Structural and functional features of the transmembrane domain of the Na,K-ATPase beta subunit revealed by tryptophan scanning.

In oligomeric P2-ATPases such as Na,K- and H,K-ATPases, beta subunits play a fundamental role in the structural and functional maturation of the catalytic alpha subunit. In the present study we performed a tryptophan scanning analysis on the transmembrane alpha-helix of the Na,K-ATPase beta1 subunit to investigate its role in the stabilization of the alpha subunit, the endoplasmic reticulum exit of alpha-beta complexes, and the acquisition of functional properties of the Na,K-ATPase. Single or multiple tryptophan substitutions in the beta subunits transmembrane domain had no significant effect on the structural maturation of alpha 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 alpha-helix containing two GXXXG transmembrane helix interaction motifs or a cysteine residue, which can be cross-linked to transmembrane helix M8 of the alpha 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, Tyr(40) and Tyr(44), 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 beta subunits transmembrane helix contribute to inter- or intrasubunit interactions and that two tyrosine residues aligned in the beta subunits transmembrane alpha-helix are determinants of intrinsic transport characteristics of Na,K-ATPase.

the Ca 2ϩ -ATPase contains 10 transmembrane (TM) 1 segments (3). 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-AT-Pases 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 ␣ subunits of Na,K-and H,K-ATPases (4 -7). Since KdpC subunits of the bacterial Kd-pFABC transporter may have a similar function (8), 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 (1). Based on topology studies on Na,K-and H,K-ATPase ␣ subunits, we have also suggested that the K ϩ 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 ␣ subunits. This has required that during evolution, K ϩ transporting ␣ subunits had to assemble with a helper protein in order to assist their correct membrane integration (9). 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 ␣ subunit with different ␤ isoforms (10) or N-terminal truncated ␤ subunits (11,12) produces Na,K-ATPases with different apparent affinities for Na ϩ and K ϩ .
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 ␣ and the ␤ subunits that are responsible for the chaperone function and/or the transportmodulating effect of the ␤ subunit. Experimental evidence suggests that ␣ and ␤ subunits interact in the extracytoplasmic, the TM, and the cytoplasmic domains. In ␣ subunits of both Na,K-ATPase (13) and H,K-ATPase (14), 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 ␣ subunit (5)(6)(7). According to results obtained using the yeast two-hybrid system, the M7 and M8 ␣-domain interacts with an extracytoplasmic region of the ␤ subunit contained within the 64 amino acids adjacent to the TM domain (13). 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 (15) or a YYPYYG sequence conserved in all known ␤ subunits (16,17) might as well participate in ␣-␤ interactions and contribute to the stabilization of the ␣ subunit. As suggested by results obtained using chimeras between different ␤ isoforms (12,18), interactions in the ectodomains of the ␣ 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 ␣ and ␤ subunits also interact in the cytoplasmic domain (11). A mutational analysis indicates that these interactions are not necessary for the structural maturation of the ␣ subunit (11,12), nor do they directly influence apparent Na ϩ or K ϩ affinities of Na,K-ATPase (12,19). On the other hand, it cannot be excluded that cytoplasmic ␣-␤ interactions contribute to some discrete steps in the catalytic cycle of Na,K-ATPase as suggested by Na ϩ occlusion and electrogenic binding assays (20).
Interactions in the TM domains of the ␣ 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 ␣ subunit (21,22), 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 ␣ 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 (23)(24)(25). 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 ␣ subunits and the stability and the transport properties of the resulting Na,K-ATPase ␣-␤ complexes were analyzed. Our results indicate that interactions in the TM domain of ␣ and ␤ subunits do not play a role in the ␣-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.

EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis-Amino acids Ile 36 to Gln 56 of Xenopus Na,K-ATPase ␤1 (␤NK) were individually replaced by tryptophan residues using the polymerase chain reaction method described by Nelson and Long (26). 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 (26). 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 (26). The mutated DNA fragments were introduced into wild type pSD5␤1NK using NheI and BamHI 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 (Met 1 -Asp 71 ) are derived from Xenopus Na,K-ATPase ␤1 and the ectodomain (Gln 76 -Lys 291 ) from rabbit, gastric H,K-ATPase ␤ subunit was prepared as previously described (27). 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 (26) 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 (26) and using pSD5␤3NK as a template. The mutated DNA fragment was introduced into wild type pSD5␤3NK using NheI and StuI restriction sites. The nucleotide sequences of all constructs were confirmed by dideoxy sequencing. cRNAs coding for Bufo Na,K-ATPase ␣1 (28), Xenopus Na,K-ATPase ␣1 (29), Xenopus ␤1 (29), ␤3 (30) subunits, and ␤ subunit mutants were obtained by in vitro transcription (31).
Expression in Xenopus Oocytes and Immunoprecipitation of ␣ and ␤ Subunits-Oocytes were obtained from Xenopus females as described (4). Routinely, 7 ng of Bufo marinus or ␣ and 0.8 ng of Xenopus ␤ subunit cRNAs were injected into oocytes. Oocytes were incubated in modified Barth's medium containing [ 35 S]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 (11) and the ␣ subunit was immunoprecipitated using a Bufo ␣1 subunit antibody (32) under nondenaturing conditions as described (11) 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 Current Measurements and Determination of Apparent K ϩ and Na ϩ Affinities-Na,K pump activity was measured as the K ϩ -induced outward current using the two-electrode voltage clamp method as described earlier (10). Current measurements were performed 3 days after injection of oocytes with Bufo ␣1 and either wild type or mutant ␤ cRNAs. One day before performing measurements, oocytes were loaded with Na ϩ in a K ϩ -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 (33).
The K ϩ activation of the Na,K pump current was determined in a Na ϩ -containing solution (80 mM sodium gluconate, 0.82 mM MgCl 2 , 0.41 mM CaCl 2 , 10 mM N-methyl-D-glucamine (NMDG)-HEPES, 5 mM BaCl 2 , 10 mM tetraethylammonium chloride, pH 7.4) or in a nominally Na ϩfree solution (sodium gluconate was replaced by 140 mM sucrose). The current induced by increasing concentrations of K ϩ (0.3, 1.0, 3.3, and 10 mM K ϩ in the presence of Na ϩ and 0.02, 0.1, 0.5, and 5.0 mM K ϩ in the absence of Na ϩ ) was measured either at Ϫ50 mV or during a series of nine 200-ms voltage steps ranging from Ϫ130 to ϩ30 mV. To determine the kinetic parameters such as maximal currents (I maxK ) and halfactivation constants (K 1/2 K ϩ ) the Hill equation was fitted to the data of the current (I) induced by various K ϩ concentrations ([K]) using a least square method: I ϭ I maxK /(1 ϩ (K 1/2 K ϩ /[K]) nH ). According to previous studies (10), the Hill coefficient (nH) was set to a value of 1.6 for experiments performed in the presence of external Na ϩ and 1.0 for experiments performed in the absence of external Na ϩ .
Measurements of the half-activation constant for internal Na ϩ was performed as previously described (12). Briefly, in addition to Na,K-ATPase ␣ and ␤ cRNAs, oocytes were injected with cRNAs coding for ␣, ␤, and ␥ subunits (0.3 ng of each subunit/oocyte) of the rat epithelial Na ϩ channel, rENaC (34), and were incubated for 3 days in a modified Barth's solution containing 10 mM Na ϩ . One day before measurements, oocytes were incubated in a Na ϩ -free solution (50 mM NMDG-Cl, 40 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 10 mM NMDG-HEPES, pH 7.4) in order to maximally reduce the internal Na ϩ concentration.
Intracellular Na ϩ 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 ϩ (5 mM sodium gluconate, 0.5 mM MgCl 2 , 2.5 mM BaCl 2 , 95 mM NMDG-Cl, 10 mM NMDG-HEPES, pH 7.4). At each intracellular Na ϩ concentration ([Na] i ) the Na,K pump K ϩ -activated current (I K ) was measured in the presence of 20 M amiloride and in the absence or presence of external Na ϩ (see above) by addition of 5 or 10 mM K ϩ . Maximal pump currents (I max,Na ) and half-activation constants for Na ϩ (K 1/2 Na ϩ int ) were determined by fitting the Hill equation [I K ϭ I max,Na /(1 ϩ (K 1/2 Na ϩ int /[Na] i ) 3 )] to the measured I K and [Na] i values. Six to eight pairs of measurements of [Na] i and of the Na,K pump K ϩ -activated current were performed successively on each oocyte at Ϫ50 mV. Between each pair of measurements, the oocytes were allowed to increase their intracellular Na ϩ concentration by exposure to a 100 mM Na ϩ solution (100 mM sodium gluconate, 1 mM MgCl 2 , 0.5 mM CaCl 2 , 10 mM Na-HEPES, pH 7.4) in the absence of amiloride and at a holding potential of Ϫ50 to Ϫ100 mV.
Measurements of the apparent affinity for external Na ϩ were performed using ouabain-sensitive Xenopus ␣1 subunits by measuring the inhibition of the K ϩ -induced current by external Na ϩ . 3 days after oocyte injection and 1 day before measurements, oocytes were loaded with Na ϩ in a K ϩ -free solution. The Na,K pump current induced by 1 mM K ϩ was measured in a nominally Na ϩ -free solution (120 mM NMDG-gluconate, 0.82 mM MgCl 2 , 0.41 mM CaCl 2 , 10 mM NMDG-HEPES, 5 mM BaCl 2 , 10 mM tetraethylammonium chloride, pH 7.4) and in the presence of 10, 30, 60, and 120 mM Na ϩ (NMDG-gluconate was replaced by sodium gluconate) during a series of nine 200-ms voltage steps ranging from Ϫ130 to ϩ30 mV. The Na,K pump was then blocked by the addition of 100 M 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 ϩ (1 mM) was chosen in order to reveal the competition of external Na ϩ with K ϩ ions for extracellular cation-binding sites. At each external Na ϩ 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 ϩ was used to determine the half-inhibition constant for external Na ϩ (K 1/2 Na ϩ ext ) by fitting the Hill equation ] to the data of the current (I) observed at each concentration of external Na ϩ . Means of the Na,K pump currents produced in oocytes expressing ␣ subunits and wild type or mutant ␤ subunits were compared by unpaired Student's t test.
Na,K-ATPase Activity Measurements-Na,K-ATPase activity was measured in microsomal fractions prepared as previously described (11) from oocytes expressing Xenopus ␣ 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. (35), 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 mM KCl, 100 mM NaCl, 4 mM ATP, and 4 mM MgCl 2 . To reduce nonspecific, mitochondrial ATPase activities, 5 mM NaN 3 were added to the reaction mixture. Activity measurements were done in the presence or absence of 10 Ϫ7 -10 Ϫ4 M orthovanadate. 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 t test. Fig. 1A shows the membrane-spanning domain of the ␤1 subunit of Xenopus Na,K-ATPase, as previously defined (19). The TM domain of the Xenopus ␤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 ␣-helical structure with at least two distinct faces (Fig. 1B). One face is characterized by three aligned residues with large lateral side chains (Tyr 40 , Tyr 44 , and Phe 51 ) (numbering of amino acids correspond to Xenopus ␤1 subunits) whereas another face contains three aligned glycine residues (Gly 45 , Gly 49 , and Gly 53 ) forming a cleft in the ␣-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 Gly 45 and/or Gly 49 . Three phenylalanine residues (Phe 39 , Phe 43 , and Phe 51 ) 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 Phe 39 . Na,K-ATPase ␤2 and ␤3 isoforms contain a fourth phenylalanine residue which is replaced by a cysteine residue (Cys 46 ) in ␤1 isoforms (Fig. 1, A  and B). In order to identify the amino acid residues in the ␤ TM domain that interact with the ␣ subunit and to determine the putative functional role of this interaction, amino acids encompassing Ile 36 to Gln 56 were substituted individually or in combination by tryptophan.

Structural Features of the Na,K-ATPase ␤ Subunits TM Domain-
Stabilization of Na,K-ATPase ␣ Subunits by ␤ Subunit Tryptophan Mutants-We first tested whether tryptophan substitutions in the TM domain of the ␤ subunit might interfere with the structural maturation of the ␣ subunit. Bufo ␣ subunits were expressed in Xenopus oocytes alone or together with Xenopus wild type or mutant ␤1 subunits and the cellular expression of the ␣-␤ complexes was followed by immunoprecipitation of metabolically labeled proteins after a pulse and various chase periods. As expected, ␣ subunits expressed without ␤ subunits were degraded during a 48-h chase period (Fig. 2,  lanes 10 and 11). Similar to wild type ␤1 subunits (Fig. 2, lanes  1-3), all ␤-mutants assembled with and stabilized ␣-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 ␣ subunits became fully glycosylated during the chase periods (lanes 5, 6, 8, and 9) indicating that the ␣-␤ complexes were able to leave the ER. Thus, these results show that introduction of tryptophan residues into the ␤1 TM domain does not significantly affect the structural maturation of the ␣ subunit.
Substitution of Tyr 40 and Tyr 44 in the ␤ Subunits TM Domain by Tryptophan Modifies the K ϩ Activation Kinetics of Na,K Pumps-To determine the functional expression and the transport properties of Na,K pumps associated with wild type or mutant ␤ subunits, K ϩ -induced outward currents were measured on intact oocytes by the two electrode voltage clamp technique. Maximal pump currents (I max ) varied but were not consistently different in oocytes expressing wild type or mutant ␣-␤ complexes (Fig. 3A), indicating that none of the ␤ tryptophan mutants affected the overall transport activity. However, measurements of the apparent affinities for external K ϩ revealed a significant increase in the half-activation constants for K ϩ (K 1/2 K ϩ ) for pumps associated with ␤1 mutants I36W, Y40W, and Y44W (Fig. 3B), compared with wild type pumps. K 1/2 K ϩ values of pumps associated with other ␤ tryptophan mutants, including the double G45W/G49W or the triple G45W/G49W/G53W mutant, were similar to those of wild type pumps.
According to the model presented in Fig. 1B, Tyr 40 and Tyr 44 are aligned with each other in the TM ␣-helix suggesting that they may cooperate in the same functional effect. To test this possibility, we measured the apparent K ϩ affinity of Na,K pumps containing ␤1 subunits in which both tyrosine residues were replaced by tryptophan. ␣-␤ Complexes containing the ␤1 double mutant Y40W/Y44W produced maximal pump currents similar to that of wild type ␣-␤ complexes (see insert Fig. 4A) and exhibited a significantly higher K 1/2 K ϩ value than that of ␣-␤ complexes containing either the Y40W or the ␤ Y44W ␤-mutant (Fig. 4A) indicating that Tyr 40 and Tyr 44 act in concert in the K ϩ effect. K 1/2 K ϩ values determined in the presence of external Na ϩ are only a reflection of the apparent affinity of K ϩ for its binding site, since this parameter is influenced by competing external Na ϩ . To obtain a more direct estimation of the effect of Tyr 40 and Tyr 44 mutations on the real binding affinities of external K ϩ to Na,K pumps we also measured K 1/2 K ϩ values in the absence of external Na ϩ . As expected, in the absence of external Na ϩ , the I max value of wild type pumps was similar but the K 1/2 K ϩ value was about 4 times lower than in the presence of external Na ϩ (Fig. 4, A and B, compare lanes 1). Significantly, only the replacement of Tyr 40 (Fig. 4B, lane 2) but not of Tyr 44 (lane 3) by tryptophan in the TM domain of ␤1 subunits significantly increased the K 1/2 K ϩ value of ␣-␤ complexes in the absence of external Na ϩ and the K ϩ effect of the Tyr 40 tryptophan substitution was less pronounced than in the presence of external Na ϩ (compare Fig. 4, A and B, lanes 1 and  2). These results indicate that tyrosine mutations produce highly specific effects on discrete steps in the transport cycle.
Since the two tyrosine residues are highly conserved in the TM domains of all ␤ subunits identified so far, we also investigated K 1/2 K ϩ values of Na,K pumps associated with tyrosine mutants of other ␤ isoforms, namely of the Na,K-ATPases ␤3 isoform. The corresponding tyrosine residues of the Xenopus ␤3 isoform, Tyr 43 and Tyr 47 , were replaced by tryptophan residues (␤3 Y43W/Y47W) and K 1/2 K ϩ values of mutant ␣-␤3 complexes were compared with those of wild type ␣-␤3 complexes, both in the presence and absence of external Na ϩ . As previously reported (10), K 1/2 K ϩ values measured in the presence, but not in the absence of external Na ϩ , were about 2-fold higher for wild type ␣1-␤3 complexes than for wild type ␣1-␤1 complexes (Fig.  4, A and B, compare lanes 1 and 5). Similar to the results obtained with the ␤1 Y40W/Y44W mutant, the ␤3 Y43W/Y47W FIG. 2. Assembly, stabilization, and intracellular transport of Na,K-ATPase ␣-␤ complexes containing tryptophan mutations in the ␤ subunit's TM domain. Xenopus oocytes were injected with 7 ng of Bufo ␣NK cRNA alone (lanes 10 and 11) or together with 0.8 ng of either Xenopus wild type (␤1wt) (lanes 1-3) or mutant ␤1 (lanes 4 -9) cRNAs. After a 6-h pulse period with [ 35 S]methionine (lanes 1, 4, 7, and 10) and after 24 h (lanes 2, 5, 8, and 11) and 72 h (lanes 3, 6, and 9) chase periods, digitonin extracts were prepared and immunoprecipitations were performed using an anti-␣ subunit antibody under nondenaturing conditions which allowed co-immunoprecipitation of associated ␤ subunits. ␣ and ␤ subunits were revealed by SDS-polyacrylamide gel electrophoresis and fluorography. One out of two to four similar experiments is shown. cg, core glycosylated ␤ subunit; fg, fully glycosylated ␤ subunit.
FIG. 3. Functional expression and K ؉ activation of wild type and mutant ␣-␤ complexes. Oocytes were injected with 7 ng of Bufo ␣NK and 0.8 ng of either Xenopus wild type (␤1wt) or mutant ␤ cRNAs. A, I max values of Na,K pumps containing wild type ␤1 subunits or ␤1 tryptophan mutants. Three days after injection, maximal K ϩ -induced Na,K pump currents (I max ) were measured at Ϫ50 mV in the presence of external Na ϩ as described under "Experimental Procedures." Measurements were performed in the presence of 200 nM ouabain which blocks endogenous, oocyte Na,K pumps but not the exogenous, moderately ouabain-resistant Bufo Na,K pumps. The I max values for Na,K pumps containing wild type ␤1 subunits (691.6 Ϯ 62 nA) or ␤1 tryptophan mutants were extrapolated from K ϩ activation curves permitting estimations of K 1/2 K ϩ values shown in B. Shown are mean Ϯ S.E. of data obtained from 14 to 28 oocytes from two to four different Xenopus females. B, apparent affinities for external K ϩ of Na,K pumps containing wild type ␤1 subunits or ␤1 tryptophan mutants. Half-activation constants for external K ϩ (K 1/2 K ϩ ) were determined at Ϫ50 mV in the presence of external Na ϩ as described under "Experimental Procedures." The K 1/2 K ϩ value for Na,K pumps containing wild type ␤1 subunits (651 Ϯ 23 M) was arbitrarily set to 1. Shown are mean Ϯ S.E. of data obtained from 14 to 28 oocytes from two to four different Xenopus females. *, p Ͻ 0.01 compared with wild type ␣-␤ complexes. mutant produced an increase in K 1/2 K ϩ values of Na,K pumps, which was greater in the presence than in the absence of external Na ϩ (Fig. 4, A and B, lanes 5 and 6).
Previous studies have shown that gastric H,K-ATPase ␤ subunits can associate with Na,K-ATPase ␣ subunits and produce ␣-␤HK complexes which exhibit higher K 1/2 K ϩ values than ␣-␤1NK complexes (10). As suggested by analysis of chimeras between ␤NK and ␤HK, the reduced apparent K ϩ affinity of ␣-␤HK complexes compared with ␣-␤NK complexes is mainly mediated by the ectodomain of ␤HK (12). To test whether the K ϩ effect of the TM domain and the ectodomain of ␤ subunits is additive, the two tyrosine residues in the TM domain of a ␤ NK/HK chimera (containing the cytoplasmic and the TM domain of ␤1NK and the ectodomain of ␤HK) were substituted by tryptophan. The ␤ NK/HK chimera, rather than the wild type ␤HK, was chosen for these studies since ␤HK only produces a small population of pumps which are functionally active (12). As previously reported (12), Na,K pumps containing ␤ NK/HK chimeras produced high I max values and the K 1/2 K ϩ value was similar to ␣-␤HK complexes and about 2.5-fold higher than that of Na,K pumps containing wild type ␤1 subunits both in the presence and absence of external Na ϩ (Fig. 4,  A and B, compare lanes 1 and 7). Compared with ␣-␤ NK⅐HK complexes, Na,K pumps which were associated with the ␤ NK/HK chimera containing the Y40W/Y44W mutations exhibited K 1/2 K ϩ values which were slightly but significantly increased in the absence of external Na ϩ (Fig. 4B, compare lanes  7 and 8). These values were more than two times higher in the presence of external Na ϩ (Fig. 4A, compare lanes 7 and 8).
These results suggest that the effect of the ␤ subunits TM domain and ectodomain on the apparent K ϩ affinity are complementary. Since the relative increase in K 1/2 K ϩ values of Na,K pumps containing double tryptophan mutants of ␤1 isoforms, ␤3 isoforms, and NK/HK chimeras was similar, our results also indicate that the K ϩ effect of the TM domain is of general functional relevance.
To further characterize the potential functional interaction of the ␤ subunit with the ␣ subunit in the TM domain, Tyr 40 and Tyr 44 were replaced by several amino acids other than tryptophan. ␣-␤ Complexes containing double phenylalanine, serine, cysteine, or alanine ␤-mutants produced similar Na,K pump currents as wild type Na,K pumps (see inset in Fig. 4C). K 1/2 K ϩ values of Na,K pumps associated with double serine, cysteine, or alanine ␤1 mutants were significantly higher than that of wild type Na,K pumps (Fig. 4C, compare lane 1 to lanes  4 -6), but were lower than that of Na,K pumps associated with the double tryptophan ␤1 mutant (compare lane 2 to lanes 4 -6). On the other hand, a double phenylalanine ␤1 mutant produced Na,K pumps with a K 1/2 K ϩ value which was similar to that of wild type pumps (compare lanes 1 and 3). These results indicate that correctly positioned aromatic side chains, present in tyrosine and phenylalanine but not in serine, cysteine, and alanine residues, are essential for correct interaction of the ␤ subunits TM domain with the ␣ subunit and the inherent Na,K pump function. The most pronounced, functional perturbations produced by the double tryptophan ␤ mutant could be due to the large side chain of tryptophan which as predicted (23) may favor disruption of helix-helix interactions FIG. 4. Apparent affinities for external K ؉ of Na,K pumps associated with ␤1 or ␤3 mutants or with ␤NK/HK chimera. A and B, K ϩ activation of wild type and mutant ␣-␤ complexes. Oocyte cRNA injection was as described in the legend to Fig. 3. Half-activation constants for external K ϩ (K 1/2 K ϩ ) were determined at Ϫ50 mV in the presence (A) or absence (B) of external Na ϩ as described under "Experimental Procedures." Shown are mean Ϯ S.E. of data obtained from 10 to 20 oocytes from two to three different Xenopus females. *, p Ͻ 0.01. Insets, maximal Na,K pump currents (I max ) extrapolated from K ϩ activation curves. C, influence on the K ϩ activation of Na,K pumps containing ␤ subunits in which Tyr 40 and Tyr 44 were replaced by various amino acids. Oocyte cRNA injection as described in the legend to Fig. 3. K 1/2 K ϩ values were determined at Ϫ50 mV in the presence of external Na ϩ . Shown are mean Ϯ S.E. of data obtained from 10 to 15 oocytes from two to three different Xenopus females. *, p Ͻ 0.01 compared with wild type ␣-␤ complexes (lane 1); NS, statistically not significantly different from wild type ␣-␤ complexes. Inset, maximal Na,K pump currents (I max ) extrapolated from the K ϩ activation curves. D, voltage dependence of K 1/2 K ϩ values. K ϩ -induced currents were measured during a series of nine 200-ms voltage steps ranging from Ϫ130 to ϩ30 mV in oocytes injected with ␣ and wild type ␤1 (squares) or ␤1 Y40W/Y44W mutant (triangles) cRNAs. K 1/2 K ϩ values were determined after each voltage step in the presence of external Na ϩ as described under "Experimental Procedures." Shown are mean Ϯ S.E. of data obtained from 14 oocytes from two different Xenopus females. *, p Ͻ 0.01 compared with wild type Na,K pumps. Inset, maximal Na,K pump currents (I max ). by inducing local structural changes.
To gain further insight into the functional effect of the ␤ subunits TM domain on the transport properties of the Na,K-ATPase ␣ subunit, we next compared the voltage dependence of Na,K pump currents in the presence of external Na ϩ and in particular of K ϩ binding to Na,K pumps associated with wild type ␤1 subunits or ␤1 Y40W/Y44W mutants. As previously observed (10), maximal Na,K pump currents of wild type Na,K pumps composed of ␣1 and ␤1 subunits are slightly voltage-dependent (Fig. 4D, inset). Na,K pumps associated with ␤1 Y40W/ Y44W mutants exhibited a stronger voltage dependence and produced lower I max values than wild type Na,K pumps, in particular at highly negative membrane potentials (Fig. 4D,  inset). K 1/2 K ϩ values of mutant Na,K pumps were significantly higher than those of wild type Na,K pumps over the whole potential range and the difference was particularly pronounced at very negative membrane potentials (Fig. 4D).
Substitution of Tyr 40 and Tyr 44 in the ␤ Subunits TM Domain by Tryptophan Modifies Na ϩ Translocation Kinetics of Na,K Pumps-Since differences in K 1/2 K ϩ values between Na,K pumps associated with wild type ␤1 subunits or ␤1 Y40W/ Y44W mutants were more pronounced when measured in the presence rather than in the absence of external Na ϩ , it is likely that mutations in the ␤ subunits TM domain not only directly influence the binding site for external K ϩ but also modify Na ϩ translocation kinetics. To verify this hypothesis we investigated the effect of Tyr 40 /Tyr 44 ␤ mutations on the apparent affinities of Na,K pumps for intra-and extracellular Na ϩ .
The apparent affinity for intracellular Na ϩ of wild type and mutant Na,K pumps was measured as described under "Experimental Procedures." Fig. 5A shows that Na,K pumps containing ␤1 Y40W/Y44W mutants had a slightly higher apparent affinity for intracellular Na ϩ than wild type Na,K pumps.
An estimate for the apparent affinity for external Na ϩ (K 1/2 Na ϩ ext ) of wild type and mutant Na,K pumps was obtained by measuring the inhibition of Na,K pump currents as a function of increasing concentrations of external Na ϩ (Fig. 5B). When measured at ϩ30 mV, inhibition of Na,K pump currents by external Na ϩ was similar for Na,K pumps associated with wild type or mutant ␤ subunits. However, when Na,K pump currents were measured at a membrane potential of Ϫ130 or Ϫ50 mV, external Na ϩ had a significantly stronger inhibitory effect on Na,K pumps containing than on those containing the wild type ␤1 subunit (Fig. 5B). These results indicate that mutant ␣-␤ complexes have a higher, voltage-dependent, apparent affinity for external Na ϩ than wild type ␣-␤ complexes.
Vanadate Sensitivity of Na,K-ATPases Containing Wild Type ␤ Subunits or ␤1Y40W/Y44W Mutants-The parallel decrease and increase in the apparent affinity for external K ϩ and internal Na ϩ , respectively, of Na,K pumps containing ␤1 Y40W/Y44W mutants compared with those containing wild type ␤1 subunits, indicates that tyrosine mutations in the ␤1 subunit may shift the E1-E2 conformational equilibrium to the E1 state. To test this hypothesis, we investigated the sensitivity to vanadate of the Na,K-ATPase activity measured in microsomes of oocytes expressing wild type ␤1 subunits or ␤1 mutants. Vanadate competes with P i for binding to the E2 conformation (36) and can be used as a conformational probe (37,38). The results shown in Fig. 6 indicate that the ␤1 Y40W/Y44W mutant significantly decreases the sensitivity of Na,K pumps to vanadate, with estimated K i values of 7 and 100 M for Na,K-ATPases containing wild type ␤1 subunits and ␤1 mutants, respectively. This result is consistent with a change in the steady-state E1/E2 distribution toward E1 of the Na,K FIG. 5. Apparent affinities for intracellular and extracellular Na ؉ of Na,K-ATPase ␣-␤ complexes containing wild type ␤1 subunits or ␤1 Y40W/Y44W mutants. A, activation of wild type and mutant Na,K pumps by intracellular Na ϩ . Oocytes were injected with Na,K-ATPase ␣ and ␤ cRNAs (7 and 0.8 ng, respectively) together with cRNAs coding for ␣, ␤, and ␥ subunits of the rat epithelial sodium channel (0.3 ng of each). Half-activation constants for intracellular Na ϩ (K 1/2 Na ϩ int ) were determined as described under "Experimental Procedures." Shown are mean Ϯ S.E. of data obtained from 11 oocytes from 3 different Xenopus females. *, p Ͻ 0.01 compared with wild type ␣-␤ complexes (lane 1). Inset, maximal Na,K pump currents (I max ). B, inhibition of wild type and mutant Na,K pumps by extracellular Na ϩ . Oocytes were injected with Xenopus ␣ cRNA and wild type ␤1 (squares) or ␤1 Y40W/Y44W mutant (triangles) cRNAs. Na,K pump currents (I) were determined at Ϫ130, Ϫ50, and ϩ30 mV in the presence of various concentrations of extracellular Na ϩ , as described under "Experimental Procedures." Shown are mean Ϯ S.E. of data obtained from 9 and 11 oocytes from two different Xenopus females for wild type and mutant pumps, respectively. *, p Ͻ 0.01 compared with wild type Na,K pumps. I values measured in the absence of external Na ϩ were arbitrarily set to 1. Half-inhibition constants for extracellular Na ϩ (K 1/2 Na ϩ ext ) estimated from the Na,K pump inhibition curves at Ϫ130, Ϫ50, and ϩ30 mV, were 37.9 Ϯ 7.3, 78 Ϯ 6.2, and 55 Ϯ 10 mM, respectively, for wild type Na,K pumps, and 9.8 Ϯ 5.7, 35.3 Ϯ 5.7, and 43.6 Ϯ 6.8 mM, respectively, for mutant Na,K pumps. pump containing the ␤1 Y40W/Y44W mutant.

DISCUSSION
By performing a tryptophan-scanning analysis we have identified two highly conserved tyrosine residues in the TM domain of Na,K-ATPase ␤ subunits which are implicated in Na,K pump function. This result provides compelling evidence for interactions of ␣ and ␤ subunits in the TM domain and highlights the role of the ␤ subunit as a determinant of intrinsic Na,K-ATPase transport properties.
Various parameters were tested to assess the functional role of the Na,K-ATPase ␤ subunits TM domain including its role in stabilizing the Na,K-ATPase ␣ subunit, in favoring a folding state that permits ER exit of ␣-␤ complexes, and in conveying particular transport properties to Na,K-ATPase.
All ␤ mutants containing individual or combined tryptophan substitutions in the TM domain were able to stabilize ␣ subunits and to permit ER exit of ␣-␤ complexes, indicating that no single amino acid, nor Tyr 40 /Tyr 44 and Gly 45 /Gly 49 /Gly 53 when mutated in combination are necessary for the structural maturation of Na,K-ATPase. These results apparently contrast with previous results obtained with chimeras between Na,Kand H,K-ATPase ␤ subunits, which showed that the presence of the TM domain of the Na,K-ATPase ␤ subunit is needed for a correct interaction with the Na,K-ATPase ␣ subunit permitting ER exit of ␣-␤ complexes (12). Although it cannot entirely be excluded that tryptophan substitutions may allow for proper ␤ assembly, it is more likely that the results obtained in this and previous studies indicate that a certain overall integrity rather than individual amino acids may be necessary for ␣-␤ interactions that mediate some specific steps in the maturation process. This conclusion is also supported by results obtained by Renaud et al. (39) who showed that deletions of 3 amino acids in the TM domain permits assembly of ␣-␤ complexes and their cell surface expression whereas deletions of 5-11 amino acids in the TM domain of ␤ subunits only allows for assembly but not for ER exit of the ␣-␤ complexes.
Tryptophan scanning of the TM domain of the Na,K-ATPase ␤1 subunit revealed that substitution of 3 amino acids, Ile 37 , Tyr 40 , and Tyr 44 , perturbs the inherent, apparent affinities for K ϩ of associated Na,K-ATPase ␣ subunits. The functional effects observed with Tyr 40 and Tyr 44 substitutions are likely to be a reflection of the disruption of a functionally active site. This is supported by the observations that the two tyrosine residues are aligned in the TM ␣-helix, they produce additive effects, and the functional effects can be reproduced with different amino acids. Moreover, the K ϩ effect produced by Tyr replacements in the ␤ subunits TM domain varies depending on experimental conditions, e.g. in the presence or absence of external Na ϩ . The cause underlying the K ϩ effect produced by substitution of Ile 37 with tryptophan is less clear. In membrane proteins, tryptophan residues are enriched at both ends of TM domains (40) and preferentially interact near the lipid carbonyl moiety (41,42). Introduction of tryptophan, in positions flanking model TM helices, tend to "push" the TM helix into the membrane (43). In the context of our result, these observations may indicate that tryptophan substitution of Ile 37 , located adjacent to Lys 36 which delimits the TM domain of ␤1 subunits, could produce a local conformational perturbation in the ␣ helix that may be transmitted to the aligned Tyr residues and thereby produce an indirect functional effect. Consistent with this idea is that the K ϩ effect of Ile 37 substitutions is observed both in the presence and absence of external Na ϩ (data not shown).
The change in the apparent K ϩ affinitiy of Na,K pumps containing ␤ subunits with Tyr 40 and Tyr 44 substitutions are more prominent when measurements are performed in the presence rather than in the absence of external Na ϩ indicating that substitution of the tyrosine residues in the ␤ subunits TM domain may not only alter the K ϩ -binding step itself, but also other discrete steps in the ion transport cycle. In both wild type and mutant Na,K pumps, a parallel exists between the decrease in the apparent affinity for external K ϩ and a gain in the apparent affinity for external Na ϩ during hyperpolarization. This result reflects the interplay between Na ϩ binding and release and K ϩ binding to external sites and the voltage dependence of these steps. Furthermore, the difference in the apparent affinities for external Na ϩ between Na,K pumps associated with ␤ subunit tyrosine mutants and those associated with wild type ␤ subunits are largest at very negative membrane potentials. This result is consistent with the idea that Tyr mutations in the ␤ subunits TM domain may favor the transition from an E2 to a Na ϩ -carrying E1 conformation which prevails during hyperpolarization. A shift of the E1-E2 equilibrium to the E1 conformation may also explain the higher apparent affinity for intracellular Na ϩ and the lower sensitivity for vanadate observed for the mutant Na,K pumps. The two tyrosine residues are highly conserved in the TM domain of all known ␤ subunits of both Na,K-and H,K-ATPases. Experimental confirmation is needed to show whether, as expected, these residues influence a basic step in the catalytic cycle common to Na,K and H,K pumps.
The tryptophan scanning analysis of the ␤1 subunits TM domain was complemented by substitution of the two functionally relevant tyrosine residues by other amino acids. An increase in K 1/2 K ϩ values was also observed in mutant Na,K pumps containing ␤ subunits in which Tyr 40 and Tyr 44 in the TM domain were replaced by serine, cysteine, or alanine residues. On the other hand, no K ϩ effect was observed by replacing the tyrosine residues with phenylalanine. These results indicate that a correctly positioned aromatic moiety present in FIG. 6. Vanadate sensitivity of Na,K-ATPase ␣-␤ complexes containing wild type ␤1 subunits or ␤1 Y40W/Y44W mutants. Oocytes were injected with Xenopus ␣ cRNA and wild type ␤1 cRNA (black bars) or ␤1 Y40W/Y44W mutant cRNA (white bars). Four days after injection, microsomes were prepared and the Na,K-ATPase activity was measured as described under "Experimental Procedures" in the absence (control) or presence of 10 Ϫ7 -10 Ϫ4 M vanadate. Data are represented as percent of control Na,K-ATPase, which amounted to 32 Ϯ 4 and 20 Ϯ 2 mol/mg of protein/h Ϫ1 for microsomes from oocytes expressing wild type ␤ subunits and ␤ mutants, respectively. Na,K-ATPase activities were 10 -30 times higher in microsomes from cRNAinjected oocytes than in microsomes from non-injected oocytes. The results shown are mean Ϯ S.E. of two independent measurements performed in triplicate on microsomes from 2 different batches of oocytes. A statistically significant difference (p Ͻ 0.01) between the vanadate sensitivity of Na,K-ATPase in microsomes from oocytes expressing wild type ␤ subunits and ␤ mutants is indicated by an asterisk. the native tyrosine and substituting phenylalanine residues, but absent in serine, cysteine, and alanine residues, may be important in producing the wild type phenotype. Since tryptophan substitutions produce the largest functional perturbations, it is also possible that the size of the side chain may be critical for a correct K ϩ effect.
The mechanistic details by which Tyr 40 and Tyr 44 in the ␤ subunits TM domain affect intrinsic transport properties of Na,K-ATPase are not known. According to our results, it is likely that the two tyrosine residues in the ␤ subunits TM domain interact with a TM segment of the ␣ subunit and produce a conformational effect. The primary interactions that specify TM helix packing which mediates subunit interactions and the correct folding of membrane proteins are still largely unknown (44). Based on energetic considerations, the possible driving forces for TM helix interactions are van der Waals interactions between closely packed helices and interhelical polar interactions. Since TM helices of membrane proteins contain only few polar amino acids and TM helix interactions can be very stable in the absence of any hydrogen bonds or salt bridges (45), van der Waals interactions may play a major role in governing interhelical interactions. However, recent work has implicated polar residues in membrane helix interactions. For instance, charged residues in the membrane domain are necessary for subunit assembly in the T-cell receptor (46). In the product of the neu oncogene, Val to Glu mutations in the TM domain result in dimer formation and constitutive activation (47). Furthermore, formation of hydrogen bonds, e.g. between Asn residues, mediates strong interactions of TM helices (48,49). Little is known on the role of tyrosine residues in mediating TM helix interactions. Aromatic amino acids such as Tyr, Phe, or Trp may undergo "amino aromatic" interactions in which neutral NH-containing groups tend to be positioned near the aromatic rings (50) and even more favorably "cation aromatic" interactions (cation-À interactions) with positively charged residues (51). Sequence analysis of TM segments of Na,K-ATPase ␣ subunits does not provide conclusive evidence for the existence of a potential interaction site matching the two tyrosine residues in the ␤ subunit. Highly conserved, neutral NH-containing amino acids are indeed present in M5, M7, and M8 of Na,K-and H,K-ATPases but they are unique or not aligned in a putative ␣-helix to permit accommodation of the two tyrosine residues of the ␤ subunits TM domain. Furthermore, interactions of Tyr 40 and Tyr 44 with positively charged residues are not very likely since arginine residues are absent in the TM domain of all Na,K-and H,K-ATPase ␣ subunits and conserved lysine residues are only present at the N-terminal border of M5 whereas a single lysine residue within M5 is present in H,K-ATPases but not in Na,K-ATPase ␣ subunits.
Interestingly, the amino acids in the ␤ subunits TM domain, which according to our analysis have an effect on the Na,K-ATPase function and therefore are likely to interact with the ␣ subunit, are located on the face of the TM ␣ helix which is opposite to the face which contains the cysteine residue that was shown to cross-link with a cysteine residue in M8 of the ␣ subunit (21,22). This result may suggest that two faces of the TM helix interact with the ␣ subunit and that the ␤ subunits TM domain is not or minimally exposed to lipids. Based on cross-linking studies and other biochemical evidence (21,22,52), possible models for the spatial organization of the transmembrane segments of Na,K-ATPases include contacts of the ␤ subunits TM domain with M9 and/or M7. Provided that the organization of the transmembrane segments of the Na,K-ATPase ␣ subunit is similar to that deduced from the crystal structure of the Ca 2ϩ -ATPase (3), and that the cysteine residue of the ␤ subunits TM is positioned toward M8 of the Ca 2ϩ -ATPase, then the two tyrosine residues would be directed toward M10 or alternatively M7 of the ␣ subunit as outlined in Fig. 7. Mutational analysis on the ␣ subunit should shed more light on the potential intersubunit interaction site mediated by the tyrosine residues in the TM domain of the ␤ subunit.
A view from the cytoplasmic side (Fig. 7) reveals that the 3 aligned glycine residues which form a grove in the ␤ TM helix (Fig. 1) may represent a third helix face. In the models presented in Fig. 7, these glycine residues are freely exposed, away from the ␣ subunit. Interestingly, the 3 glycine residues are part of a TM helix interaction motif which was initially identified in glycophorin A (53) but which occurs frequently in other helical membrane proteins (54). This motif consists of a LIXXGXXXGXXXT sequence where GXXXG was found to be necessary and sufficient for dimerization and corect packing of glycophorin A into a right-handed pair of ␣ helices (55). A LIXXGXXXGXXXG motif is present in the TM domain of all Na,K-ATPase ␤1 isoforms and the basic GXXXG motif in that of Xenopus and Bufo ␤3 isoforms, strongly suggesting that the TM domains of these ␤ subunits interact with another TM ␣-helix containing a similar motif. Since TM domains of Na,K-ATPase ␣ subunits lack GXXXG motifs, there is the potential for dimer formation via this motif, with a yet unknown protein or with the ␤ subunit itself. The possibility exists that the GXXXG motif mediates the formation of an 2␣Ϫ2␤ structure, proposed by several studies to be the functional unit of Na,K-ATPases (56,57), by forming a ␣-␤-␤-␣ complex. However, if this would be the case, monomeric and dimeric ␣-␤ complexes must exhibit very discrete differences in transport properties since in this study, we could not detect any impairment of the Na,K pump function after disrupting the putative ␤-␤ interaction site.
In conclusion, modeling of the TM helix accompanied by tryptophan scanning reveal that the TM domain of ␤1 subunits of Na,K-ATPase contain two distinct helix faces which are probably both buried in the interior of the protein and partic- FIG. 7. Possible positioning and orientation of the Na,K-ATPase ␤1 subunits TM helix with respect to the TM domain of P2-ATPases. Shown are views from the cytoplasmic side of the transmembrane helix of the Na,K-ATPase ␤1 subunit, and of the 10 transmembrane helices (M1 to M10) of the sarcoplasmic Ca pump ␣ subunit as deduced from the crystal structure by Toyoshima et al. (3). The Na,K-ATPase ␤1 subunits TM helix was oriented with Cys 46 directing toward M8 of the ␣ subunit as suggested by cross-linking studies (21,22). In this model, Tyr 40 and Tyr 44 in the ␤ subunits TM domain, which contribute to intrinsic transport properties of Na,K pumps, are directed toward M10 or M7 of the ␣ subunit and the putative TM helix dimerization motif, GXXXGXXXG is freely exposed. ipate in membrane protein-protein interactions. Whereas no functional role could be attributed to TM ␣-␤ interactions in the face of the ␤ subunits TM helix containing the cysteine residue which is closely located to M8 of the ␣ subunit, putative TM interactions in another face of the ␤ subunits TM helix, which are mediated by two highly conserved tyrosine residues, contribute to intrinsic functional properties of oligomeric P-type ATPases. Finally, a putative third face, containing a basic GXXXG motif, may be of potential functional relevance, in permitting ␤1 subunit dimerization.