-type and the single mutant E820K. A, shown is the Western blot analysis of plasma membrane (PP, upper panel) and total membrane fractions (TP, lower panel) isolated from oocytes co-expressing the wild-type H,K-ATPase β-subunit together with the indicated α-subunit constructs. Detection used anti-H,K α-antibody HK12.18. One representative Western blot of three similar ones is shown. B, Densitometric analysis is shown of Western blot bands from corresponding total membrane or plasma fraction (PP) preparations as shown in A, normalized to the band intensity of the TP fraction of the WT protein. Data are expressed as means ± S.D. from three experiments. a.u., absorbance units. C, shown is H,K-ATPase-mediated Rb+ uptake at 5 mm RbCl in the absence (hatched bars) or presence (black bars) of 100 μm SCH28080. Results from uninjected control oocytes, oocytes injected with the wild-type β-subunit, and either reference construct HKαS806C or HKαS806C-constructs with the indicated additional mutations are shown. Data are the means ± S.E. from two experiments with 15–20 oocytes, normalized to Rb+ uptake of the wild-type construct HKαS806C/βwt (corresponding to 14.4 and 29.8 pmol/oocyte/min, respectively). a.u., arbitrary units.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Apart from this proposed function as a pKa-modulating molecular device, Lys-791 is possibly also a major determinant for the electroneutral transport mode characteristic for H,K-ATPases, as suggested by mutagenesis studies on the non-gastric H,K-ATPase expressed in Xenopus oocytes. Upon mutation of the corresponding Lys-800 to Ala or Glu, the normally electroneutral Na+/K+ exchange of wild-type toad bladder H,K-ATPase (sometimes also termed X,K-ATPase to differentiate it from the “gastric” H,K-ATPase) becomes electrogenic, thus, generating positive pump currents in the presence of extracellular K+. Vice versa, a charge-introducing mutation of the respective serine to a positively charged arginine in toad Na,K-ATPase resulted in a loss of pump currents with minor effects on Rb+ transport activity (24Burnay M. Crambert G. Kharoubi-Hess S. Geering K. Horisberger J.D. J. Biol. Chem. 2003; 278: 19237-19244Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). However, the interpretation of these ion transport studies on the non-gastric H,K-ATPase was complicated by the fact that both Na+ and H+ ions are transported in exchange for K+ and that the H+(Na+)/K+ transport stoichiometry is not known. Because the non-gastric H,K-ATPase is more similar to Na,K-ATPases in terms of transported ions (25Cougnon M. Planelles G. Crowson M.S. Shull G.E. Rossier B.C. Jaisser F. J. Biol. Chem. 1996; 271: 7277-7280Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 26Cougnon M. Bouyer P. Planelles G. Jaisser F. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 6516-6520Crossref PubMed Scopus (79) Google Scholar) and the sensitivity toward ouabain (27Rajendran V.M. Sangan P. Geibel J. Binder H.J. J. Biol. Chem. 2000; 275: 13035-13040Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and SCH28080 (28Jaisser F. Horisberger J.D. Geering K. Rossier B.C. J. Cell Biol. 1993; 123: 1421-1429Crossref PubMed Scopus (92) Google Scholar, 29Buffin-Meyer B. Younes-Ibrahim M. Barlet-Bas C. Cheval L. Marsy S. Doucet A. Am. J. Physiol. 1997; 272: F124-F131PubMed Google Scholar), the implications of these findings for the gastric enzyme are unclear so far. These limitations prompted us to investigate the functional relevance of Lys-791 for electroneutral transport of a bona fide H+-transporting H,K-ATPase, the rat gastric H,K-ATPase expressed in Xenopus oocytes. We took advantage of a variant gastric H,K-ATPase α-subunit with a single cysteine replacement S806C in the extracellular TM5/TM6 loop (see Fig. 1) that enabled us to study several Lys-791 mutants also by voltage clamp fluorometry (VCF) upon site-specific labeling with tetramethylrhodamine-6-maleimide (TMRM), as described previously (30Dürr K.L. Abe K. Tavraz N.N. Friedrich T. J. Biol. Chem. 2009; 284: 20147-20154Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 31Dürr K.L. Tavraz N.N. Dempski R.E. Bamberg E. Friedrich T. J. Biol. Chem. 2009; 284: 3842-3854Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 32Dürr K.L. Tavraz N.N. Zimmermann D. Bamberg E. Friedrich T. Biochemistry. 2008; 47: 4288-4297Crossref PubMed Scopus (19) Google Scholar). In addition to the information regarding steady-state transport, which was provided by Rb+ uptake measurements, this method allows the study of voltage-dependent conformational changes under presteady-state conditions. Moreover, VCF not only reveals the voltage-dependent distribution between E1P/E2P states but also the kinetics of the E1P ↔ E2P conformational transition. The cDNAs of the rat gastric H,K-ATPase β-subunit and a modified form of the α-subunit with a single cysteine replacement in the TM5/TM6 extracellular loop (S806C, see Fig. 1) were subcloned into vector pTLN (33Lorenz C. Pusch M. Jentsch T.J. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13362-13366Crossref PubMed Scopus (215) Google Scholar). This cysteine replacement is homologous to the N790C mutation in the TM5/TM6 loop of the Na,K-ATPase α-subunit (34Geibel 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: 31-38Crossref PubMed Scopus (20) Google Scholar, 35Geibel S. Kaplan J.H. Bamberg E. Friedrich T. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 964-969Crossref PubMed Scopus (48) Google Scholar). It has been shown previously that the S806C mutation enables site-specific labeling of H,K-ATPase with the environmentally sensitive fluorophore TMRM, and rubidium uptake measurements confirmed that the αS806C mutation did not affect the transport properties of gastric H,K-ATPase (32Dürr K.L. Tavraz N.N. Zimmermann D. Bamberg E. Friedrich T. Biochemistry. 2008; 47: 4288-4297Crossref PubMed Scopus (19) Google Scholar). Additional amino acid replacements at positions Lys-791 and Glu-820 were introduced into the αS806C reference construct (which is referred as “wild-type” or WT throughout the current study) using the QuikChange XL site-directed mutagenesis kit (Agilent Technologies) and verified by DNA sequencing (Eurofins MWG Operon). Xenopus oocytes were obtained by collagenase treatment after partial ovarectomy from Xenopus laevis females. cRNAs were prepared using the SP6 mMessage mMachine kit (Applied Biosystems). A 50-nl aliquot containing 20–25 ng of H,K-ATPase α-subunit cRNA and 5 ng of H,K-ATPase β-subunit was 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 2 days. Before all experiments, which were usually carried out 2–3 days after injection at 21–24 °C, oocytes were preincubated in solutions containing 100 μm ouabain (Sigma) to inhibit the endogenous Xenopus Na,K-ATPase. Oocytes were incubated for 15 min in Rb+ flux buffer (5 mm RbCl, 85 mm tetramethylammonium chloride, 20 mm tetraethylammonium chloride, 5 mm BaCl2, 5 mm NiCl2, 10 mm MES, pH 5.5, 100 μm ouabain). After 3 washing steps in Rb+-free washing buffer (90 mm tetramethylammonium chloride or NaCl, 20 mm tetraethylammonium chloride, 5 mm BaCl2, 5 mm NiCl2, 10 mm MES, pH 5.5) and 1 wash in water, each individual oocyte was homogenized in 1 ml of Millipore water. For inhibition experiments, the K+-competitive inhibitor SCH28080 (Sigma) was added to the preincubation solution and Rb+ flux buffer (to final concentrations as indicated by the respective figure legends to Figs. 3, 6, and 8). For vanadate inhibition experiments, the respective oocytes were injected with 50 nl of a solution containing 100 mm sodium orthovanadate (buffered with 10 mm Hepes at pH 7.4) ∼30–40 min before Rb+ uptake measurements. Assuming an oocyte volume of about 1 μl, this corresponds to a final intracellular concentration of ∼5 mm. Whereas the inhibitor vanadate interacts specifically" @default.
• W2000341993 created "2016-06-24" @default.
• W2000341993 creator A5036783911 @default.
• W2000341993 creator A5066662505 @default.
• W2000341993 creator A5069837459 @default.
• W2000341993 date "2010-12-01" @default.
• W2000341993 modified "2023-10-01" @default.
• W2000341993 title "Deceleration of the E1P-E2P Transition and Ion Transport by Mutation of Potentially Salt Bridge-forming Residues Lys-791 and Glu-820 in Gastric H+/K+-ATPase" @default.
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