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• W2036799673 abstract "In oligomeric P2-ATPases such as Na,K- and H,K-ATPases, % subunits play a fundamental role in the structural and functional maturation of the catalytic α subunit. In the present study we performed a tryptophan scanning analysis on the transmembrane α-helix of the Na,K-ATPase %1 subunit to investigate its role in the stabilization of the α subunit, the endoplasmic reticulum exit of α-% complexes, and the acquisition of functional properties of the Na,K-ATPase. Single or multiple tryptophan substitutions in the % subunits transmembrane domain had no significant effect on the structural maturation of α subunits expressed in Xenopus oocytes nor on the level of expression of functional Na,K pumps at the cell surface. Furthermore, tryptophan substitutions in regions of the transmembrane α-helix containing two GXXXG transmembrane helix interaction motifs or a cysteine residue, which can be cross-linked to transmembrane helix M8 of the α subunit, had no effect on the apparent K+ affinity of Na,K-ATPase. On the other hand, substitutions by tryptophan, serine, alanine, or cysteine, but not by phenylalanine of two highly conserved tyrosine residues, Tyr40 and Tyr44, on another face of the transmembrane helix, perturb the transport kinetics of Na,K pumps in an additive way. These results indicate that at least two faces of the % subunits transmembrane helix contribute to inter- or intrasubunit interactions and that two tyrosine residues aligned in the % subunits transmembrane α-helix are determinants of intrinsic transport characteristics of Na,K-ATPase. In oligomeric P2-ATPases such as Na,K- and H,K-ATPases, % subunits play a fundamental role in the structural and functional maturation of the catalytic α subunit. In the present study we performed a tryptophan scanning analysis on the transmembrane α-helix of the Na,K-ATPase %1 subunit to investigate its role in the stabilization of the α subunit, the endoplasmic reticulum exit of α-% complexes, and the acquisition of functional properties of the Na,K-ATPase. Single or multiple tryptophan substitutions in the % subunits transmembrane domain had no significant effect on the structural maturation of α subunits expressed in Xenopus oocytes nor on the level of expression of functional Na,K pumps at the cell surface. Furthermore, tryptophan substitutions in regions of the transmembrane α-helix containing two GXXXG transmembrane helix interaction motifs or a cysteine residue, which can be cross-linked to transmembrane helix M8 of the α subunit, had no effect on the apparent K+ affinity of Na,K-ATPase. On the other hand, substitutions by tryptophan, serine, alanine, or cysteine, but not by phenylalanine of two highly conserved tyrosine residues, Tyr40 and Tyr44, on another face of the transmembrane helix, perturb the transport kinetics of Na,K pumps in an additive way. These results indicate that at least two faces of the % subunits transmembrane helix contribute to inter- or intrasubunit interactions and that two tyrosine residues aligned in the % subunits transmembrane α-helix are determinants of intrinsic transport characteristics of Na,K-ATPase. transmembrane N-methyl-d-glucamine endoplasmic reticulum P-type ATPases represent a family of ubiquitous transporters which are characterized by the formation of a phosphorylated intermediate during the catalytic cycle and which are mainly involved in cation homeostasis. Over 200 members of this family have been identified (1Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (735) Google Scholar). Of these, only animal Na,K- and H,K-ATPase isozymes and bacterial K-ATPase isozymes, are oligomeric with 1 and 3 subunits, respectively, in addition to the catalytic subunit. Na,K- and H,K-ATPase α subunits have the highest sequence identity among the P-type ATPases. Based on the membrane topology, the catalytic α subunits of Na,K- and H,K-ATPases belong to the group of P2-ATPases (2Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (414) Google Scholar) which according to the crystal structure of the Ca2+-ATPase contains 10 transmembrane (TM)1 segments (3Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1594) Google Scholar). The % subunits associated with Na,K- and H,K-ATPases are type II proteins with one TM segment, a short cytoplasmic tail, and a large ectodomain containing several sugar chains and 3 disulfide bridges. So far, three Na,K-ATPase % isoforms and one H,K-ATPase % subunit have been identified. The unique presence of % subunits in Na,K- and H,K-ATPases remains intriguing both from a functional and evolutionary point of view. At present, we know that the % subunit has two main functions. Of primary importance is its role as a specific chaperone which favors the correct membrane insertion and hence the resistance against proteolysis and cellular degradation of the newly synthesized α subunits of Na,K- and H,K-ATPases (4Geering K. Theulaz I. Verrey F. Häuptle M.T. Rossier B.C. Am. J. Physiol. 1989; 257: C851-C858Crossref PubMed Google Scholar, 5Béguin P. Hasler U. Beggah A. Horisberger J.D. Geering K. J. Biol. Chem. 1998; 273: 24921-24931Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 6Béguin P. Hasler U. Staub O. Geering K. Mol. Biol. Cell. 2000; 11: 1657-1672Crossref PubMed Scopus (52) Google Scholar, 7Beggah A.T. Béguin P. Bamberg K. Sachs G. Geering K. J. Biol. Chem. 1998; 274: 8217-8223Abstract Full Text Full Text PDF Scopus (42) Google Scholar). Since KdpC subunits of the bacterial KdpFABC transporter may have a similar function (8Altendorf K. Gassel M. Puppe W. Mollenkamp T. Zeeck A. Boddien C. Fendler K. Bamberg E. Drose S. Acta Physiol. Scand. Suppl. 1998; 643: 137-146PubMed Google Scholar), it has been speculated that the % subunits of Na,K- and H,K-ATPases may be remnants of the bacterial KdpC subunit that have been eliminated in other P2-ATPases (1Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (735) Google Scholar). Based on topology studies on Na,K- and H,K-ATPase α subunits, we have also suggested that the K+ transport function common to all oligomeric P-type ATPases, is associated with a particular amino acid composition that is not compatible with efficient membrane insertion of the α subunits. This has required that during evolution, K+ transporting α subunits had to assemble with a helper protein in order to assist their correct membrane integration (9Geering K. J. Membr. Biol. 2000; 174: 181-190Crossref PubMed Scopus (25) Google Scholar). In addition to their chaperone function, % subunits also influence the cation sensitivity of oligomeric P-type ATPases expressed at the cell surface. The association of the α subunit with different % isoforms (10Jaisser F. Jaunin P. Geering K. Rossier B.C. Horisberger J.-D. J. Gen. Physiol. 1994; 103: 605-623Crossref PubMed Scopus (130) Google Scholar) or N-terminal truncated % subunits (11Geering K. Beggah A. Good P. Giradet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar, 12Hasler U. Wang X. Crambert G. Béguin P. Jaisser F. Horisberger J.-D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) produces Na,K-ATPases with different apparent affinities for Na+ and K+. An important issue concerning the structure-function relationship of Na,K- and H,K-ATPases is the identification of the matching interaction sites in the α and the % subunits that are responsible for the chaperone function and/or the transport-modulating effect of the % subunit. Experimental evidence suggests that α and % subunits interact in the extracytoplasmic, the TM, and the cytoplasmic domains. In α subunits of both Na,K-ATPase (13Colonna T.E. Huynh L. Fambrough D.M. J. Biol. Chem. 1997; 272: 12366-12372Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and H,K-ATPase (14Melle-Milovanovic D. Milovanovic M. Nagpal S. Sachs G. Shin J.M. J. Biol. Chem. 1998; 273: 11075-11081Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), the most clearly defined interaction site is located in the extracytoplasmic loop between the TM segment M7 and M8. Interaction of the % subunit with this region was shown to be important for the correct membrane insertion and the structural maturation of the Na,K- and H,K-ATPase α subunit (5Béguin P. Hasler U. Beggah A. Horisberger J.D. Geering K. J. Biol. Chem. 1998; 273: 24921-24931Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 6Béguin P. Hasler U. Staub O. Geering K. Mol. Biol. Cell. 2000; 11: 1657-1672Crossref PubMed Scopus (52) Google Scholar, 7Beggah A.T. Béguin P. Bamberg K. Sachs G. Geering K. J. Biol. Chem. 1998; 274: 8217-8223Abstract Full Text Full Text PDF Scopus (42) Google Scholar). According to results obtained using the yeast two-hybrid system, the M7 and M8 α-domain interacts with an extracytoplasmic region of the % subunit contained within the 64 amino acids adjacent to the TM domain (13Colonna T.E. Huynh L. Fambrough D.M. J. Biol. Chem. 1997; 272: 12366-12372Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). However, mutational and immunological studies suggest that other regions in the extracytoplasmic domain of the % subunit such as the 10 most C-terminal amino acids (15Beggah A.T. Béguin P. Jaunin P. Peitsch M.C. Geering K. Biochemistry. 1993; 32: 14117-14124Crossref PubMed Scopus (39) Google Scholar) or a YYPYYG sequence conserved in all known % subunits (16Geering K. Jaunin P. Jaisser F. Merillat A.M. Horisberger J.D. Mathews P.M. Lemas V. Fambrough D.M. Rossier B.C. Am. J. Physiol. 1993; 265: C1169-1174Crossref PubMed Google Scholar, 17Okamoto C.T. Chow D.C. Forte J.G. Am. J. Physiol. 2000; 278: C727-C738Crossref Google Scholar) might as well participate in α-% interactions and contribute to the stabilization of the α subunit. As suggested by results obtained using chimeras between different % isoforms (12Hasler U. Wang X. Crambert G. Béguin P. Jaisser F. Horisberger J.-D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar,18Wang S.-G. Eakle K.A. Levenson R. Farley R.A. Am. J. Physiol. 1997; 272: C923-C930Crossref PubMed Google Scholar), interactions in the ectodomains of the α and the % subunit might also be responsible for the % subunits effects on the cation sensitivity of Na,K-ATPase. Controlled proteolysis assays performed on Na,K-ATPase % subunits, indicate that α and % subunits also interact in the cytoplasmic domain (11Geering K. Beggah A. Good P. Giradet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar). A mutational analysis indicates that these interactions are not necessary for the structural maturation of the α subunit (11Geering K. Beggah A. Good P. Giradet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar,12Hasler U. Wang X. Crambert G. Béguin P. Jaisser F. Horisberger J.-D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), nor do they directly influence apparent Na+ or K+ affinities of Na,K-ATPase (12Hasler U. Wang X. Crambert G. Béguin P. Jaisser F. Horisberger J.-D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 19Hasler U. Greasley P.J. von Heijne G. Geering K. J. Biol. Chem. 2000; 275: 29011-29022Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). On the other hand, it cannot be excluded that cytoplasmic α-% interactions contribute to some discrete steps in the catalytic cycle of Na,K-ATPase as suggested by Na+ occlusion and electrogenic binding assays (20Shainskaya A. Scneeberger A. Appell H-J. Karlish S.J.D. J. Biol. Chem. 2000; 275: 2019-2028Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Interactions in the TM domains of the α and % subunits are the least well understood both in molecular and functional terms. So far, cross-linking experiments have provided evidence that the TM domain of the % subunit may be in contact with M8 of the α subunit (21Ivanov A. Zhao H. Modyanov N.N. Biochemistry. 2000; 39: 9778-9785Crossref PubMed Scopus (19) Google Scholar, 22Or E. Goldshleger R. Karlish S.J.D. J. Biol. Chem. 1999; 274: 2802-2809Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), but nothing is known on the functional implications of this possible intersubunit interaction. In this study, we have aimed to identify amino acid residues in the % TM domain that interact with the α subunit and to determine the putative functional role of this interaction by using a tryptophan scanning analysis. Tryptophan scanning has previously been used to determine structural features of integral membrane proteins (23Sharp L.L. Zhou J. Blair D.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7946-7950Crossref PubMed Scopus (96) Google Scholar, 24Sharp L.L. Zhou J. Blair D.F. Biochemistry. 1995; 34: 9166-9171Crossref PubMed Scopus (98) Google Scholar, 25Braun P. Persson B. Kaback H.R. von Heijne G. J. Biol. Chem. 1997; 272: 29566-29571Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Tryptophan was chosen because of its moderately hydrophobic properties and its large size. The results of previous studies are consistent with the expectations that if tryptophan is introduced at several positions in a membrane segment, its large side chain is tolerated when facing the lipid bilayer but, when positioned inside the protein, it may disrupt function by breaking helix-helix interactions. Based on these predictions, we aimed to get structural and functional information on the TM domain of the Na,K-ATPase % subunit by replacing amino acids individually or in combination by tryptophan. % Subunit mutants were expressed in Xenopus oocytes together with α subunits and the stability and the transport properties of the resulting Na,K-ATPase α-% complexes were analyzed. Our results indicate that interactions in the TM domain of α and % subunits do not play a role in the α-stabilizing effect of the % subunit. On the other hand, mutations of two tyrosine residues, highly conserved in the TM domain of all known % subunits, significantly modulate the transport kinetics of the Na,K-ATPase. The results of this study thus provide evidence that the TM domain of the % subunit contributes to intrinsic functional properties of oligomeric P-type ATPases as opposed to the transport-modulating interactions in the ectodomain observed for % isoforms. Amino acids Ile36 to Gln56 of Xenopus Na,K-ATPase %1 (%NK) were individually replaced by tryptophan residues using the polymerase chain reaction method described by Nelson and Long (26Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar). Briefly, fragments of %1NK contained in a pSD5 vector (pSD5%1NK) were first amplified by polymerase chain reaction using sense oligonucleotides containing mutated sequences coding for tryptophan and an antisense oligonucleotide consisting of nucleotides 628–648 tailed by primer D of Nelson and Long (26Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar). The amplified fragments were then used as primers to elongate the inverse DNA strand which in turn was amplified using a sense oligonucleotide encoding the SP6 sequence of pSD5%NK and primer D of Nelson and Long (26Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar). The mutated DNA fragments were introduced into wild type pSD5%1NK using NheI andBamHI restriction sites. The Y40W mutant was used as a template for the preparation of Y40W/Y44W. G45W was used as a template for the preparation of G45W/G49W which in turn was used for the preparation of G45W/G49W/G53W. Y40F/Y44F, Y40S/Y44S, Y40C/Y44C, and Y40A/Y44A were prepared using sense oligonucleotides containing both mutated sequences. The chimera NK/HK in which the cytoplasmic and TM domains (Met1-Asp71) are derived fromXenopus Na,K-ATPase %1 and the ectodomain (Gln76-Lys291) from rabbit, gastric H,K-ATPase % subunit was prepared as previously described (27Jaunin P. Jaisser F. Beggah A.T. Takeyasu K. Mangeat P. Rossier B.C. Horisberger J.-D. Geering K. J. Cell Biol. 1993; 123: 1751-1759Crossref PubMed Scopus (74) Google Scholar). NK/HK Y40W/Y44W was prepared by amplifying a fragment of %1NK as described above using a sense oligonucleotide containing both mutated sequences, an antisense oligonucleotide encoding nucleotides 420–440 tailed by primer D of Nelson and Long (26Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar) and using pSD5 NK/HK as a template.NheI and PvuII restriction sites were used to introduce the amplified fragment into the pSD5 vector containing NK/HK. %3 Y43W/Y47W was prepared by amplifying a fragment of %3NK contained in a pSD5 vector (pSD5%3NK) using a sense oligonucleotide containing both mutated sequences, an antisense oligonucleotide encoding nucleotides 301–320 tailed by primer D of Nelson and Long (26Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar) and using pSD5%3NK as a template. The mutated DNA fragment was introduced into wild type pSD5%3NK using NheI andStuI restriction sites. The nucleotide sequences of all constructs were confirmed by dideoxy sequencing. cRNAs coding forBufo Na,K-ATPase α1 (28Jaisser J. Canessa C.M. Horisberger J.-D. Rossier B.C. J. Biol. Chem. 1992; 267: 16895-16903Abstract Full Text PDF PubMed Google Scholar), Xenopus Na,K-ATPase α1 (29Verrey F. Kairouz P. Schaerer E. Fuentes P. Geering K. Rossier B.C. Kraehenbuhl J.-P. Am. J. Physiol. 1989; 256: F1034-F1043PubMed Google Scholar), Xenopus %1 (29Verrey F. Kairouz P. Schaerer E. Fuentes P. Geering K. Rossier B.C. Kraehenbuhl J.-P. Am. J. Physiol. 1989; 256: F1034-F1043PubMed Google Scholar), %3 (30Good P.J. Richter K. Dawid I.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9088-9092Crossref PubMed Scopus (92) Google Scholar) subunits, and % subunit mutants were obtained by in vitro transcription (31Melton D.A. Krieg P.A. Rebagliati M.R. Maniatis T. Zinn K. Green M.R. Nucleic Acids Res. 1984; 12: 7035-7056Crossref PubMed Scopus (4042) Google Scholar). Oocytes were obtained from Xenopusfemales as described (4Geering K. Theulaz I. Verrey F. Häuptle M.T. Rossier B.C. Am. J. Physiol. 1989; 257: C851-C858Crossref PubMed Google Scholar). Routinely, 7 ng of Bufo marinus or α and 0.8 ng of Xenopus % subunit cRNAs were injected into oocytes. Oocytes were incubated in modified Barth's medium containing [35S]methionine (0.5 mCi/ml) for 6 h and then subjected to 24 and 72 h chase periods in the presence of 10 mm cold methionine. Digitonin extracts were prepared as described (11Geering K. Beggah A. Good P. Giradet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar) and the α subunit was immunoprecipitated using aBufo α1 subunit antibody (32Girardet M. Geering K. Frantes J.M. Geser D. Rossier B.C. Kraehenbuhl J.-P. Bron C. Biochememistry. 1981; 20: 6684-6691Crossref PubMed Scopus (435) Google Scholar) under nondenaturing conditions as described (11Geering K. Beggah A. Good P. Giradet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar) allowing co-immunoprecipitation of the associated % subunit. The dissociated immune complexes were separated by SDS-polyacrylamide gel electrophoresis and labeled proteins were detected by fluorography. Quantification of immunoprecipitated bands was performed with a laser densitometer (LKB Ultrascan 2202). Na,K pump activity was measured as the K+-induced outward current using the two-electrode voltage clamp method as described earlier (10Jaisser F. Jaunin P. Geering K. Rossier B.C. Horisberger J.-D. J. Gen. Physiol. 1994; 103: 605-623Crossref PubMed Scopus (130) Google Scholar). Current measurements were performed 3 days after injection of oocytes withBufo α1 and either wild type or mutant % cRNAs. One day before performing measurements, oocytes were loaded with Na+ in a K+-free solution containing 200 nm ouabain, a concentration that inhibits the endogenous Na,K pumps but not the ouabain-resistant exogenous Bufo Na,K pumps (33Wang X.Y. Jaisser F. Horisberger J.-D. J. Physiol. 1996; 491: 579-594Crossref PubMed Scopus (28) Google Scholar). The K+ activation of the Na,K pump current was determined in a Na+-containing solution (80 mm sodium gluconate, 0.82 mm MgCl2, 0.41 mmCaCl2, 10 mm N-methyl-d-glucamine (NMDG)-HEPES, 5 mm BaCl2, 10 mm tetraethylammonium chloride, pH 7.4) or in a nominally Na+-free solution (sodium gluconate was replaced by 140 mm sucrose). The current induced by increasing concentrations of K+ (0.3, 1.0, 3.3, and 10 mm K+ in the presence of Na+ and 0.02, 0.1, 0.5, and 5.0 mmK+ in the absence of Na+) was measured either at −50 mV or during a series of nine 200-ms voltage steps ranging from −130 to +30 mV. To determine the kinetic parameters such as maximal currents (ImaxK) and half-activation constants (K1/2K+) the Hill equation was fitted to the data of the current (I) induced by various K+ concentrations ([K]) using a least square method: I = ImaxK/(1 + (K1/2K+/[K])nH). According to previous studies (10Jaisser F. Jaunin P. Geering K. Rossier B.C. Horisberger J.-D. J. Gen. Physiol. 1994; 103: 605-623Crossref PubMed Scopus (130) Google Scholar), the Hill coefficient (nH) was set to a value of 1.6 for experiments performed in the presence of external Na+ and 1.0 for experiments performed in the absence of external Na+. Measurements of the half-activation constant for internal Na+ was performed as previously described (12Hasler U. Wang X. Crambert G. Béguin P. Jaisser F. Horisberger J.-D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Briefly, in addition to Na,K-ATPase α and % cRNAs, oocytes were injected with cRNAs coding for α, %, and γ subunits (0.3 ng of each subunit/oocyte) of the rat epithelial Na+ channel, rENaC (34Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.-D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1739) Google Scholar), and were incubated for 3 days in a modified Barth's solution containing 10 mm Na+. One day before measurements, oocytes were incubated in a Na+-free solution (50 mm NMDG-Cl, 40 mm KCl, 1 mmCaCl2, 1 mm MgCl2, 10 mm NMDG-HEPES, pH 7.4) in order to maximally reduce the internal Na+ concentration. Intracellular Na+ concentrations were calculated from the reversal potential of the amiloride-sensitive current obtained from a pair of I-V curves recorded with and without amiloride in a solution containing 5 mm Na+ (5 mm sodium gluconate, 0.5 mm MgCl2, 2.5 mmBaCl2, 95 mm NMDG-Cl, 10 mmNMDG-HEPES, pH 7.4). At each intracellular Na+concentration ([Na]i) the Na,K pump K+-activated current (IK) was measured in the presence of 20 μm amiloride and in the absence or presence of external Na+ (see above) by addition of 5 or 10 mmK+. Maximal pump currents (Imax,Na) and half-activation constants for Na+(K1/2Na+int) were determined by fitting the Hill equation [IK =Imax,Na/(1 + (K1/2Na+int/[Na]i)3)] to the measured IK and [Na]i values. Six to eight pairs of measurements of [Na]i and of the Na,K pump K+-activated current were performed successively on each oocyte at −50 mV. Between each pair of measurements, the oocytes were allowed to increase their intracellular Na+concentration by exposure to a 100 mm Na+solution (100 mm sodium gluconate, 1 mmMgCl2, 0.5 mm CaCl2, 10 mm Na-HEPES, pH 7.4) in the absence of amiloride and at a holding potential of −50 to −100 mV. Measurements of the apparent affinity for external Na+ were performed using ouabain-sensitive Xenopus α1 subunits by measuring the inhibition of the K+-induced current by external Na+. 3 days after oocyte injection and 1 day before measurements, oocytes were loaded with Na+ in a K+-free solution. The Na,K pump current induced by 1 mm K+ was measured in a nominally Na+-free solution (120 mm NMDG-gluconate, 0.82 mm MgCl2, 0.41 mmCaCl2, 10 mm NMDG-HEPES, 5 mm BaCl2, 10 mm tetraethylammonium chloride, pH 7.4) and in the presence of 10, 30, 60, and 120 mm Na+ (NMDG-gluconate was replaced by sodium gluconate) during a series of nine 200-ms voltage steps ranging from −130 to +30 mV. The Na,K pump was then blocked by the addition of 100 μm ouabain and the same series of measurements was repeated. Currents specific for the Na,K pump could be deduced by subtracting the currents observed in the presence of ouabain from those observed in its absence. A nonsaturating concentration of K+ (1 mm) was chosen in order to reveal the competition of external Na+ with K+ ions for extracellular cation-binding sites. At each external Na+concentration, the averaged endogenous, ouabain-sensitive Na,K pump current was subtracted from total ouabain-sensitive currents measured in oocytes expressing exogenous Na,K pumps. The decrease in the Na,K pump current produced by exposure to external Na+ was used to determine the half-inhibition constant for external Na+(K1/2Na+ext) by fitting the Hill equation [I = Imax,Na(1–1/(1 + (K1/2Na+ext/[Na])nH)] to the data of the current (I) observed at each concentration of external Na+. Means of the Na,K pump currents produced in oocytes expressing α subunits and wild type or mutant % subunits were compared by unpaired Student'st test. Na,K-ATPase activity was measured in microsomal fractions prepared as previously described (11Geering K. Beggah A. Good P. Giradet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar) from oocytes expressing Xenopus α subunits and wild type %1 subunits or %1 Y40W/Y44W mutants. Before activity measurements, samples were freeze/thawed twice in liquid nitrogen. Na,K-ATPase activity was measured in triplicate by an enzyme-linked assay, according to Schoner et al. (35Schoner W. von Ilberg C. Kramer K. Seubert W. Eur. J. Biochem. 1967; 1: 334-343Crossref PubMed Scopus (246) Google Scholar), in which the resynthesis of ATP consumed by the ATPase is coupled by the pyruvate and lactate dehydrogenase reactions to NADH oxidation. The oxidation rate of NADH was recorded at 340 nm wavelength in the automated enzyme kinetic accessory of a DU-64 spectrophotometer (Beckman Instruments). The substrate concentrations of the reaction mixture were 5 mmKCl, 100 mm NaCl, 4 mm ATP, and 4 mm MgCl2. To reduce nonspecific, mitochondrial ATPase activities, 5 mm NaN3 were added to the reaction mixture. Activity measurements were done in the presence or absence of 10−7-10−4morthovanadate. The specific enzyme activity was calculated as the difference between samples incubated in the presence or absence of 1 mm ouabain. In the absence of vanadate, ouabain-sensitive activities represented 20–30% of the total enzyme activity. Statistical analysis was done by unpaired Student's ttest. Fig. 1 A shows the membrane-spanning domain of the %1 subunit of XenopusNa,K-ATPase, as previously defined (19Hasler U. Greasley P.J. von Heijne G. Geering K. J. Biol. Chem. 2000; 275: 29011-29022Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The TM domain of theXenopus %1 subunit comprises 23 amino acids and shows a sequence identity of 43% with Xenopus %2, 65% with %3, and 43% with the % subunit of gastric H,K-ATPase. Secondary structure prediction on the %1 TM domain revealed an α-helical structure with at least two distinct faces (Fig. 1 B). One face is characterized by three aligned residues with large lateral side chains (Tyr40, Tyr44, and Phe51) (numbering of amino acids correspond to Xenopus %1 subunits) whereas another face contains three aligned glycine residues (Gly45, Gly49, and Gly53) forming a cleft in the α-helix. The two tyrosine residues are conserved in all known % subunits whereas the three glycine residues are only found in Na,K-ATPase %1 isoforms and certain %3 isoforms. Other Na,K-ATPase % isoforms and H,K-ATPase % subunits contain 1 or 2 glycine residues corresponding to either Gly45 and/or Gly49. Three phenylalanine residues (Phe39, Phe43, and Phe51) are present in the TM domain of all Na,K-ATPase % isoforms whereas the H,K-ATPase % subunit contains a conservative substitution of tyrosine for Phe39. Na,K-ATPase %2 and %3 isoforms contain a fourth phenylalanine residue which is replaced by a cysteine residue (Cys46) in %1 isoforms (Fig. 1, A and B). In order to identify the amino acid residues in the % TM domain that interact with the α subunit and to determine the putative functional role of this interaction, amino acids encompassing Ile36 to Gln56 were substituted individually or in combination by tryptophan. We first tested whether tryptophan substitutions in the TM domain of the % subunit might interfere with the structural maturation of the α subunit. Bufo α subunits were expressed in Xenopus oocytes alone or together withXenopus wild type or mutant %1 subunits and the cellular expression of the α-% complexes was followed by immunoprecipitation of metabolically labeled proteins after a pulse and various chase periods. As expected, α subunits expressed without % subunits were degraded during a 48-h chase period (Fig.2, lanes 10 and11). Similar to wild type %1 subunits (Fig. 2, lanes 1–3), all %-mutants assembled with and stabilized α-subunits as illustrated for the triple glycine mutant G45W/G49W/G53W (lanes 4–6) and the double tyrosine mutant Y40W/Y44W (lanes 7–9). In addition, all % subunit mutants assembled with α subunits became full" @default.
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