Valine 904, Tyrosine 898, and Cysteine 908 in Na,K-ATPase α Subunits Are Important for Assembly with β Subunits*

A 26-amino acid sequence in an extracellular loop of the Na,K-ATPase α subunit between membrane-spanning segments 7 and 8 has been shown to bind to the β subunit of Na,K-ATPase and to promote αβ assembly (Lemas, M. V., Hamrick, M., Takeyasu, K., and Fambrough, D. M. (1994) J. Biol. Chem. 269, 8255–8259) When this 26-amino acid sequence of the rat Na,K-ATPase α3 subunit was replaced by the corresponding sequence of the rat gastric H,K-ATPase α subunit, the chimeric α subunit assembled preferentially with the rat gastric H,K-ATPase β subunit (Wang, S.-G., Eakle, K. A., Levenson, R., and Farley, R. A. (1997)Am. J. Physiol. 272, C923–C930). In the present study, these 26 amino acids (Asn886–Ala911) of rat Na,K-ATPase α3 were replaced by the corresponding amino acids Asn908–Ala933 of rat distal colon H,K-ATPase. Site-directed mutagenesis of the chimeric α subunits and Na,K-ATPase α3 showed that Val904, Tyr898, and Cys908 in the Na,K-ATPase α3 subunit are key residues in αβ subunit interactions. The V904Q mutation in Na,K-ATPase α3 reduced the B max for ouabain binding and the ATPase activity of α3β1 complexes by ∼95%, and Y898R reduced theB max and ATPase activity by ∼60%. The complementary mutations Q904V and R898Y increased the amount of ouabain bound by yeast membranes expressing the chimera with the colon H,K-ATPase sequence. The amount of ouabain bound by complexes assembled between Na,K-ATPase α3 containing the Y898R,C908G mutations and gastric H,K-ATPase β was less than 10% of wild type Na,K-ATPase α3 expressed with the same β subunit. The R898Y,G908C mutations in the chimeric α subunits also increased ouabain binding.

molecular mass between 70 and 200 kDa, and the potassiumtransporting ATPases such as HK and NK also require a second, smaller glycosylated ␤ subunit (30 -55 kDa) for their enzymatic functions. The ␣ subunit has multiple transmembrane segments and contains all of the amino acids thus far identified with the enzymatic functions of ATP hydrolysis and cation transport. The ␤ subunit is a glycoprotein with one transmembrane segment and most of its mass located on the noncytoplasmic side of the membrane. The role of the ␤ subunit in active ion transport is not fully understood.
Lemas et al. (1) have identified 26 amino acids in NK␣, predicted to be located in an extracellular loop between transmembrane segments 7 and 8, which mediate interactions between ␣ and ␤ subunits. These 26 amino acids correspond to Asn 886 -Ala 911 of the rat NK␣3 subunit. Wang et al. (2) examined ␣␤ assembly using a chimeric ␣ subunit (NGH26) formed by replacement of Asn 886 -Ala 911 of rat NK␣3 with the corresponding amino acids Gln 905 -Val 930 of rat gastric HK␣. When NGH26 was expressed in yeast cells with HK␤, the number of ouabain binding sites was the same as for NK␣3 expressed with either NK␤1 or HK␤. In contrast, only about 10% as many complexes were formed between NGH26 and NK␤1. Wang et al. concluded that some amino acids within the sequence Gln 905 -Val 930 of rat gastric HK␣ probably destabilize ␣␤ complexes formed with NK␤, leading to a reduction in the number of steady-state pumps. This conclusion is consistent with the observation that a chimeric ␣ subunit with amino acids 1-519 from NK␣ and amino acids 519 -1033 from gastric HK␣ did not form stable complexes with NK␤1 (3).
Unlike gastric HK␣, other HK␣ subunits do not appear to discriminate between different ␤ subunits. For example, functional pumps are assembled between a distal colon HK␣ subunit and either NK␤1 or HK␤ subunits. Codina et al. (4) showed that 86 Rb uptake into Xenopus oocytes increased when colon HK␣ was expressed with either NK␤1 or gastric HK␤, and Cougnon et al. (5) observed an increase in the 86 Rb uptake rate of oocytes injected with cRNA for colon HK␣ and an amphibian HK␤. A human HK␣ subunit (ATP1AL1) with 86% amino acid sequence identity to rat colon HK␣ was cloned by Modyanov et al. (6), who observed that any of several different ␤ subunits could be coimmunoprecipitated with ATP1AL1 when expressed in Xenopus oocytes. Expression of rabbit gastric HK␤ with ATP1AL1 in Xenopus oocytes resulted in a 3-fold increase in 86 Rb uptake compared with uninjected cells (7).
To identify amino acids that are involved in interactions between the ␣ and ␤ subunits, a new chimeric ␣ subunit (NCH26) was made by replacing the 26-amino acid sequence Asn 886 -Ala 911 of the rat NK␣3 subunit with the corresponding sequence Asn 908 -Ala 933 of rat distal colon HK ␣ subunit. A series of mutations was introduced into NK␣3 or the chimeric NGH26 and NCH26 subunits, and the presence of functional ␣␤ complexes was measured by ouabain binding or ATPase activity after expression of the ␣ polypeptides in yeast with either NK␤1 or gastric HK␤. Stability of the complexes was estimated from the ability of each ␣␤ complex to bind ouabain at elevated temperatures. As a result of these measurements, Val 904 , Tyr 898 , and Cys 908 in NK␣3 have been identified as important amino acids for assembly of ␣ subunits and ␤ subunits.

MATERIALS AND METHODS
Construction of the Chimeric ␣ Subunit NCH26 -The plasmid pRD-CHK containing the cDNA of the rat colon HK ␣ subunit was a gift of Dr. Gary Shull (University of Cincinnati). A 102-base pair fragment of pRD-CHK, encoding positions 2711-2812, was amplified by polymerase chain reaction, and ClaI and HpaI restriction sites were introduced at the same time. For construction of plasmid pNCH26m, the polymerase chain reaction fragment was digested with ClaI and HpaI and was ligated into the corresponding region of clone pNGH26m (2) whose ClaI-HpaI fragment (79 base pairs) had been removed. Three mutations (N886D, 2 A911V, and F912N), resulting from the introduction of ClaI and HpaI sites, were corrected by polymerase chain reaction. The resultant plasmid pNCH26 encodes the rat NK␣3 subunit with the region (Asn 886 -Ala 911 ) replaced by the 26 amino acids (Asn 908 -Ala 933 ) of the rat colon HK␣ subunit. The AflII-BglII fragment (1,486 base pairs) of the yeast expression plasmid YEpNGH26 (2) was replaced by the corresponding AflII-BglII fragment of pNCH26. The final plasmid (YEpNCH26) was analyzed by restriction digestion and by DNA sequencing.
Site-directed Mutagenesis-Mutations in the cDNA were made using the polymerase chain reaction, as described previously (2). Pfu DNA polymerase (Stratagene) was used to perform the site-directed mutagenesis, and the resultant mutants were screened by restriction enzymes and were confirmed by DNA sequencing using the Sequenase version 2.0 (U. S. Biochemical Corp.).
Expression of NCH26 or ␣ Mutants with ␤ Subunits in Yeast Cells-The yeast strain 30-4 (MAT ␣, trp1, ura3, Vn2, GALϩ) obtained from R. Hitzeman (Genentech, South San Francisco) was transformed with different combinations of the chimeric ␣ subunit or ␣ mutant expression plasmid and one of the ␤ subunit expression plasmids pG1T-R␤1 and pG1T-HK␤ (9) by the method of Elble (8). After identification of transformants containing ␣ and ␤ subunits on selective medium, frozen glycerol stocks from four different clones were made and were stored at Ϫ80°C. Cultures for experiments described in this report were started from these glycerol stocks. A membrane fraction of transformed yeast cells was prepared as described previously (9). Membranes were extracted with 0.1% (w/v) SDS as described previously (10).
[ 3 H]Ouabain Binding-Ouabain binding to yeast membranes was done as described previously (2). Experiments shown in Figs. 5 and 7 were done using approximately 20 nM [ 3 H]ouabain. To determine the number of steady-state pump complexes (B max ) and the ouabain dissociation constant (K d ), binding data were fit by a self-competition model (11) within the ouabain concentration range 0 -1,000 nM. For determination of the effects of heat on ␣␤ stability, 3 mg of yeast microsomal membrane protein was dissolved in 400 l of 25 mM imidazole-HCl, 1 mM EDTA (sodium-free), pH 7.4, and was heated at different temperatures (40 -50°C) for 90 s. Membranes were placed in ice for 15-30 min, and the amount of ouabain bound at 37°C was measured. The amount of ouabain bound by membranes without heating was used as 100%.
SDS-Polyacrylamide Gel Electrophoresis and Immunoblots-100 g of yeast microsomal membrane protein was separated on 10% SDSpolyacrylamide gels and then was transferred to Immobilon-P membranes (Millipore). The blots were first incubated with monoclonal antibody ␣5 (D. Fambrough, Johns Hopkins University), then incubated with the alkaline phosphatase-conjugated goat anti-mouse IgG (Calbiochem). The ␣ subunits were visualized with 5-bromo-4-chloro-3indolyl phosphate (Sigma) and nitro blue tetrazolium (Sigma). The density of each ␣ band was determined by scanning with a Bio-Rad scanner (Imaging Densitometer, model GS-670). The average of the expression levels of NCH26 ϩ NK␤1 and NCH26 ϩ HK␤ with three different clones was determined and compared with the NK␣3 ϩ NK␤1 and NK␣3 ϩ HK␤ controls.

RESULTS
Expression Levels of NCH26 with NK␤1 or HK␤-The sequence Asn 886 -Ala 911 of rat NK␣3 was replaced by the corresponding sequence Asn 908 -Ala 933 of rat colon HK␣, and this chimeric ␣ subunit NCH26 was expressed in yeast cells with either NK␤1 and gastric HK␤. Fig. 1A shows an immunoblot of membranes prepared from three different clones expressing either NK␣3 or NCH26 with either NK␤1 or HK␤. Three clones expressing only NK␣3 are also shown. Because the antibody ␣5 recognizes the same epitope on both NK␣3 and NCH26, the relative abundance of each ␣ subunit can be compared directly. The abundance of NCH26 expressed with NK␤1 is 82 Ϯ 19% of NK␣3 expressed with NK␤1, and the amount of NCH26 expressed with HK␤ is 87 Ϯ 11% of NK␣3 expressed with HK␤ ( Fig. 1B). When expressed in the absence of a ␤ subunit, NK␣3 is present at only 10 Ϯ 10% of NK␣3 levels found with NK␤1. In the absence of the ␤ subunit, the ␣ subunit is degraded rapidly, and the higher steady-state abundance of ␣ subunits expressed with either NK␤1 or HK␤ reflects stabilization of the ␣ subunit by association with a ␤ subunit (13). These results show that the steady-state level of NCH26 expressed in yeast with NK␤1 or HK␤ is not significantly different from NK␣3 (p Ͼ 0.05).
Ouabain Binding by NCH26 -Functional NCH26 ϩ ␤ complexes were quantitated by ouabain binding, and the B max and K d values for NK␣3 or NCH26 expressed with different ␤ subunits were determined for three different clones each. The results presented in Fig. 2 demonstrate that functional complexes are formed equally well in yeast between NK␣3 and either NK␤ or HK␤. Although the NCH26 polypeptide is present at the same level as NK␣3, fewer functional complexes are formed between NCH26 and either NK␤1 or HK␤ than with NK␣3. Yeast membranes containing NCH26 ϩ NK␤1 form about 40% of the number of functional pumps as NK␣3 ϩ NK␤1, and the number of functional NCH26 ϩ HK␤ complexes is about 20% of NK␣3 ϩ HK␤. The K d for ouabain binding by NCH26 ϩ NK␤1 is 50 Ϯ 13 nM, and for NCH26 ϩ HK␤ it is 206 Ϯ 95 nM. These values are 8-and 27-fold higher than the K d FIG. 1. Expression levels of NK␣3 and NCH26 expressed in yeast with NK␤1 or HK␤. Panel A, yeast membranes containing 100 g of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The blot was probed sequentially with monoclonal antibody ␣5 and alkaline phosphatase-conjugated goat anti-mouse IgG. Three different clones were used to determine the expression level of ␣ subunit of each ␣␤ complex. Panel B, relative expression levels were measured by densitometry. The averages of three clones of NK␣3 expressed with NK␤1 or HK␤ are the 100% controls, and the expression levels of NCH26 expressed with NK␤1 or HK␤ were normalized to the controls. Bars show means Ϯ S.D.
The number of ouabain-binding complexes formed by NCH26 and HK␤ is only 20 -40% of the number formed by either NK␣3 ϩ HK␤ or NGH26 ϩ HK␤ (2). This result might be explained if residues within the sequence Asn 908 -Ala 933 of rat colon HK␣ are less suitable for assembly with gastric HK␤ than corresponding residues of rat NK␣3 or rat gastric HK␣. A comparison of the amino acid sequences Asn 886 -Ala 911 of rat NK␣3, Gln 905 -Val 930 of rat gastric HK␣, and Asn 908 -Ala 933 of rat colon HK␣ shows that the three sequences have identical residues or conservative substitutions in all positions except at amino acids 898, 908, and 909. In both NK␣3 and rat gastric HK␣, a tyrosine or a phenylalanine is located at position 898, and a cysteine is located at position 908. In rat colon HK␣ these residues are arginine and glycine, respectively. In position 909, the amino acids are different for all three sequences. To see whether the amino acids in positions 898 and 908 are important for the assembly with HK␤, mutations R898Y and/or G908C were introduced into NCH26, and the amount of ouabain bound by the mutants expressed in yeast with different ␤ subunits was measured.
Ouabain Binding by NCH26 Mutants-The maximum amount of ouabain bound (B max ) by the R898Y, G908C, and R898Y,G908C mutants of NCH26 expressed in yeast cells with either NK␤1 or gastric HK␤ was used to indicate the number of functional ␣␤ complexes (Fig. 3). The mutation R898Y increases the B max of NCH26 ϩ NK␤1 complexes to the same value as NK␣3 ϩ NK␤1. When expressed with HK␤, the B max of the R898Y mutant increases 2.7 times higher than that of NCH26 ϩ HK␤ but is still less than that of NK␣3 ϩ HK␤. The mutation G908C does not affect the number of NCH26 ϩ NK␤1 complexes. When expressed with HK␤, however, the mutation G908C reduces the B max of NCH26 from 22% to 3% of NK␣3 ϩ HK␤. When the two mutations R898Y and G908C are made in NCH26, the B max for ouabain binding is the same as NK␣3 ϩ HK␤ (p Ͼ 0.05), and is 1.8 times higher than that of NCH26/ R898Y ϩ HK␤.
Ouabain Binding by NGH26 Mutants-Wang et al. studied the chimeric NGH26 ␣ subunit and concluded that amino acids of gastric HK␣ between Gln 905 and Val 930 interact more stably with the extracellular domain of HK␤ than NK␤ (2). Charged amino acids have been implicated in the assembly of some membrane proteins (14,15), and within the 26 residues that were exchanged during chimera formation, the charged amino acids Lys 902 and Glu 905 are conserved among all of the NK␣ subunits and also in the colon HK␣ subunits. In the gastric HK␣ subunits, these amino acids are leucine and glutamine, respectively. Because NGH26 forms fewer functional pumps with NK␤1 than with HK␤, charged residues in positions 902 and 905 may be important for interactions between ␣ subunits and NK␤1. To test this possibility, the mutations L902K and Q905E were introduced separately or together into NGH26, and the ␣ subunits were expressed in yeast with either NK␤1 or HK␤. Only the Q905E mutation reduced the ouabain binding capacity of yeast membranes containing the mutant ␣ subunits assembled with HK␤ (p Ͻ 0.05). Introduction of the double mutation (NGH26-KE), however, restored the binding capacity and reduced the K d for ouabain binding by NGH26 from 72.7 to 23 nM (Table I).
Site-directed Mutagenesis of NGH26-KE-Although the mutations L902K and Q905E make the amino acid sequence of NGH26 more nearly like that of NK␣3, the number of pumps is not increased above the NGH26 ϩ NK␤1 level. This may be a result of the presence in NGH26 and NGH26-KE of amino acids whose side chains are sterically or electrostatically incompatible with assembly with NK␤1or because of the absence in these ␣ subunits of amino acids whose side chains are important for specific interactions with NK␤1. There are 10 amino acid differences between the NK␣3 sequence and NGH26-KE (Fig. 4). Each of these amino acids was changed in NGH26-KE, either individually or in pairs, to those amino acids in NK␣3, and ouabain binding was used to identify amino acids that are important for assembly of NK␣3 with NK␤1.
Ouabain Binding by NGH26-KE and Related Mutants-Yeast membranes containing the ␣ subunit mutants derived  from NGH26-KE together with either NK␤1 or gastric HK␤ were equilibrated with 20 nM [ 3 H]ouabain, and specific binding was measured as before. As shown in Fig. 5, when the double mutation Y903V,Q904V is made in NGH26-KE and this mutant ␣ subunit is expressed with NK␤1, yeast membranes bind 10 times more ouabain than when NGH26-KE is expressed with NK␤1. In addition, the double mutation Y903V,Q904V in NGH26-KE increases the amount of ouabain bound by when expressed in yeast with HK␤. The amount of ouabain bound by the rest of the mutants when expressed with either NK␤1 or HK␤ is not significantly different from that of NGH26-KE ϩ NK␤ or NGH26-KE ϩ HK␤ (p Ͼ 0.05), respectively.
Ouabain Binding by NK␣3 Mutants-The mutations Y903V,Q904V in the chimera NGH26-KE and R898Y,G908C in the chimera NCH26 lead to large increases in the amount of ouabain bound by yeast membranes containing these chimeric ␣ subunits and either NK␤1 or HK␤. These increases could be due to changes in the affinity of the mutants for ouabain and/or to increased stability of the ␣␤ complexes. If amino acids in positions 898, 903, 904, and/or 908 participate in interactions between ␣ and ␤ subunits, then the reverse mutations in NK␣ should reduce the amount of ouabain bound by yeast membranes expressing each ␣␤ complex. Thus, the mutations V904Q, Y898R, C908G, and Y898R,C908G were made in NK␣3 and the ouabain binding affinity and capacity of each ␣␤ complex were measured after isolation of yeast membranes. Nonpolar amino acid side chains are conserved among NK␣ subunits in position 904 but not in position 903, and so the effect of amino acid substitutions in position 903 was not tested. The left panel of Fig. 6 shows the B max values of the NK␣3 mutants expressed with NK␤1, and the right panel shows the B max values for NK␣3 mutants expressed with HK␤. The B max of NK␣3/V904Q ϩ NK␤1 is only 5% of the B max for NK␣3 ϩ NK␤1, and the B max of NK␣3/V904Q ϩ HK␤ is 18% of NK␣3 ϩ HK␤, confirming that Val 904 is important for the functional assembly of NK␣ with both ␤ subunits. The B max of NK␣3/ Y898R ϩ NK␤1 is 42% of NK␣3 ϩ NK␤1, and the B max of NK␣3/Y898R ϩ HK␤ is not significantly different from that of NK␣3 ϩ HK␤. For the mutation C908G in NK␣3, no significant increase or decrease in the number of ␣␤ complexes was seen when assembled with either subunit. Even though the individual mutation Y898R or C908G has no significant effect on the number of NK␣3 ϩ HK␤ complexes, the double mutation Y898R,C908G was associated with a reduction in the B max for ouabain binding to about 10% of NK␣3 ϩ HK␤ levels. Table II shows that for the V904Q mutation, no change in ouabain affinity was observed when the mutant NK␣3 subunit was expressed with either NK␤1 or HK␤.
ATPase Activity of NK␣3 Mutants Expressed with NK␤1-Yeast membranes containing the different ␣ ϩ NK␤1 complexes were extracted with SDS (16) to see whether reduction in the number of pump complexes caused by mutation of residues in NK␣3 is accompanied by a similar reduction in ATPase activity. When expressed with NK␤1, the ATPase activities of the mutants V904Q, Y898R, C908G, and the double mutant Y898R,C908G were reduced to the same extent as the B max values (Table III). As shown previously (9), the ␣ ϩ HK␤ complexes are unstable in SDS, and the influence of the mutations on the ATPase activity of these complexes could not be determined.
Thermal Stability-The stability of the NK␣3 mutants expressed in yeast with NK␤1 or HK␤ was investigated by heating the membranes at different temperatures (40 -50°C) for 90 s and then measuring the amount of ouabain bound at 37°C. Fig. 7 (upper panel) shows that all of the mutants are as stable as NK␣3 ϩ NK␤1 when expressed with NK␤1. In contrast, when expressed with HK␤, both NK␣3 and the mutants denature at much lower temperatures (lower panel). Compared with NK␣3 ϩ HK␤, the ␣ ϩ HK␤ complexes containing the mutations Y898R and C908G are significantly less stable. The mutant containing the double mutation Y898R,C908G is extremely unstable when expressed with HK␤. Ouabain binding by complexes of this mutant expressed with HK␤ was not detected after the membranes were heated at 45°C for 90 s (data not shown). In contrast to Y898R and C908G, the mutation V904Q in NK␣3 has no effect on the thermal stability of the functional ␣ ϩ HK␤ complexes. Glutamine is conserved in all gastric HK␣ subunits in the position corresponding to Val 904 in NK␣. DISCUSSION It has been shown previously that both ␣ and ␤ subunits are required by NK and gastric HK to catalyze active ion transport (17)(18)(19). NK␣ subunits assemble equally well with NK␤1 and gastric HK␤ (9), but gastric HK␣ does not form functional pumps with NK␤ (3). In contrast to gastric HK␣, coexpression of colon HK␣ in Xenopus oocytes with either gastric HK␤ or NK␤ (4) or with toad urinary bladder HK␤ (5) leads to functional ion pumps. This result indicates that interactions between ␤ subunits and colon HK␣ or gastric HK␣ are mediated by amino acids that are different in the two ␣ subunits. Wang et al. (2) have shown that substitution of amino acids Gln 905 -Val 930 from rat gastric HK␣ for the corresponding sequence Asn 886 -Ala 911 of rat NK␣3 reduces the assembly of this chimeric ␣ subunit (NGH26) with NK␤1 compared with assembly with HK␤. In the experiments reported here, a chimeric ␣ subunit (NCH26) was formed by replacing the sequence Asn 886 -Ala 911 of rat NK␣3 with the corresponding sequence Asn 908 -Ala 933 of the rat colon HK␣ subunit to examine the structural basis for the selectivity of ␣ subunits for assembly with different ␤ subunits. The formation of functional pumps was compared when NK␣3, NCH26, or NGH26 was expressed in yeast with either NK␤1 or gastric HK␤, and amino acids in the ␣ subunits that are important for interactions between ␣ and ␤ subunits were identified after site-directed mutagenesis.
The steady-state abundance of the NCH26 polypeptides expressed in yeast with NK␤1 or HK␤ is the same as that of NK␣3 (Fig. 1A). About 40% of the NCH26 ϩ NK␤1 complexes are functional, as determined by the ability to bind ouabain, and about 20% of the NCH26 ϩ HK␤ complexes are functional (Fig. 2). This result is consistent with the observation of Codina et al. (4) that the colon HK␣ is capable of functional assembly with either NK␤1 or gastric HK␤. The result also suggests that some NCH26 ϩ ␤ complexes are inactive in the yeast membranes.
Codina et al. (4) reported that high concentrations of ouabain inhibited colon HK␣ expressed in Xenopus oocytes with either NK␤1 or HK␤ (IC 50 ϭ 400 -600 M in the presence of 1 mM external KCl). In the absence of KCl, the K d for ouabain binding to NK␣3 expressed in yeast with either NK␤1 or HK␤ is 6 -8 nM (Tables 1 and 2). The K d for ouabain binding to NCH26 ϩ NK␤1 is 50 nM, and for NCH26 ϩ gastric HK␤ the K d is greater than 200 nM. It is likely, therefore, that the low affinity of colon HK␣ for ouabain is due at least in part to amino acids Asn 908 -Ala 933 . These amino acids are found in a loop predicted from hydropathy analysis to be located on the non-cytoplasmic side of the cell membrane, between transmembrane segments 7 and 8. The loop between these transmembrane segments of NK␣ has been implicated in ouabain binding by Schultheis et al. (20), who observed that mutations in Arg 880 led to a reduction in the affinity of the sodium pump for ouabain.
The chimeric ␣ subunit NGH26 contains amino acids Gln 905 -Val 930 from rat gastric HK␣ substituted for Asn 886 -Ala 911 of rat NK␣3. When this chimera was expressed in yeast, about 10 times as many pumps were assembled with gastric HK␤ as with NK␤1 (2). The difference in the number of pumps assembled in yeast from NK␣, from gastric HK␣, or from the chimeric polypeptides NCH26 and NGH26 and either NK␤1 or HK␤ probably reflects differences in assembly or in the stability of the different ␣␤ complexes. Consequently, mutations were made in the chimeras NGH26 and NCH26 and also in NK␣3 to identify amino acid side chains that might mediate ␣␤ subunit interactions. Introduction of charged amino acids in positions 902 and 905 of NGH26 (L902K and Q905E) increased the affinity of the pump for ouabain but did not increase the number of pumps assembled with HK␤ (Table I). Because the single mutations alone did not influence the dissociation constant of ouabain binding of NGH26 ϩ HK␤, it is likely that neither Leu 902 nor Gln 905 interacts directly with ouabain. The lower K d of the double mutant is probably caused by an induced tertiary structure in the ouabain binding site of the double mutant similar to that of the NK␣3 subunit. Because neither the single mutations (L902K and Q905E) nor the double mutation (L902K,Q905E) increase the B max of NGH26 expressed with either NK␤1 or HK␤, these amino acids also are probably not located in the ␣␤ interaction interface.
The chimera containing the L902K and Q905E mutations (NGH26-KE) was used as a template for additional mutations that changed amino acids in the chimera to those found in NK␣3. Most of the mutations in NGH26-KE did not affect ouabain binding by the chimera; however, the mutations Y903V and Q904V led to a significant increase in the amount of ouabain bound when the mutant/chimeric ␣ subunit was expressed with NK␤1 (Fig. 5). This result suggests that one or both of these small hydrophobic amino acids may be important for complex formation with NK␤1. The valine at position 903 is not conserved among NK␣ subunits. Polar amino acids including threonine, glutamine, and glutamic acid are also found at this position in some isoforms or species. A non-polar amino acid such as valine, leucine, or isoleucine is conserved at position 904 in all NK␣ subunits, and a glutamine is found at this position in all gastric HK␣ subunits. Because gastric HK␣ subunits do not assemble with NK␤ subunits, the mutation V904Q was made in NK␣3 to test whether the glutamine in position 904 of gastric HK␣ could be the reason that gastric HK␣ does not assemble with NK␤1. The V904Q mutation in NK␣3 caused a reduction in both the number of functional ␣␤ complexes (Fig. 6) and the ATPase activity (Table III) to only 5% of NK␣3 ϩ NK␤1, without affecting the affinity of the mutant for ouabain (Table II). This result shows that the presence of glutamine at position 904 in gastric HK␣ subunits is sufficient to prevent assembly of HK␣ subunits with NK␤1. It also suggests that valine or another small hydrophobic amino acid in position 904 in NK␣ subunits is important for assembly with NK␤1.
In addition to Val 904 , Tyr 898 also appears to be important for assembly of ␣ subunits with NK␤1. The mutation R898Y in NCH26 caused a 2-fold increase in the number of functional ␣ ϩ NK␤1 complexes (Fig. 3), and the reverse mutation Y898R in NK␣3 caused a 50% reduction in the number of functional pumps when assembled with NK␤1 (Fig. 6). Arginine is conserved in colon HK␣ subunits at the position corresponding to amino acid 898 of NK␣3, and the positive charge may limit assembly of colon HK␣ subunits with NK␤ subunits. Interestingly, the Y898R mutation did not lead to a decrease in the number of pumps formed with gastric HK␤ (Fig. 6).
The thermal stability of pumps containing the V904Q or Y898R mutation in NK␣3 is comparable to that of nonmutated NK␣3 (Fig. 7). This result indicates that the reduced number of functional pumps containing these mutations is probably not the consequence of unstable ␣␤ complexes. The limiting factor may be the initial assembly of the two polypeptides, such that the V904Q or Y898R mutation in NK␣3 prevents the majority of the two subunits from forming functional complexes. Beggah et al. (21) reported that mutations to hydrophobic amino acids near the carboxyl terminus of NK␤3 interfere with ␣␤ complex formation in Xenopus oocytes (21). In particular, the double mutation V269N,F271N abolished the cellular accumulation of ␣ subunits, which is an indication of ␣␤ complex formation. The finding here that Val 904 and Tyr 898 in NK␣3 are important for assembly with NK␤1 is intriguing in this context. Perhaps the valine-aromatic amino acid pair on each subunit interacts with one another to provide a stable contact between the subunits.
The double mutation Y898R,C908G in NK␣3 caused a reduction in the number of functional pumps assembled with HK␤ by 90%, despite the absence of an effect of either mutation alone (Fig. 6). This effect of the double mutation on the assembly of NK␣3 with HK␤ is consistent with the observation that the reciprocal mutations R898Y,G908C in NCH26 led to a 4-fold increase in the amount of ouabain bound when when this chimeric/mutant ␣ subunit was expressed in yeast with HK␤. The thermal stability profile (Fig. 7) demonstrates that NK␣3 with the double mutation Y898R,C908G is extremely unstable when assembled with HK␤. Thus, the small number of pumps assembled either from NK␣3 containing either these mutations or from NCH26, and HK␤, may be the consequence of instability in ␣ ϩ HK␤ complexes caused by arginine and glycine at these positions. The double mutations Y898R,C908G in NK␣3 also caused a 13-fold reduction in the ouabain affinity of pumps assembled with NK␤1 (Table II) with little effect on complex stability (Fig. 7). Because neither Y898R nor C908G alone affected ouabain binding, it is likely that neither Tyr 898 nor Cys 908 is located within the ouabain binding site, and the double mutation reduces ouabain affinity by indirectly affecting the ouabain binding site.