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Functional Role of Oxygen-Containing Residues in the Fifth Transmembrane Segment of the Na,K-ATPase α Subunit
Archives of Biochemistry and Biophysics Vol. 364, No. 2, April 15, pp. 254 –263, 1999 Article ID abbi.1999.1124, available online at http://www.idealibrary.com on
Functional Role of Oxygen-Containing Residues in the Fifth Transmembrane Segment of the Na,K-ATPase a Subunit 1 Jose´ M. Argu¨ello,* ,2,3 Jeffrey Whitis,† Man C. Cheung,* and Jerry B. Lingrel† *Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609; and †Department of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0524
Received November 20, 1998, and in revised form January 19, 1999
The functional roles of Tyr771, Thr772, and Asn776 in the fifth transmembrane segment of the Na, KATPase a subunit were studied using site-directed mutagenesis, expression, and kinetics analysis. Nonconservative replacements Thr772Tyr and Asn776Ala led to reduced Na,K-ATPase turnover. Replacements at these positions (Asn776Ala, Thr772Leu, and Thr772Tyr) also led to high Na-ATPase activity (in the absence of K 1). However, Thr772- and Asn776-substituted enzymes showed only small alterations in the apparent Na 1 and K 1 affinities (K 1/2 for Na,K-ATPase activation). Thus, the high Na-ATPase activity does not appear related to cation-binding alterations. It is probably associated with conformational alterations which lead to an acceleration of enzyme dephosphorylation by Na 1 acting at the extracellular space (Argu ¨ ello et al. J. Biol. Chem. 271, 24610 –24616, 1996). Nonconservative substitutions at position 771 (Tyr771Ala and Tyr771Ser) produced a significant decrease of enzyme turnover. Enzyme–Na 1 interaction was greatly changed in these enzymes, while their activation by K 1 did not appear affected. Although the Na 1 K 1/2 for Na,K-ATPase stimulation was unchanged (Tyr771Ala, Tyr771Ser), the activation by this cation showed no cooperativity (Tyr771Ala, n Hill 5 0.75; Tyr771Ser, n Hill 5 0.92; Control, n Hill 5 2.28). Substitution Tyr771Phe did not lead to a significant reduction in the cooperativity of the ATPase Na 1 dependence (n Hill 5 1.91). All Tyr771-substituted enzymes showed low steady-state levels of phosphoenzyme during Na-activated phosphorylation by ATP. Phosphorylation levels were not increased by oligomycin, although the drug bound and inactivated Tyr771-substituted enzymes. No E1 7 E2 equilibrium 1 This work was supported by National Institute of Health Grant HL28573 (J.B.L.). 2 J.M.A. is a recipient of a Research Development Award for Minority Faculty, HL 03373, from the National Institute of Health. 3 To whom correspondence should be addressed. Fax: (508) 8315933. E-mail: [email protected].
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alterations were detected using inhibition by vanadate as a probe. The data suggest that Tyr771 might play a central role in Na 1 binding and occlusion without participating in K 1– enzyme interactions. © 1999 Academic Press
The Na,K-ATPase transports Na 1 and K 1 ions across the plasma membrane of eukaryotic cells against their electrochemical gradients. The enzyme consists of two major subunits, a (M r 5 112,000) and b (M r 5 35,000 for the protein component). The catalytic a subunit contains the nucleotide and cation-binding domains. In recent years a topological model of the a subunit involving 10 transmembrane segments has emerged based on hydropathy analysis, topological studies, and analogies with other similar ATPases (1– 3). Nevertheless, the exact identity of some transmembrane segments is questioned; for instance, the location of the membrane interfacial boundaries and extent of the fifth and sixth transmembrane segments (H5 4 and H6, respectively) is still debated (2, 4, 5). Several reports have indicated that the H5–H6 transmembrane region is key to cation binding and transport. Studies directed to locate residues involved in cation coordination during binding and transport have suggested that amino acids Asp804 and Asp808, located in H6, are essential for enzyme function as they appear to be cation-coordinating residues (6 – 8) (Fig. 1A). Close to these amino acids but in H5, Glu779 seems involved in determining the enzyme voltage dependence (9); however, there is controversy regarding its role either as a cation-coordinating residue or as necessary to stabilize the occluded-cation conformation 4 Abbreviations used: RD a1, sheep a1 carrying substitutions Gln111Arg and Asn122Asp; H5, fifth transmembrane segment; H6, sixth transmembrane segment.
0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
FUNCTIONAL ROLES OF Y771, T772, AND N776 IN THE Na,K-ATPase
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Asn122Asp to encode a form of the enzyme with low affinity for ouabain (RD a1) (14, 21, 22). Nucleotide substitutions were made to produce the following amino acid replacements: Tyr771Ala, Tyr771Ser, Tyr771Phe, Thr772Ala, Thr772Leu, Thr772Tyr, Asn776Ala, Asn776Gln, and Asn776Leu. Transfection and selection of ouabain-resistant HeLa cell lines expressing substituted RD a1 isoforms (22, 23), cell selection in high external KCl (14), isolation of crude plasma membranes (24, 25), protein determinations using bovine serum albumin as standard (26), quantification of the expressed enzyme using purified sheep Na,K-ATPase as standard and sheep a1-specific antibody M8-P1-A3 (14, 27), Na,K-ATPase and Na-ATPase activity determinations (9, 14), phosphorylation assays (9, 14, 28), and K 1 transport assays using 86Rb 1 as congener (15, 29) were all performed using previously described methods. FIG. 1. (A) Proposed topology of the H5-H6 domain. Primary structure positions of amino acids targeted in this study are indicated. (B) Helical wheel diagram of H5. Variable residues are circled. Primary structure positions of those oxygen-containing residues located in the “polar face” of the segment are indicated. Radial lines indicate suggested boundaries between hydrophobic and hydrophilic sides.
(8, 10 –13). We described that Ser775, also in H5, is likely part of the K 1-binding site, while it does not seem to participate in the binding of Na 1 (14, 15). Furthermore, the selective loss of the H5–H6 hairpin from the membrane following K 1 removal from a posttryptic preparation also supports the involvement of this region in cation coordination (16). The importance of the H5–H6 domain is also highlighted by the putative role it plays in ouabain binding and inactivation (16) and in the energy transduction between the cation and ATP-binding domains (9 –11, 17). The participation of amino acids in H5 and H6 of the sarcoplasmic reticulum Ca-ATPase in Ca 12 binding and transport is also well documented (18 –20). The analysis of helical wheel models of the putative H5 of the Na,K-ATPase a subunit (extending from Ser769 to Ala789) shows a clear polarity in the position of conserved– hydrophilic residues (Fig. 1B). In addition to residues that appear to play a central role in the enzyme function (Ser775, Glu779, and Phe786) (9, 14, 17), the “polar” phase contains three other well-conserved oxygen-containing residues, Tyr771, Thr772 and Asn776. Located close to the cytoplasmic end of H5 and among residues involved in cation coordination, these residues seemed logical targets for site-directed mutagenesis studies directed to understand structure– function relationships in the Na,K-ATPase. We describe in this report the functional characterization of Tyr771-, Thr772-, or Asn776-substituted enzymes. Our results indicate the putative role of Tyr771 in Na 1– enzyme interactions and the importance of the structural integrity of this region for the conformational transitions that occur in the catalytic phosphorylation domain upon cation binding. EXPERIMENTAL PROCEDURES Site-directed mutagenesis was performed as previously described using an a1 subunit cDNA modified by substitutions Gln111Arg and
RESULTS
When HeLa cells transfected with ouabain-resistant RD a1 isoforms are grown in tissue culture medium containing 1 mM ouabain, expression of functional exogenous enzyme enables cell growth. We have previously observed that substitutions Asn776Ala and Tyr771Ala yield enzymes that can not support HeLa cell growth (30). Various replacements at positions 771, 772, and 776 (Tyr771Ala, Tyr771Ser, Thr772Tyr, Asn776Ala, and Asn776Leu) were also unable to support cell growth under normal tissue culture conditions (5.4 mM KCl) (Fig. 2). This was considered a preliminary indication of the altered functionality of these enzymes. We have previously observed that increasing KCl concentration in the culture medium rescued cells transfected with some apparently nonfunctional substituted enzymes (14). This experimental maneuver was again successful allowing the survival of cells expressing Tyr771Ala-, Tyr771Ser-, Thr772Tyr-, and Asn776Ala-substituted enzymes. Cell clones expressing these substitutions were subsequently maintained at the external K 1 concentration at which they were selected. Asn776Leu-substituted enzyme was unable to support cell growth under any of the tested conditions. Characterization of Thr772- and Asn776-substituted enzymes. The variants that led to stable cell lines were further characterized. Table I shows the functional biochemical characterization of Thr772- and Asn776-substituted enzymes in terms of expression levels, Na,K-ATPase activity, Na-ATPase activity (activity in the absence of K 1), turnover number, and cation activation. Values for RD Control enzyme were similar to those obtained in previous studies (9, 14). Substitutions at position 776 led to reduced Na,KATPase activities and turnover numbers (21–27% of control enzymes). This was presumably compensated by a higher expression of the enzyme (Table I); i.e., it is apparent that only high-expressing clones were able to generate the necessary Na 1 and K 1 electrochemical gradients. The nonconservative replacement Asn776Ala leads to a high Na-ATPase activity (in the absence of K 1). This phenomena has been previously observed in
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FIG. 2. Number of ouabain-resistant colonies resulting from the expression of RD Control and Try771-, Thr772-, and Asn776-substituted enzymes, after selection in growth medium containing different K 1 concentrations. Two days after transfection HeLa cells (5 3 10 5 cells/plate) were subjected to ouabain-selective pressure by inclusion of 1 mM ouabain in the Dulbecco’s modified Eagle’s (10% calf serum) culture medium. Selection was stopped after 2 weeks in cells transfected with RD Control cDNA and after 4 weeks in the case of cells transfected with mutated cDNA. Colonies were stained with Methylene Blue and counted. The values are means 6 SE of six independent experiments. External KCl concentration: 5.4 mM (white); 10 mM (pattern); 20 mM (gray); 5.4 mM 1 15 mM choline chloride (black).
enzymes carrying substitutions Glu779Ala (9, 12) and Asn324Leu (31). In the case of Asn776Ala-substituted enzymes the Na 1 dependence of this activity follows a pattern similar to that previously described for Glu779Ala-substituted enzymes but reaches a higher V max (83% of control compared to 60% for Glu779Ala and 32% for Asn324Leu) (Fig. 3). Replacement of Thr772 produced a significant decrease in Na,K-ATPase activity and turnover only in the case of Thr772Tyr; however, no major increase in expression level of this enzyme was detected (Table I). As in the case of Asn776, Glu779, and Asn324 substitutions, nonconservative changes of Thr772 (to leucine and tyrosine) led to a significant increase in the NaATPase activities. The Na 1 dependence of these activities again roughly followed an apparent single exponential kinetic (not shown). Thr772- and Asn776-substituted enzymes showed only small alterations in the cation activation of Na,KATPase activity. Table I shows the Na 1 K 1/2 and K 1 K 1/2 for enzyme activation together with the corresponding Hill coefficients obtained by fitting Na,K-ATPase activ-
ity vs cation concentration curves (not shown). K 1 interaction with the enzyme seemed unaffected, while the Na 1 apparent affinity was only slightly decreased (two times increase in K 1/2) by substitution of Asn776. These data suggest that these Thr772 and Asn776 are likely not directly involved in cation coordination. Na 1-activated phosphorylation by ATP of Thr772and Asn776-substituted enzymes was significantly reduced in those substitutions that have high Na-ATPase activity (Thr772Leu, Thr772Tyr, and Asn776Ala) (Table II). Previous studies have shown that substitution Glu779Ala leads to high Na-ATPase activity associated with low phosphoenzyme levels (9, 12). We observed that these alterations are due to fast dephosphorylation by Na 1 acting at extracellular sites with normal affinity (9, 32). Phosphorylation levels similar to RD Control enzyme have been detected in the Glu779Ala enzyme by inclusion of oligomycin in the phosphorylation medium (9, 12). This inhibitor prevents enzyme dephosphorylation by stabilizing the Na 1-bound forms of the enzyme (33, 3). As expected, Thr772Leu-, Thr772Tyr-, and Asn776Ala-substituted enzymes
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FUNCTIONAL ROLES OF Y771, T772, AND N776 IN THE Na,K-ATPase TABLE I
Expression Level, Na,K-ATPase Activity, Na-ATPase Activity, Turnover No., and ATPase Cation Activation Parameters of RD Control and Thr772- and Asn776-Substituted Enzymes
Expression (mg/mg) Na,K-ATPase activity (mmol/min/mg) c Na-ATPase activity (% of total Na,K-ATPase) d Turnover No. (1/min) e Na 1 activation K 1/2 (mM) f n Hill K 1 activation K 1/2 (mM) n Hill a
RD Control
Asn776Ala
Asn776Gln
Thr772Ala
Thr772Leu
Thr772Tyr
34 6 8 6.81 6 1.75
110 6 40 1.76 6 0.28
72 6 2 1.53 6 0.26
37 6 7 4.80 6 2.17
28 6 7 6.65 6 1.04
44 6 6 1.98 6 0.53
17.8 6 4.3
83.2 6 6.1
18.3 6 9.1
20.0 6 1.5
71.3 6 1.5
63.3 6 7.0
9533 6 1481
2576 6 46
2013 6 362
5708 6 1505
6325 6 865
2709 6 532
6.61 6 0.43 2.28 6 0.16
13.99 6 1.5 1.39 6 0.16
14.25 6 0.89 2.04 6 0.21
4.70 6 0.64 1.89 6 0.47
4.34 6 0.67 1.70 6 0.51
6.26 6 0.83 1.40 6 0.27
0.43 6 0.06 1.29 6 0.09
0.30 6 0.05 1.24 6 0.13
0.42 6 0.03 1.15 6 0.10
0.38 6 0.08 1.08 6 0.06
0.61 6 0.18 1.00 6 0.07
0.84 6 0.31 0.69 6 0.07
b
a
Micrograms of heterologously expressed Na,K-ATPase per mg of total protein in the membrane preparation. Values are means 6 SE of n 5 4 independent clones. Each clone was assayed at least in duplicate. c Maximum activity as mmol of hydrolyzed ATP per min per mg of heterologously expressed Na,K-ATPase measured at 37°C in a medium containing 130 mM NaCl, 20 mM KCl, 50 mM choline-Cl, 3 mM MgCl 2, 3 mM ATP, 0.5 mM ethylene glycol bis(b-aminoethyl ether)-N,N9tetraacetic acid 50 mM imidazole, pH 7.2, 0.3 mg/ml bovine serum albumin, 1 mg/ml of membrane protein. The activity of heterologous protein was calculated as the difference in activity measured in presence of 10 mM and 10 mM ouabain (14). d Na-ATPase activity measured in presence of 200 mM NaCl, 0 mM KCl. e The turnover number was calculated independently for each preparation as the ratio of ATPase activity to the phosphoenzyme level (measured in the presence of 100 mg/ml oligomycin). f Na 1 and K 1 K 1/2 and Hill coefficients for enzyme activation were obtained by fitting Na,K-ATPase activity vs cation concentration curves to the equation: v 5 V maxL n/L n 1 K 1/2. b
showed phosphoenzyme levels comparable to RD Control when oligomycin was included in the phosphorylation media (Table II). Characterization of Tyr771-substituted enzymes. Replacement of Tyr771 has marked effects on the functionality of the Na,K-ATPase (Table III). Enzymes carrying substitutions Tyr771Ala and Tyr771Ser have very low Na,K-ATPase activity (13 and 18% of control enzyme, respectively). This low activity disturbed the analysis of these mutants; for instance, their NaATPase activity could not be evaluated with confidence. The replacement Tyr771Phe, maintaining the aromatic character in this location, reduced the Na,KATPase to a lesser extent (46% of control enzyme) and increased the Na-ATPase activity (twice the level of control). Because of uncertainty in their maximal phosphorylation level (see below), we were unable to calculate turnover numbers for these variants. The K 1 apparent affinity (measured as K 1/2 for Na,KATPase activation) was not affected by replacements Tyr771Ser or Tyr771Phe (Table III, Fig. 4). Low Na,KATPase activity prevented us from accurately measuring this parameter for Tyr771Ala enzyme. To assess the interaction of K 1 with this latter mutant and confirm our findings for Tyr771Ser or Tyr771Phe enzymes, the K 1 dependence of K 1 ( 86Rb 1) uptake was measured (curves not shown). These determinations also showed no alteration in the K 1 K 1/2 for enzyme activation (Table III). Therefore, it appears that re-
placement of Tyr771 did not affect K 1– enzyme interactions. Figure 5 shows that Tyr771Ala- and Tyr771Ser-substituted enzymes had altered Na 1 activation kinetics. The K 1/2 for Na 1 activation of these enzymes’ Na,KATPase activity was close to that of the RD Control. However, they presented a largely reduced n Hill, suggesting an alteration in the cooperative Na 1 binding to the enzyme (Table III). Tyr771Phe-substituted enzyme had a reduced apparent affinity for Na 1, but still showed close to normal cooperativity for Na 1 activation. Thus, these results suggest that Na 1 interaction with the enzyme was significantly affected by replacement of Tyr771. Try771 replacement also altered Na 1-activated phosphorylation by ATP. These enzymes showed reduced (9 –32% of control enzyme) steady-state phosphorylation levels (Table IV). Contrary to that observed with other substituted enzymes that show reduced phosphorylation (see Tables I and II), the presence of oligomycin in the phosphorylation medium did not increase Tyr771-substituted enzymes phosphorylation to levels similar to RD Control enzyme (Table IV). The maximum level of phosphorylated enzyme (in presence of oligomycin) is assumed to be equivalent to the number of functional enzymes and used to calculate the enzyme turnover numbers (7–9, 12–15). Thus, the low phosphoenzyme levels detected in Tyr771-sub-
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FIG. 3. Na 1 dependence of Na-ATPase activity of RD Control and Asn776Ala-substituted enzymes. Na-ATPase activity was determined at 37°C in medium containing 3 mM MgCl 2, 3 mM ATP, 0.5 mM ethylene glycol bis(b-aminoethyl ether)-N,N9-tetraacetic acid, 50 mM imidazole, pH 7.2, 0.3 mg/ml bovine serum albumin, approximately 1 mg/ml of membrane protein, 0.01 mM ouabain, and various concentrations of NaCl and choline chloride (total [monovalent cation] 5 200 mM). Background activity measured in the presence of 10 mM ouabain was subtracted. The Na-ATPase activity is expressed as percentage of the maximal Na,K-ATPase activity (Table I). The values are means 6 SE of results obtained with membrane preparations from four independent clones, each one measured in duplicate. RD enzyme (E); Asn776Ala substituted enzyme (F).
stituted enzymes led to problems in establishing the number of functional enzymes. Assuming the phosphoenzyme level is an accurate indication of this number, in the case of Tyr771 mutants, only a small pool of protein would be functional (10% of Tyr771Ala, 47% of Tyr771Ser, and 35% of Tyr771Phe, based on the percentages of phosphorylation with respect to the control). However, using these phosphorylation data to calculate turnover numbers, the following values were obtained: Tyr771Ala, 11,950 min 21; Tyr771Ser 3,361 min 21; and Tyr771Phe 13,551 min 21 (compared to RD
Control, 9533 min 21). Aside from Tyr771Ser, the values obtained for the Tyr771Ser and Tyr771Phe substitutions are incompatible with the concept that the larger fraction of these enzymes appears nonfunctional. Based on the implausibility of these figures, we did not considered the calculated turnover numbers an indication of the enzyme functionality. Different alterations in enzyme kinetic or ligand– enzyme interaction might explain the low phosphoenzyme levels. In an attempt to clarify this point we measured different characteristics of the Tyr771
TABLE II
Phosphorylation of RD Control and Thr772- and Asn776-Substituted Enzymes Phosphorylation (nmol/mg of heterologous enzyme) a
2 Oligomycin 1 Oligomycin
RD Control
Asn776Ala
Asn776Gln
Thr772Ala
Thr772Leu
Thr772Tyr
0.58 6 0.10 0.70 6 0.10
0.37 6 0.11 0.79 6 0.14
0.50 6 0.14 0.76 6 0.16
0.55 6 0.09 0.84 6 0.23
0.23 6 0.07 0.92 6 0.06
0.27 6 0.04 0.84 6 0.10
b
The Na-activated ATP phosphorylation was carried out at 0°C in a medium containing 1 mM MgCl 2, 5 mM [g- 32P]ATP, 0.04 mM ethylene glycol bis(b-aminoethyl ether)-N,N9-tetraacetic acid, 75 mM Hepes/imidazole, pH 7.2, 0.01 mM ouabain, and 0.15 mg/ml membrane protein, in the presence of either 50 mM NaCl or 50 mM NaCl plus 100 mg/ml oligomycin. Background activity measured in the presence of 50 mM KCl (0 mM Na 1) was subtracted. b The values are means 6 SE of results obtained with membrane preparations from three independent clones, each one measured in triplicate. a
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FUNCTIONAL ROLES OF Y771, T772, AND N776 IN THE Na,K-ATPase TABLE III
Expression Level, Na,K-ATPase Activity, Na-ATPase Activity, Turnover No., and ATPase Cation Activation Parameters of RD Control and Tyr771-Substituted Enzymes
Expression (mg/mg) Na,K-ATPase activity (mmol/min/mg) Na-ATPase activity (% of total Na,K-ATPase) Na 1 activation K 1/2 (mM) n Hill 1 K activation (ATPase activity) K 1/2 (mM) n Hill K 1 activation (K 1 uptake) K 1/2 (mM) n Hill a
a
RD Control
Tyr771Ala
Tyr771Ser
Tyr771Phe
34 6 8 6.81 6 1.75
69 6 8 0.89 6 0.29
75 6 13 1.21 6 0.38
72 6 21 3.70 6 0.31
17.8 6 4.3
nd
nd
38.0 6 2.3
6.61 6 0.43 2.28 6 0.16
4.92 6 0.98 0.75 6 0.12
8.83 6 0.75 0.92 6 0.08
23.49 6 3.21 1.91 6 0.29
0.43 6 0.06 1.29 6 0.09
nd nd
0.70 6 0.12 0.81 6 0.09
0.57 6 0.02 1.39 6 0.25
0.91 6 0.17 1.14 6 0.13
1.09 6 0.23 1.13 6 0.13
1.36 6 0.22 1.38 6 0.14
0.98 6 0.18 1.06 6 0.20
Assay conditions for measurements described in this table are similar to those described in the notes to Table I. nd, not determined.
mutants phosphorylation by ATP. The ATP binding to the enzyme did not seem affected by Tyr771 replacement. The substitutions did not increase the ATP K m for the Na 1 -activated phosphorylation of the
FIG. 4. K 1 dependence of Na,K-ATPase activity of RD Control and Tyr771-substituted enzymes. The Na,K-ATPase activity was measured in the medium described in Table I, in the presence of 100 mM NaCl and various concentrations of KCl and choline chloride (total [monovalent cation] 5 200 mM). The Na,K-ATPase activities corresponding to 100% were similar to the values presented in Table III. The values are means of results obtained with membrane preparations from four independent clones, each one measured in duplicate. Standard errors were between 5 and 10% for Tyr771-substituted enzymes and lower than 5% for the RD Control; error bars were not plotted for simplicity. Curve-fitting parameters (K 1/2 and Hill coefficients) are listed in Table III. RD Control (E), Tyr771Ala (F), Tyr771Ser (Œ), Tyr771Phe (}).
enzyme; on the contrary, a small decrease of this K m was evident in the Tyr771Phe-substituted enzyme (Fig. 6A). Na 1 dependence of phosphorylation by ATP showed relatively small increases in Na 1 K 1/2 (Fig. 6B). It is known that Na 1 activates phosphorylation in a noncooperative fashion (34). Consequently the alterations in cooperativity detected in the Na 1 stimulation of the Na,K-ATPase (Fig. 5) were not expected in these determinations. Steadystate conditions were verified by measuring phosphorylation levels at 30, 60, and 120 s, and no differences were detected by extending the phosphorylation time (not shown). Low starting phosphoenzyme levels prevented us from measuring dephosphorylation rates. However, it is unlikely that the low steady-state phosphorylation would be associated with high dephosphorylation rates, since the Na 1 dependence of phosphorylation does not show the bell-shaped curve (reduction at high Na 1 ) observed in mutants with high dephosphorylation rates (9). Furthermore, these low phosphoenzyme levels are detectable even in the presence of oligomycin (Table IV). This observation is valid assuming that the inhibitor is able to bind to these enzymes, stabilize the Na 1 -bound forms, and consequently block the E1P 7 E2P conversion (33). To verify this point, we determined that oligomycin is able to fully inhibit the Na,K-ATPase activity of Tyr771-substituted enzymes with an IC 50 similar to that of RD Control (curves not shown) (IC 50 (mg/ml): RD Control, 0.42 6 0.05; Tyr771Ala 0.29 6 0.10; Tyr771Ser, 0.42 6 0.05; and Tyr771Phe, 0.19 6 0.05). Consequently, it is apparent that the Tyr771-substituted enzymes can bind the inhibitor. In an attempt to determine if the alterations in Na 1 stimulation of ATPase activity and the low
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FIG. 5. Na 1 dependence of Na,K-ATPase activity of RD Control and Tyr771-substituted enzymes. The Na,K-ATPase activity was measured in the medium described in the notes to Table I, in the presence of 20 mM KCl and various concentrations of NaCl and choline chloride (total [monovalent cation] 5 200 mM). The Na,K-ATPase activities corresponding to 100% were similar to the values presented in Table III. Curve-fitting parameters (K 1/2 and Hill coefficients) are listed in Table III. The values are means of results obtained with membrane preparations from four independent clones, each one measured in duplicate. Standard errors were between 5 and 10% for Tyr771-substituted enzymes and lower than 5% for the RD Control; error bars were not plotted for simplicity. RD Control (E), Tyr771Ala (F), Tyr771Ser (Œ), Tyr771Phe (}).
steady-state levels were associated with alterations in the equilibrium among different enzyme conformations the effect of vanadate on Tyr771 enzymes was tested. The concentration dependence of vanadate inhibition for Tyr771-substituted enzymes was similar to that of RD Control enzyme (curves not shown) (IC 50 (mM) were: RD Control, 0.81 6 0.09; Tyr771Ala 2.50 6 0.52; Tyr771Ser, 1.63 6 0.34; and Tyr771Phe, 1.21 6 0.16). This suggests that Tyr771substituted enzymes would not behave differently than the control in their tendency to remain in either E1 or E2 conformation.
DISCUSSION
Effects of replacement of amino acids Thr772 and Asn776. Replacement of residues at positions 772 and 776 led to significant reductions in enzyme activity. Expression of some of these substitutions was achieved by increasing the external [K 1]. We have hypothesized in the case of substitutions of Ser775 (which lead to a dramatic decrease in K 1 affinity) that the higher concentration of the cation helped cell survival by increasing enzyme turnover via saturation of the external cation-binding sites (14). Substitutions of
TABLE IV
Phosphorylation of RD Control and Tyr771-Substituted Enzymes Phosphorylation (nmol/mg of heterologous enzyme) a
2 Oligomycin 1 Oligomycin a
RD Control
Tyr771Ala
Tyr771Ser
Tyr771Phe
0.58 6 0.10 0.70 6 0.10
0.053 6 0.011 0.075 6 0.002
0.18 6 0.10 0.36 6 0.09
0.17 6 0.02 0.27 6 0.04
b
Assay conditions are similar to those described of Table II. The values are means 6 SE of results obtained with membrane preparations from three independent clones, each one measured in triplicate. b
FUNCTIONAL ROLES OF Y771, T772, AND N776 IN THE Na,K-ATPase
261
FIG. 6. Kinetic and ligand stimulation of phosphorylation by ATP of RD Control and Tyr771-substituted enzymes. In these sets of experiments Na-activated ATP phosphorylation was carried out in the absence of oligomycin in the medium described in the notes to Table II. [ATP] vs phosphorylation curves (A) were fit to a simple Michaelis kinetics, and ATP K m (mM) were as follows: RD Control, 1.12 6 0.25; Tyr771Ser, 1.06 6 0.20; and Tyr771Phe, 0.46 6 0.17. Na 1 dependence (B) was measured by variable [Na 1] while ionic strength was kept constant with choline chloride. [Na 1] vs phosphorylation curves (B) were fit to the equation v 5 V maxL n/L n 1 K 1/2. The Na 1 K 1/2 (mM) and Hill coefficients values were as follows: RD Control, 0.52 6 0.10, n 5 0.83; Tyr771Ala, 3.81 6 0.81, n 5 0.87; Tyr771Ser, 4.43 6 1.10, n 5 0.78; and Tyr771Phe, 2.28 6 0.17, n 5 0.86. The values are means of results obtained with membrane preparations from three independent clones, each one measured in triplicate. Standard errors were between 5 and 15% and error bars were not plotted for simplicity. RD Control (E), Tyr771Ala (F), Tyr771Ser (Œ), Tyr771Phe (}).
Thr772 and Asn776, as well as Tyr771, did not have significant effects on the binding of K 1 to the enzyme. Thus, another mechanism must explain the observed effect of extracellular [K 1]. Considering the dependence of the Na,K-ATPase on the membrane potential, a possible simple explanation is that the partial depolarization produced by increasing external [K 1] increased the activity of mutants with slow turnover leading to levels compatible with maintaining the required Na 1 and K 1 electrochemical gradients. Replacement of Thr772 or Asn776 had no major effects on the apparent affinities of Na 1 and K 1. Substitutions of Asn776 increased the Na 1 K 1/2 for Na,KATPase activation; however, the magnitude of this effect was small when compared to that observed when residues probably participating in cation coordination were replaced (Ser775, Asp804, and Asp808) (6, 14). Thus, these results suggest that Thr772 and Asn776 are not directly involved in the coordination of Na 1 or K 1. The most significant alteration resulting from replacing Thr772 and Asn776 was a large increase in the Na-ATPase activity. This was accompanied by a reduction in the steady-state phosphorylation levels that could be reversed by including oligomycin in the phosphorylation medium. Previous studies have reported similar findings after nonconservative replacement of amino acids Asn324 and Glu779 (6, 12, 31). It appears from studies of Glu779Ala-substituted enzymes that
these replacements increase the dephosphorylation rates upon binding of Na 1 to external cation-binding sites (9). It was later shown that the apparent affinity for external Na 1 is not modified by the Glu779Ala substitution (32). Thr772, Asn776, and Glu779 are located in the fifth transmembrane segments of the a subunit, in direct connection with the hydrolytic domain of the enzyme. The putative a helical structure of H5 suggests that these residues would be positioned in consecutive turns of the helix with similar orientation with respect to the helical backbone, likely interacting with the same adjacent transmembrane segment (Fig. 1). This hypothetical organization might explain the similar alterations observed upon nonconservative replacement of any of these amino acids. In contrast, replacement of Tyr771 or Ser775, located in the same region but with a different orientation with respect to the backbone of H5, does not lead to effects similar to those produced by substitutions of Thr772, Asn772, or Glu779. It has been proposed that H5 provides a way in which information about ATP binding and phosphorylation in the major cytoplasmic loop is transmitted to the intramembrane cation sites during the reaction cycle (9, 11, 16). The reverse information flux is also required by the enzyme mechanism, for instance, in the E 2P dephosphorylation upon K 1 binding. The functional alterations observed after Thr772 or Asn776 replacement support these ideas since they suggest
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¨ ELLO ET AL. ARGU
that particular structural modifications in H5 lead to changes in phosphoenzyme stability upon external Na 1 binding. Effects of replacing Tyr771. Substitution of Tyr771 had significant effects on the enzyme kinetics and in the interaction of Na 1 with the enzyme. Although Na 1 apparent affinities did not decrease significantly (based on the K 1/2 for activation of ATPase activity or phosphorylation), the Na 1 dependence of the Na,KATPase was no longer cooperative in enzymes carrying Tyr771Ala and Tyr771Ser replacements. Na 1 activation of phosphorylation by ATP did not show this effect because it normally follows a single exponential dependence (34). The changes in cooperativity were not observed in Tyr771Phe-substituted enzyme, which would suggest that the aromatic group is the relevant characteristic at this position. The involvement of aromatic groups in cation–protein interactions has been previously proposed (35, 36). The alterations in the cooperativity of the Na 1 activation might be interpreted as a change in the affinities of the different Na 1 binding sites such that kinetically all appear equivalent. This effect might be due to a general conformational alteration produced by these substitutions. However, Tyr771 replacement did not appear to affect K 1 binding or the E1 7 E2 equilibrium. Therefore, the alterations in Na 1 binding would be a direct consequence of Tyr771 substitution. Significant reductions in the cooperativity of Na 1 interactions with the enzyme have been previously observed in Asp804 and Asp808 substituted enzymes. These aspartyl residues appear involved in the coordination of both Na 1 and K 1 ions (6). The second major effect of replacing Tyr771 is the low steady-state phosphorylation level observed in the modified enzymes. The presence of oligomycin in the medium, although it increased phosphorylation, did not lead to levels similar to control. A simple explanation for these results could be that in fact the substitutions yielded a smaller pool of functional enzyme that reach phosphoenzyme levels similar to the control. However, because this hypothesis would imply turnover numbers (for the functional pools) higher than that of control enzyme we consider it unlikely. These high turnover numbers would be incompatible with functionally altered enzymes. Oligomycin did not stimulate phosphorylation of Tyr771-substituted enzymes to levels similar to those of RD Control enzymes as in the case of Thr772, Asn776, or Glu779 substituted enzymes. This observation could be due to a decreased oligomycin binding; however, oligomycin was able to inhibit the substituted enzymes in a way similar to the controls. This suggests a normal interaction of the inhibitor with the enzymes. We also considered that replacement of Tyr771 might alter the phosphorylation rate. However, extended phosphorylation times did not lead to higher phosphor-
ylation levels. Furthermore, since replacement of Tyr771 apparently did not decrease the affinity for ATP and only slightly increased the Na 1 K 1/2 for activation, substrates were saturating in our phosphorylation determinations. Consequently, from these observations, one possible explanation of the observed low phosphorylation levels is a displacement of the E1Na 3ATP 7 E1P(3Na) 1 ADP equilibrium toward the dephosphorylated form of the enzyme. This would not be inconsistent with the lack of oligomycin effect on the phosphorylation level, since this drug seems to influence phosphorylation via stabilizing the Na 1bound forms of the enzyme (33). Furthermore, the small increase in ATP affinity detected in Tyr771Phe enzyme might be associated to alterations of this equilibrium. Finally, we should point out that although the importance of Tyr771 for reaction steps associated with Na 1 binding and occlusion is plausible, the relationship between the changes in cooperativity and the reduced phosphorylation is not clear at this moment. Substitution of Tyr773 of the sarcoplasmic reticulum Ca-ATPase (equivalent to Tyr771 in the Na,K-ATPase) for glycine produces the uncoupling of the ATP hydrolysis from Ca 21 transport (19). This interesting phenotype, although not observed in the Tyr763Thr mutant (20), also indicates a fundamental role of Tyr771 on the cytoplasmic side of H5 of P-type ATPases. Functional relevance of the fifth transmembrane segment. The importance of the polar face of H5 is evident when considering those residues that appear to play a central role in enzyme function. Located in H5, Ser775 participates in K 1 coordination (14, 15), Glu779 appears involved in determining the voltage dependence of the enzyme (9), and Phe786 is involved in ouabain binding (17). Furthermore, conformational changes of H5 during the catalytic cycle have been proposed (14, 16). Based on this information we have proposed a structural–functional model where the energy from ATP hydrolysis is transferred to the cationbinding site (formed in part by the H5–H6 hairpin) through a conformational change of H5. Ouabain binding to the extracellular half of the H5–H6 region probably blocks this conformational change, therefore inhibiting the enzyme (17). This study provides further evidence of the functional importance of the fifth transmembrane segment of the a subunit of Na,K-ATPase. The present information supports the concept that this region of the enzyme is involved in the transfer of information between the phosphorylation and cation binding domains. In addition, it points out the importance of Tyr771 as a required component for normal Na 1– enzyme interactions. ACKNOWLEDGMENTS We thank Dr. J. Feng for constructing the pKC4 vector carrying the Tyr771Ala, Thr772Ala, and Gln776Ala substitutions and Dr. J. Ball for supplying the antibody M8-P1-A3.
FUNCTIONAL ROLES OF Y771, T772, AND N776 IN THE Na,K-ATPase Note added in proof. During this manuscript review, a report was published by Pedersen et al. (Biochemistry 37, 1818 –1827, 1998). This paper also describes effects of substitutions of Tyr771, Thr772, and Asn776 in the pig a subunit expressed in yeast.
REFERENCES 1. Lingrel, J. B., and Kuntzweiler, T. (1994) J. Biol. Chem. 269, 19659 –19662. 2. Jørgensen, P. L. (1992) in Molecular Aspects of Transport Proteins (De Pont, J. J., Ed.), pp. 1–26, Elsevier Science Pub. B.V. 3. Horisberger, J-.D. (1994) The Na,K-ATPase: Structure–Function Relationship, Landes, Austin, TX. 4. Mohraz, M., Arystarkhova, E., and Sweadner, K. J. (1993) J. Biol. Chem. 269, 2929 –2936. 5. Canfield, V. A., Norbeck, L., and Levenson, R. (1996) Biochemistry 32, 13782–13786. 6. Kuntzweiler, T., Argu¨ello, J. M., and Lingrel, J. B. (1996) J. Biol. Chem. 271, 29682–29687. 7. Pedersen, P. A., Rasmussen, J. H., Nielsen, J. M., and Jørgensen, P. L. (1997) FEBS Lett. 400, 206 –210. 8. Nielsen, J. M., Pedersen, P. A., Karlish, S. J. D., and Jørgensen, P. L. (1998) Biochemistry 37, 1961–1968. 9. Argu¨ello, J. M., Peluffo, R. D., Feng, J., Lingrel, J. B., and Berlin, J. R. (1996) J. Biol. Chem. 271, 24610 –24616. 10. Argu¨ello, J. M., and Kaplan, J. H. (1991) J. Biol. Chem. 266, 14627–14635. 11. Argu¨ello, J. M., and Kaplan, J. H. (1994) J. Biol. Chem. 269, 6892– 6899. 12. Vilsen, B. (1995) Biochemistry 34, 1455–1463. 13. Koster, J. C., Blanco, G., Mills, P. B., and Mercer, R. W. (1996) J. Biol. Chem. 271, 2413–2421. 14. Argu¨ello, J. M., and Lingrel, J. B. (1995) J. Biol. Chem. 270, 22764 –22771. 15. Blostein, R., Wylczynska, A., Karlish, S. J. D., Argu¨ello, J. M., and Lingrel, J. B. (1997) J. Biol. Chem. 272, 24987–24993.
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16. Lutsenko, S., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. USA 92, 7936 –7940. 17. Palasis, M., Kuntzweiler, T., Argu¨ello, J. M., and Lingrel, J. B. (1996) J. Biol. Chem. 271, 14176 –14183. 18. Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1990) Nature 339, 476 – 478. 19. Andersen, J. P. (1995) J. Biol. Chem. 270, 908 –914. 20. Rice, W. J., and MacLennan, D. H. (1996) J. Biol. Chem. 271, 31412–31419. 21. Kunkel, T. A. (1985) Proc. Nat. Acad. Sci. USA 82, 488 – 492. 22. Price, E. M., and Lingrel, J. B. (1988) Biochemistry 27, 8400 – 8408. 23. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745–2752. 24. Lane, L. K., Copenhaver, J. H., Jr., Lindenmayer, G. E., and Schwartz, A. (1973) J. Biol. Chem. 248, 7197–7200. 25. Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925–16930. 26. Bradford, M. M. (1976) Anal. Biochem. 72, 248 –254. 27. Malik, B., Jamieson, G. A., Jr., and Ball, W., Jr. (1993) Protein Sci. 2, 2103–2111. 28. Blostein, R. (1988) Methods Enzymol. 156, 171–178. 29. Munzer, J. S., Daly, S. E., Jewell-Motz, E. A., Lingrel, J. B., and Blostein, R. (1994) J. Biol. Chem. 269, 16668 –16676. 30. Feng, J., and Lingrel, J. B. (1994) Biochemistry 33, 4218 – 4224. 31. Vilsen, B. (1995) FEBS Lett. 363, 179 –183. 32. Peluffo, R. D., Argu¨ello, J. M., Lingrel, J. B., and Berlin, J. R. (1998) Biophys. J. 74, A171. 33. Arato-Oshima, T., Matsui, H., Wkizaka, A., and Homareda, H. (1996) J. Biol. Chem. 271, 25604 –25610. 34. Mardh, S., and Post, R. L. (1977) J. Biol. Chem. 252, 633– 638. 35. Heginbotham, L., and MacKinnon, R. (1992) Neuron 8, 483– 491. 36. Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69 –76.
Functional Role of Oxygen-Containing Residues in the Fifth Transmembrane Segment of the Na,K-ATPase a Subunit 1 Jose´ M. Argu¨ello,* ,2,3 Jeffrey Whitis,† Man C. Cheung,* and Jerry B. Lingrel† *Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609; and †Department of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0524
Received November 20, 1998, and in revised form January 19, 1999
The functional roles of Tyr771, Thr772, and Asn776 in the fifth transmembrane segment of the Na, KATPase a subunit were studied using site-directed mutagenesis, expression, and kinetics analysis. Nonconservative replacements Thr772Tyr and Asn776Ala led to reduced Na,K-ATPase turnover. Replacements at these positions (Asn776Ala, Thr772Leu, and Thr772Tyr) also led to high Na-ATPase activity (in the absence of K 1). However, Thr772- and Asn776-substituted enzymes showed only small alterations in the apparent Na 1 and K 1 affinities (K 1/2 for Na,K-ATPase activation). Thus, the high Na-ATPase activity does not appear related to cation-binding alterations. It is probably associated with conformational alterations which lead to an acceleration of enzyme dephosphorylation by Na 1 acting at the extracellular space (Argu ¨ ello et al. J. Biol. Chem. 271, 24610 –24616, 1996). Nonconservative substitutions at position 771 (Tyr771Ala and Tyr771Ser) produced a significant decrease of enzyme turnover. Enzyme–Na 1 interaction was greatly changed in these enzymes, while their activation by K 1 did not appear affected. Although the Na 1 K 1/2 for Na,K-ATPase stimulation was unchanged (Tyr771Ala, Tyr771Ser), the activation by this cation showed no cooperativity (Tyr771Ala, n Hill 5 0.75; Tyr771Ser, n Hill 5 0.92; Control, n Hill 5 2.28). Substitution Tyr771Phe did not lead to a significant reduction in the cooperativity of the ATPase Na 1 dependence (n Hill 5 1.91). All Tyr771-substituted enzymes showed low steady-state levels of phosphoenzyme during Na-activated phosphorylation by ATP. Phosphorylation levels were not increased by oligomycin, although the drug bound and inactivated Tyr771-substituted enzymes. No E1 7 E2 equilibrium 1 This work was supported by National Institute of Health Grant HL28573 (J.B.L.). 2 J.M.A. is a recipient of a Research Development Award for Minority Faculty, HL 03373, from the National Institute of Health. 3 To whom correspondence should be addressed. Fax: (508) 8315933. E-mail: [email protected].
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alterations were detected using inhibition by vanadate as a probe. The data suggest that Tyr771 might play a central role in Na 1 binding and occlusion without participating in K 1– enzyme interactions. © 1999 Academic Press
The Na,K-ATPase transports Na 1 and K 1 ions across the plasma membrane of eukaryotic cells against their electrochemical gradients. The enzyme consists of two major subunits, a (M r 5 112,000) and b (M r 5 35,000 for the protein component). The catalytic a subunit contains the nucleotide and cation-binding domains. In recent years a topological model of the a subunit involving 10 transmembrane segments has emerged based on hydropathy analysis, topological studies, and analogies with other similar ATPases (1– 3). Nevertheless, the exact identity of some transmembrane segments is questioned; for instance, the location of the membrane interfacial boundaries and extent of the fifth and sixth transmembrane segments (H5 4 and H6, respectively) is still debated (2, 4, 5). Several reports have indicated that the H5–H6 transmembrane region is key to cation binding and transport. Studies directed to locate residues involved in cation coordination during binding and transport have suggested that amino acids Asp804 and Asp808, located in H6, are essential for enzyme function as they appear to be cation-coordinating residues (6 – 8) (Fig. 1A). Close to these amino acids but in H5, Glu779 seems involved in determining the enzyme voltage dependence (9); however, there is controversy regarding its role either as a cation-coordinating residue or as necessary to stabilize the occluded-cation conformation 4 Abbreviations used: RD a1, sheep a1 carrying substitutions Gln111Arg and Asn122Asp; H5, fifth transmembrane segment; H6, sixth transmembrane segment.
0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
FUNCTIONAL ROLES OF Y771, T772, AND N776 IN THE Na,K-ATPase
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Asn122Asp to encode a form of the enzyme with low affinity for ouabain (RD a1) (14, 21, 22). Nucleotide substitutions were made to produce the following amino acid replacements: Tyr771Ala, Tyr771Ser, Tyr771Phe, Thr772Ala, Thr772Leu, Thr772Tyr, Asn776Ala, Asn776Gln, and Asn776Leu. Transfection and selection of ouabain-resistant HeLa cell lines expressing substituted RD a1 isoforms (22, 23), cell selection in high external KCl (14), isolation of crude plasma membranes (24, 25), protein determinations using bovine serum albumin as standard (26), quantification of the expressed enzyme using purified sheep Na,K-ATPase as standard and sheep a1-specific antibody M8-P1-A3 (14, 27), Na,K-ATPase and Na-ATPase activity determinations (9, 14), phosphorylation assays (9, 14, 28), and K 1 transport assays using 86Rb 1 as congener (15, 29) were all performed using previously described methods. FIG. 1. (A) Proposed topology of the H5-H6 domain. Primary structure positions of amino acids targeted in this study are indicated. (B) Helical wheel diagram of H5. Variable residues are circled. Primary structure positions of those oxygen-containing residues located in the “polar face” of the segment are indicated. Radial lines indicate suggested boundaries between hydrophobic and hydrophilic sides.
(8, 10 –13). We described that Ser775, also in H5, is likely part of the K 1-binding site, while it does not seem to participate in the binding of Na 1 (14, 15). Furthermore, the selective loss of the H5–H6 hairpin from the membrane following K 1 removal from a posttryptic preparation also supports the involvement of this region in cation coordination (16). The importance of the H5–H6 domain is also highlighted by the putative role it plays in ouabain binding and inactivation (16) and in the energy transduction between the cation and ATP-binding domains (9 –11, 17). The participation of amino acids in H5 and H6 of the sarcoplasmic reticulum Ca-ATPase in Ca 12 binding and transport is also well documented (18 –20). The analysis of helical wheel models of the putative H5 of the Na,K-ATPase a subunit (extending from Ser769 to Ala789) shows a clear polarity in the position of conserved– hydrophilic residues (Fig. 1B). In addition to residues that appear to play a central role in the enzyme function (Ser775, Glu779, and Phe786) (9, 14, 17), the “polar” phase contains three other well-conserved oxygen-containing residues, Tyr771, Thr772 and Asn776. Located close to the cytoplasmic end of H5 and among residues involved in cation coordination, these residues seemed logical targets for site-directed mutagenesis studies directed to understand structure– function relationships in the Na,K-ATPase. We describe in this report the functional characterization of Tyr771-, Thr772-, or Asn776-substituted enzymes. Our results indicate the putative role of Tyr771 in Na 1– enzyme interactions and the importance of the structural integrity of this region for the conformational transitions that occur in the catalytic phosphorylation domain upon cation binding. EXPERIMENTAL PROCEDURES Site-directed mutagenesis was performed as previously described using an a1 subunit cDNA modified by substitutions Gln111Arg and
RESULTS
When HeLa cells transfected with ouabain-resistant RD a1 isoforms are grown in tissue culture medium containing 1 mM ouabain, expression of functional exogenous enzyme enables cell growth. We have previously observed that substitutions Asn776Ala and Tyr771Ala yield enzymes that can not support HeLa cell growth (30). Various replacements at positions 771, 772, and 776 (Tyr771Ala, Tyr771Ser, Thr772Tyr, Asn776Ala, and Asn776Leu) were also unable to support cell growth under normal tissue culture conditions (5.4 mM KCl) (Fig. 2). This was considered a preliminary indication of the altered functionality of these enzymes. We have previously observed that increasing KCl concentration in the culture medium rescued cells transfected with some apparently nonfunctional substituted enzymes (14). This experimental maneuver was again successful allowing the survival of cells expressing Tyr771Ala-, Tyr771Ser-, Thr772Tyr-, and Asn776Ala-substituted enzymes. Cell clones expressing these substitutions were subsequently maintained at the external K 1 concentration at which they were selected. Asn776Leu-substituted enzyme was unable to support cell growth under any of the tested conditions. Characterization of Thr772- and Asn776-substituted enzymes. The variants that led to stable cell lines were further characterized. Table I shows the functional biochemical characterization of Thr772- and Asn776-substituted enzymes in terms of expression levels, Na,K-ATPase activity, Na-ATPase activity (activity in the absence of K 1), turnover number, and cation activation. Values for RD Control enzyme were similar to those obtained in previous studies (9, 14). Substitutions at position 776 led to reduced Na,KATPase activities and turnover numbers (21–27% of control enzymes). This was presumably compensated by a higher expression of the enzyme (Table I); i.e., it is apparent that only high-expressing clones were able to generate the necessary Na 1 and K 1 electrochemical gradients. The nonconservative replacement Asn776Ala leads to a high Na-ATPase activity (in the absence of K 1). This phenomena has been previously observed in
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¨ ELLO ET AL. ARGU
FIG. 2. Number of ouabain-resistant colonies resulting from the expression of RD Control and Try771-, Thr772-, and Asn776-substituted enzymes, after selection in growth medium containing different K 1 concentrations. Two days after transfection HeLa cells (5 3 10 5 cells/plate) were subjected to ouabain-selective pressure by inclusion of 1 mM ouabain in the Dulbecco’s modified Eagle’s (10% calf serum) culture medium. Selection was stopped after 2 weeks in cells transfected with RD Control cDNA and after 4 weeks in the case of cells transfected with mutated cDNA. Colonies were stained with Methylene Blue and counted. The values are means 6 SE of six independent experiments. External KCl concentration: 5.4 mM (white); 10 mM (pattern); 20 mM (gray); 5.4 mM 1 15 mM choline chloride (black).
enzymes carrying substitutions Glu779Ala (9, 12) and Asn324Leu (31). In the case of Asn776Ala-substituted enzymes the Na 1 dependence of this activity follows a pattern similar to that previously described for Glu779Ala-substituted enzymes but reaches a higher V max (83% of control compared to 60% for Glu779Ala and 32% for Asn324Leu) (Fig. 3). Replacement of Thr772 produced a significant decrease in Na,K-ATPase activity and turnover only in the case of Thr772Tyr; however, no major increase in expression level of this enzyme was detected (Table I). As in the case of Asn776, Glu779, and Asn324 substitutions, nonconservative changes of Thr772 (to leucine and tyrosine) led to a significant increase in the NaATPase activities. The Na 1 dependence of these activities again roughly followed an apparent single exponential kinetic (not shown). Thr772- and Asn776-substituted enzymes showed only small alterations in the cation activation of Na,KATPase activity. Table I shows the Na 1 K 1/2 and K 1 K 1/2 for enzyme activation together with the corresponding Hill coefficients obtained by fitting Na,K-ATPase activ-
ity vs cation concentration curves (not shown). K 1 interaction with the enzyme seemed unaffected, while the Na 1 apparent affinity was only slightly decreased (two times increase in K 1/2) by substitution of Asn776. These data suggest that these Thr772 and Asn776 are likely not directly involved in cation coordination. Na 1-activated phosphorylation by ATP of Thr772and Asn776-substituted enzymes was significantly reduced in those substitutions that have high Na-ATPase activity (Thr772Leu, Thr772Tyr, and Asn776Ala) (Table II). Previous studies have shown that substitution Glu779Ala leads to high Na-ATPase activity associated with low phosphoenzyme levels (9, 12). We observed that these alterations are due to fast dephosphorylation by Na 1 acting at extracellular sites with normal affinity (9, 32). Phosphorylation levels similar to RD Control enzyme have been detected in the Glu779Ala enzyme by inclusion of oligomycin in the phosphorylation medium (9, 12). This inhibitor prevents enzyme dephosphorylation by stabilizing the Na 1-bound forms of the enzyme (33, 3). As expected, Thr772Leu-, Thr772Tyr-, and Asn776Ala-substituted enzymes
257
FUNCTIONAL ROLES OF Y771, T772, AND N776 IN THE Na,K-ATPase TABLE I
Expression Level, Na,K-ATPase Activity, Na-ATPase Activity, Turnover No., and ATPase Cation Activation Parameters of RD Control and Thr772- and Asn776-Substituted Enzymes
Expression (mg/mg) Na,K-ATPase activity (mmol/min/mg) c Na-ATPase activity (% of total Na,K-ATPase) d Turnover No. (1/min) e Na 1 activation K 1/2 (mM) f n Hill K 1 activation K 1/2 (mM) n Hill a
RD Control
Asn776Ala
Asn776Gln
Thr772Ala
Thr772Leu
Thr772Tyr
34 6 8 6.81 6 1.75
110 6 40 1.76 6 0.28
72 6 2 1.53 6 0.26
37 6 7 4.80 6 2.17
28 6 7 6.65 6 1.04
44 6 6 1.98 6 0.53
17.8 6 4.3
83.2 6 6.1
18.3 6 9.1
20.0 6 1.5
71.3 6 1.5
63.3 6 7.0
9533 6 1481
2576 6 46
2013 6 362
5708 6 1505
6325 6 865
2709 6 532
6.61 6 0.43 2.28 6 0.16
13.99 6 1.5 1.39 6 0.16
14.25 6 0.89 2.04 6 0.21
4.70 6 0.64 1.89 6 0.47
4.34 6 0.67 1.70 6 0.51
6.26 6 0.83 1.40 6 0.27
0.43 6 0.06 1.29 6 0.09
0.30 6 0.05 1.24 6 0.13
0.42 6 0.03 1.15 6 0.10
0.38 6 0.08 1.08 6 0.06
0.61 6 0.18 1.00 6 0.07
0.84 6 0.31 0.69 6 0.07
b
a
Micrograms of heterologously expressed Na,K-ATPase per mg of total protein in the membrane preparation. Values are means 6 SE of n 5 4 independent clones. Each clone was assayed at least in duplicate. c Maximum activity as mmol of hydrolyzed ATP per min per mg of heterologously expressed Na,K-ATPase measured at 37°C in a medium containing 130 mM NaCl, 20 mM KCl, 50 mM choline-Cl, 3 mM MgCl 2, 3 mM ATP, 0.5 mM ethylene glycol bis(b-aminoethyl ether)-N,N9tetraacetic acid 50 mM imidazole, pH 7.2, 0.3 mg/ml bovine serum albumin, 1 mg/ml of membrane protein. The activity of heterologous protein was calculated as the difference in activity measured in presence of 10 mM and 10 mM ouabain (14). d Na-ATPase activity measured in presence of 200 mM NaCl, 0 mM KCl. e The turnover number was calculated independently for each preparation as the ratio of ATPase activity to the phosphoenzyme level (measured in the presence of 100 mg/ml oligomycin). f Na 1 and K 1 K 1/2 and Hill coefficients for enzyme activation were obtained by fitting Na,K-ATPase activity vs cation concentration curves to the equation: v 5 V maxL n/L n 1 K 1/2. b
showed phosphoenzyme levels comparable to RD Control when oligomycin was included in the phosphorylation media (Table II). Characterization of Tyr771-substituted enzymes. Replacement of Tyr771 has marked effects on the functionality of the Na,K-ATPase (Table III). Enzymes carrying substitutions Tyr771Ala and Tyr771Ser have very low Na,K-ATPase activity (13 and 18% of control enzyme, respectively). This low activity disturbed the analysis of these mutants; for instance, their NaATPase activity could not be evaluated with confidence. The replacement Tyr771Phe, maintaining the aromatic character in this location, reduced the Na,KATPase to a lesser extent (46% of control enzyme) and increased the Na-ATPase activity (twice the level of control). Because of uncertainty in their maximal phosphorylation level (see below), we were unable to calculate turnover numbers for these variants. The K 1 apparent affinity (measured as K 1/2 for Na,KATPase activation) was not affected by replacements Tyr771Ser or Tyr771Phe (Table III, Fig. 4). Low Na,KATPase activity prevented us from accurately measuring this parameter for Tyr771Ala enzyme. To assess the interaction of K 1 with this latter mutant and confirm our findings for Tyr771Ser or Tyr771Phe enzymes, the K 1 dependence of K 1 ( 86Rb 1) uptake was measured (curves not shown). These determinations also showed no alteration in the K 1 K 1/2 for enzyme activation (Table III). Therefore, it appears that re-
placement of Tyr771 did not affect K 1– enzyme interactions. Figure 5 shows that Tyr771Ala- and Tyr771Ser-substituted enzymes had altered Na 1 activation kinetics. The K 1/2 for Na 1 activation of these enzymes’ Na,KATPase activity was close to that of the RD Control. However, they presented a largely reduced n Hill, suggesting an alteration in the cooperative Na 1 binding to the enzyme (Table III). Tyr771Phe-substituted enzyme had a reduced apparent affinity for Na 1, but still showed close to normal cooperativity for Na 1 activation. Thus, these results suggest that Na 1 interaction with the enzyme was significantly affected by replacement of Tyr771. Try771 replacement also altered Na 1-activated phosphorylation by ATP. These enzymes showed reduced (9 –32% of control enzyme) steady-state phosphorylation levels (Table IV). Contrary to that observed with other substituted enzymes that show reduced phosphorylation (see Tables I and II), the presence of oligomycin in the phosphorylation medium did not increase Tyr771-substituted enzymes phosphorylation to levels similar to RD Control enzyme (Table IV). The maximum level of phosphorylated enzyme (in presence of oligomycin) is assumed to be equivalent to the number of functional enzymes and used to calculate the enzyme turnover numbers (7–9, 12–15). Thus, the low phosphoenzyme levels detected in Tyr771-sub-
¨ ELLO ET AL. ARGU
258
FIG. 3. Na 1 dependence of Na-ATPase activity of RD Control and Asn776Ala-substituted enzymes. Na-ATPase activity was determined at 37°C in medium containing 3 mM MgCl 2, 3 mM ATP, 0.5 mM ethylene glycol bis(b-aminoethyl ether)-N,N9-tetraacetic acid, 50 mM imidazole, pH 7.2, 0.3 mg/ml bovine serum albumin, approximately 1 mg/ml of membrane protein, 0.01 mM ouabain, and various concentrations of NaCl and choline chloride (total [monovalent cation] 5 200 mM). Background activity measured in the presence of 10 mM ouabain was subtracted. The Na-ATPase activity is expressed as percentage of the maximal Na,K-ATPase activity (Table I). The values are means 6 SE of results obtained with membrane preparations from four independent clones, each one measured in duplicate. RD enzyme (E); Asn776Ala substituted enzyme (F).
stituted enzymes led to problems in establishing the number of functional enzymes. Assuming the phosphoenzyme level is an accurate indication of this number, in the case of Tyr771 mutants, only a small pool of protein would be functional (10% of Tyr771Ala, 47% of Tyr771Ser, and 35% of Tyr771Phe, based on the percentages of phosphorylation with respect to the control). However, using these phosphorylation data to calculate turnover numbers, the following values were obtained: Tyr771Ala, 11,950 min 21; Tyr771Ser 3,361 min 21; and Tyr771Phe 13,551 min 21 (compared to RD
Control, 9533 min 21). Aside from Tyr771Ser, the values obtained for the Tyr771Ser and Tyr771Phe substitutions are incompatible with the concept that the larger fraction of these enzymes appears nonfunctional. Based on the implausibility of these figures, we did not considered the calculated turnover numbers an indication of the enzyme functionality. Different alterations in enzyme kinetic or ligand– enzyme interaction might explain the low phosphoenzyme levels. In an attempt to clarify this point we measured different characteristics of the Tyr771
TABLE II
Phosphorylation of RD Control and Thr772- and Asn776-Substituted Enzymes Phosphorylation (nmol/mg of heterologous enzyme) a
2 Oligomycin 1 Oligomycin
RD Control
Asn776Ala
Asn776Gln
Thr772Ala
Thr772Leu
Thr772Tyr
0.58 6 0.10 0.70 6 0.10
0.37 6 0.11 0.79 6 0.14
0.50 6 0.14 0.76 6 0.16
0.55 6 0.09 0.84 6 0.23
0.23 6 0.07 0.92 6 0.06
0.27 6 0.04 0.84 6 0.10
b
The Na-activated ATP phosphorylation was carried out at 0°C in a medium containing 1 mM MgCl 2, 5 mM [g- 32P]ATP, 0.04 mM ethylene glycol bis(b-aminoethyl ether)-N,N9-tetraacetic acid, 75 mM Hepes/imidazole, pH 7.2, 0.01 mM ouabain, and 0.15 mg/ml membrane protein, in the presence of either 50 mM NaCl or 50 mM NaCl plus 100 mg/ml oligomycin. Background activity measured in the presence of 50 mM KCl (0 mM Na 1) was subtracted. b The values are means 6 SE of results obtained with membrane preparations from three independent clones, each one measured in triplicate. a
259
FUNCTIONAL ROLES OF Y771, T772, AND N776 IN THE Na,K-ATPase TABLE III
Expression Level, Na,K-ATPase Activity, Na-ATPase Activity, Turnover No., and ATPase Cation Activation Parameters of RD Control and Tyr771-Substituted Enzymes
Expression (mg/mg) Na,K-ATPase activity (mmol/min/mg) Na-ATPase activity (% of total Na,K-ATPase) Na 1 activation K 1/2 (mM) n Hill 1 K activation (ATPase activity) K 1/2 (mM) n Hill K 1 activation (K 1 uptake) K 1/2 (mM) n Hill a
a
RD Control
Tyr771Ala
Tyr771Ser
Tyr771Phe
34 6 8 6.81 6 1.75
69 6 8 0.89 6 0.29
75 6 13 1.21 6 0.38
72 6 21 3.70 6 0.31
17.8 6 4.3
nd
nd
38.0 6 2.3
6.61 6 0.43 2.28 6 0.16
4.92 6 0.98 0.75 6 0.12
8.83 6 0.75 0.92 6 0.08
23.49 6 3.21 1.91 6 0.29
0.43 6 0.06 1.29 6 0.09
nd nd
0.70 6 0.12 0.81 6 0.09
0.57 6 0.02 1.39 6 0.25
0.91 6 0.17 1.14 6 0.13
1.09 6 0.23 1.13 6 0.13
1.36 6 0.22 1.38 6 0.14
0.98 6 0.18 1.06 6 0.20
Assay conditions for measurements described in this table are similar to those described in the notes to Table I. nd, not determined.
mutants phosphorylation by ATP. The ATP binding to the enzyme did not seem affected by Tyr771 replacement. The substitutions did not increase the ATP K m for the Na 1 -activated phosphorylation of the
FIG. 4. K 1 dependence of Na,K-ATPase activity of RD Control and Tyr771-substituted enzymes. The Na,K-ATPase activity was measured in the medium described in Table I, in the presence of 100 mM NaCl and various concentrations of KCl and choline chloride (total [monovalent cation] 5 200 mM). The Na,K-ATPase activities corresponding to 100% were similar to the values presented in Table III. The values are means of results obtained with membrane preparations from four independent clones, each one measured in duplicate. Standard errors were between 5 and 10% for Tyr771-substituted enzymes and lower than 5% for the RD Control; error bars were not plotted for simplicity. Curve-fitting parameters (K 1/2 and Hill coefficients) are listed in Table III. RD Control (E), Tyr771Ala (F), Tyr771Ser (Œ), Tyr771Phe (}).
enzyme; on the contrary, a small decrease of this K m was evident in the Tyr771Phe-substituted enzyme (Fig. 6A). Na 1 dependence of phosphorylation by ATP showed relatively small increases in Na 1 K 1/2 (Fig. 6B). It is known that Na 1 activates phosphorylation in a noncooperative fashion (34). Consequently the alterations in cooperativity detected in the Na 1 stimulation of the Na,K-ATPase (Fig. 5) were not expected in these determinations. Steadystate conditions were verified by measuring phosphorylation levels at 30, 60, and 120 s, and no differences were detected by extending the phosphorylation time (not shown). Low starting phosphoenzyme levels prevented us from measuring dephosphorylation rates. However, it is unlikely that the low steady-state phosphorylation would be associated with high dephosphorylation rates, since the Na 1 dependence of phosphorylation does not show the bell-shaped curve (reduction at high Na 1 ) observed in mutants with high dephosphorylation rates (9). Furthermore, these low phosphoenzyme levels are detectable even in the presence of oligomycin (Table IV). This observation is valid assuming that the inhibitor is able to bind to these enzymes, stabilize the Na 1 -bound forms, and consequently block the E1P 7 E2P conversion (33). To verify this point, we determined that oligomycin is able to fully inhibit the Na,K-ATPase activity of Tyr771-substituted enzymes with an IC 50 similar to that of RD Control (curves not shown) (IC 50 (mg/ml): RD Control, 0.42 6 0.05; Tyr771Ala 0.29 6 0.10; Tyr771Ser, 0.42 6 0.05; and Tyr771Phe, 0.19 6 0.05). Consequently, it is apparent that the Tyr771-substituted enzymes can bind the inhibitor. In an attempt to determine if the alterations in Na 1 stimulation of ATPase activity and the low
¨ ELLO ET AL. ARGU
260
FIG. 5. Na 1 dependence of Na,K-ATPase activity of RD Control and Tyr771-substituted enzymes. The Na,K-ATPase activity was measured in the medium described in the notes to Table I, in the presence of 20 mM KCl and various concentrations of NaCl and choline chloride (total [monovalent cation] 5 200 mM). The Na,K-ATPase activities corresponding to 100% were similar to the values presented in Table III. Curve-fitting parameters (K 1/2 and Hill coefficients) are listed in Table III. The values are means of results obtained with membrane preparations from four independent clones, each one measured in duplicate. Standard errors were between 5 and 10% for Tyr771-substituted enzymes and lower than 5% for the RD Control; error bars were not plotted for simplicity. RD Control (E), Tyr771Ala (F), Tyr771Ser (Œ), Tyr771Phe (}).
steady-state levels were associated with alterations in the equilibrium among different enzyme conformations the effect of vanadate on Tyr771 enzymes was tested. The concentration dependence of vanadate inhibition for Tyr771-substituted enzymes was similar to that of RD Control enzyme (curves not shown) (IC 50 (mM) were: RD Control, 0.81 6 0.09; Tyr771Ala 2.50 6 0.52; Tyr771Ser, 1.63 6 0.34; and Tyr771Phe, 1.21 6 0.16). This suggests that Tyr771substituted enzymes would not behave differently than the control in their tendency to remain in either E1 or E2 conformation.
DISCUSSION
Effects of replacement of amino acids Thr772 and Asn776. Replacement of residues at positions 772 and 776 led to significant reductions in enzyme activity. Expression of some of these substitutions was achieved by increasing the external [K 1]. We have hypothesized in the case of substitutions of Ser775 (which lead to a dramatic decrease in K 1 affinity) that the higher concentration of the cation helped cell survival by increasing enzyme turnover via saturation of the external cation-binding sites (14). Substitutions of
TABLE IV
Phosphorylation of RD Control and Tyr771-Substituted Enzymes Phosphorylation (nmol/mg of heterologous enzyme) a
2 Oligomycin 1 Oligomycin a
RD Control
Tyr771Ala
Tyr771Ser
Tyr771Phe
0.58 6 0.10 0.70 6 0.10
0.053 6 0.011 0.075 6 0.002
0.18 6 0.10 0.36 6 0.09
0.17 6 0.02 0.27 6 0.04
b
Assay conditions are similar to those described of Table II. The values are means 6 SE of results obtained with membrane preparations from three independent clones, each one measured in triplicate. b
FUNCTIONAL ROLES OF Y771, T772, AND N776 IN THE Na,K-ATPase
261
FIG. 6. Kinetic and ligand stimulation of phosphorylation by ATP of RD Control and Tyr771-substituted enzymes. In these sets of experiments Na-activated ATP phosphorylation was carried out in the absence of oligomycin in the medium described in the notes to Table II. [ATP] vs phosphorylation curves (A) were fit to a simple Michaelis kinetics, and ATP K m (mM) were as follows: RD Control, 1.12 6 0.25; Tyr771Ser, 1.06 6 0.20; and Tyr771Phe, 0.46 6 0.17. Na 1 dependence (B) was measured by variable [Na 1] while ionic strength was kept constant with choline chloride. [Na 1] vs phosphorylation curves (B) were fit to the equation v 5 V maxL n/L n 1 K 1/2. The Na 1 K 1/2 (mM) and Hill coefficients values were as follows: RD Control, 0.52 6 0.10, n 5 0.83; Tyr771Ala, 3.81 6 0.81, n 5 0.87; Tyr771Ser, 4.43 6 1.10, n 5 0.78; and Tyr771Phe, 2.28 6 0.17, n 5 0.86. The values are means of results obtained with membrane preparations from three independent clones, each one measured in triplicate. Standard errors were between 5 and 15% and error bars were not plotted for simplicity. RD Control (E), Tyr771Ala (F), Tyr771Ser (Œ), Tyr771Phe (}).
Thr772 and Asn776, as well as Tyr771, did not have significant effects on the binding of K 1 to the enzyme. Thus, another mechanism must explain the observed effect of extracellular [K 1]. Considering the dependence of the Na,K-ATPase on the membrane potential, a possible simple explanation is that the partial depolarization produced by increasing external [K 1] increased the activity of mutants with slow turnover leading to levels compatible with maintaining the required Na 1 and K 1 electrochemical gradients. Replacement of Thr772 or Asn776 had no major effects on the apparent affinities of Na 1 and K 1. Substitutions of Asn776 increased the Na 1 K 1/2 for Na,KATPase activation; however, the magnitude of this effect was small when compared to that observed when residues probably participating in cation coordination were replaced (Ser775, Asp804, and Asp808) (6, 14). Thus, these results suggest that Thr772 and Asn776 are not directly involved in the coordination of Na 1 or K 1. The most significant alteration resulting from replacing Thr772 and Asn776 was a large increase in the Na-ATPase activity. This was accompanied by a reduction in the steady-state phosphorylation levels that could be reversed by including oligomycin in the phosphorylation medium. Previous studies have reported similar findings after nonconservative replacement of amino acids Asn324 and Glu779 (6, 12, 31). It appears from studies of Glu779Ala-substituted enzymes that
these replacements increase the dephosphorylation rates upon binding of Na 1 to external cation-binding sites (9). It was later shown that the apparent affinity for external Na 1 is not modified by the Glu779Ala substitution (32). Thr772, Asn776, and Glu779 are located in the fifth transmembrane segments of the a subunit, in direct connection with the hydrolytic domain of the enzyme. The putative a helical structure of H5 suggests that these residues would be positioned in consecutive turns of the helix with similar orientation with respect to the helical backbone, likely interacting with the same adjacent transmembrane segment (Fig. 1). This hypothetical organization might explain the similar alterations observed upon nonconservative replacement of any of these amino acids. In contrast, replacement of Tyr771 or Ser775, located in the same region but with a different orientation with respect to the backbone of H5, does not lead to effects similar to those produced by substitutions of Thr772, Asn772, or Glu779. It has been proposed that H5 provides a way in which information about ATP binding and phosphorylation in the major cytoplasmic loop is transmitted to the intramembrane cation sites during the reaction cycle (9, 11, 16). The reverse information flux is also required by the enzyme mechanism, for instance, in the E 2P dephosphorylation upon K 1 binding. The functional alterations observed after Thr772 or Asn776 replacement support these ideas since they suggest
262
¨ ELLO ET AL. ARGU
that particular structural modifications in H5 lead to changes in phosphoenzyme stability upon external Na 1 binding. Effects of replacing Tyr771. Substitution of Tyr771 had significant effects on the enzyme kinetics and in the interaction of Na 1 with the enzyme. Although Na 1 apparent affinities did not decrease significantly (based on the K 1/2 for activation of ATPase activity or phosphorylation), the Na 1 dependence of the Na,KATPase was no longer cooperative in enzymes carrying Tyr771Ala and Tyr771Ser replacements. Na 1 activation of phosphorylation by ATP did not show this effect because it normally follows a single exponential dependence (34). The changes in cooperativity were not observed in Tyr771Phe-substituted enzyme, which would suggest that the aromatic group is the relevant characteristic at this position. The involvement of aromatic groups in cation–protein interactions has been previously proposed (35, 36). The alterations in the cooperativity of the Na 1 activation might be interpreted as a change in the affinities of the different Na 1 binding sites such that kinetically all appear equivalent. This effect might be due to a general conformational alteration produced by these substitutions. However, Tyr771 replacement did not appear to affect K 1 binding or the E1 7 E2 equilibrium. Therefore, the alterations in Na 1 binding would be a direct consequence of Tyr771 substitution. Significant reductions in the cooperativity of Na 1 interactions with the enzyme have been previously observed in Asp804 and Asp808 substituted enzymes. These aspartyl residues appear involved in the coordination of both Na 1 and K 1 ions (6). The second major effect of replacing Tyr771 is the low steady-state phosphorylation level observed in the modified enzymes. The presence of oligomycin in the medium, although it increased phosphorylation, did not lead to levels similar to control. A simple explanation for these results could be that in fact the substitutions yielded a smaller pool of functional enzyme that reach phosphoenzyme levels similar to the control. However, because this hypothesis would imply turnover numbers (for the functional pools) higher than that of control enzyme we consider it unlikely. These high turnover numbers would be incompatible with functionally altered enzymes. Oligomycin did not stimulate phosphorylation of Tyr771-substituted enzymes to levels similar to those of RD Control enzymes as in the case of Thr772, Asn776, or Glu779 substituted enzymes. This observation could be due to a decreased oligomycin binding; however, oligomycin was able to inhibit the substituted enzymes in a way similar to the controls. This suggests a normal interaction of the inhibitor with the enzymes. We also considered that replacement of Tyr771 might alter the phosphorylation rate. However, extended phosphorylation times did not lead to higher phosphor-
ylation levels. Furthermore, since replacement of Tyr771 apparently did not decrease the affinity for ATP and only slightly increased the Na 1 K 1/2 for activation, substrates were saturating in our phosphorylation determinations. Consequently, from these observations, one possible explanation of the observed low phosphorylation levels is a displacement of the E1Na 3ATP 7 E1P(3Na) 1 ADP equilibrium toward the dephosphorylated form of the enzyme. This would not be inconsistent with the lack of oligomycin effect on the phosphorylation level, since this drug seems to influence phosphorylation via stabilizing the Na 1bound forms of the enzyme (33). Furthermore, the small increase in ATP affinity detected in Tyr771Phe enzyme might be associated to alterations of this equilibrium. Finally, we should point out that although the importance of Tyr771 for reaction steps associated with Na 1 binding and occlusion is plausible, the relationship between the changes in cooperativity and the reduced phosphorylation is not clear at this moment. Substitution of Tyr773 of the sarcoplasmic reticulum Ca-ATPase (equivalent to Tyr771 in the Na,K-ATPase) for glycine produces the uncoupling of the ATP hydrolysis from Ca 21 transport (19). This interesting phenotype, although not observed in the Tyr763Thr mutant (20), also indicates a fundamental role of Tyr771 on the cytoplasmic side of H5 of P-type ATPases. Functional relevance of the fifth transmembrane segment. The importance of the polar face of H5 is evident when considering those residues that appear to play a central role in enzyme function. Located in H5, Ser775 participates in K 1 coordination (14, 15), Glu779 appears involved in determining the voltage dependence of the enzyme (9), and Phe786 is involved in ouabain binding (17). Furthermore, conformational changes of H5 during the catalytic cycle have been proposed (14, 16). Based on this information we have proposed a structural–functional model where the energy from ATP hydrolysis is transferred to the cationbinding site (formed in part by the H5–H6 hairpin) through a conformational change of H5. Ouabain binding to the extracellular half of the H5–H6 region probably blocks this conformational change, therefore inhibiting the enzyme (17). This study provides further evidence of the functional importance of the fifth transmembrane segment of the a subunit of Na,K-ATPase. The present information supports the concept that this region of the enzyme is involved in the transfer of information between the phosphorylation and cation binding domains. In addition, it points out the importance of Tyr771 as a required component for normal Na 1– enzyme interactions. ACKNOWLEDGMENTS We thank Dr. J. Feng for constructing the pKC4 vector carrying the Tyr771Ala, Thr772Ala, and Gln776Ala substitutions and Dr. J. Ball for supplying the antibody M8-P1-A3.
FUNCTIONAL ROLES OF Y771, T772, AND N776 IN THE Na,K-ATPase Note added in proof. During this manuscript review, a report was published by Pedersen et al. (Biochemistry 37, 1818 –1827, 1998). This paper also describes effects of substitutions of Tyr771, Thr772, and Asn776 in the pig a subunit expressed in yeast.
REFERENCES 1. Lingrel, J. B., and Kuntzweiler, T. (1994) J. Biol. Chem. 269, 19659 –19662. 2. Jørgensen, P. L. (1992) in Molecular Aspects of Transport Proteins (De Pont, J. J., Ed.), pp. 1–26, Elsevier Science Pub. B.V. 3. Horisberger, J-.D. (1994) The Na,K-ATPase: Structure–Function Relationship, Landes, Austin, TX. 4. Mohraz, M., Arystarkhova, E., and Sweadner, K. J. (1993) J. Biol. Chem. 269, 2929 –2936. 5. Canfield, V. A., Norbeck, L., and Levenson, R. (1996) Biochemistry 32, 13782–13786. 6. Kuntzweiler, T., Argu¨ello, J. M., and Lingrel, J. B. (1996) J. Biol. Chem. 271, 29682–29687. 7. Pedersen, P. A., Rasmussen, J. H., Nielsen, J. M., and Jørgensen, P. L. (1997) FEBS Lett. 400, 206 –210. 8. Nielsen, J. M., Pedersen, P. A., Karlish, S. J. D., and Jørgensen, P. L. (1998) Biochemistry 37, 1961–1968. 9. Argu¨ello, J. M., Peluffo, R. D., Feng, J., Lingrel, J. B., and Berlin, J. R. (1996) J. Biol. Chem. 271, 24610 –24616. 10. Argu¨ello, J. M., and Kaplan, J. H. (1991) J. Biol. Chem. 266, 14627–14635. 11. Argu¨ello, J. M., and Kaplan, J. H. (1994) J. Biol. Chem. 269, 6892– 6899. 12. Vilsen, B. (1995) Biochemistry 34, 1455–1463. 13. Koster, J. C., Blanco, G., Mills, P. B., and Mercer, R. W. (1996) J. Biol. Chem. 271, 2413–2421. 14. Argu¨ello, J. M., and Lingrel, J. B. (1995) J. Biol. Chem. 270, 22764 –22771. 15. Blostein, R., Wylczynska, A., Karlish, S. J. D., Argu¨ello, J. M., and Lingrel, J. B. (1997) J. Biol. Chem. 272, 24987–24993.
263
16. Lutsenko, S., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. USA 92, 7936 –7940. 17. Palasis, M., Kuntzweiler, T., Argu¨ello, J. M., and Lingrel, J. B. (1996) J. Biol. Chem. 271, 14176 –14183. 18. Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1990) Nature 339, 476 – 478. 19. Andersen, J. P. (1995) J. Biol. Chem. 270, 908 –914. 20. Rice, W. J., and MacLennan, D. H. (1996) J. Biol. Chem. 271, 31412–31419. 21. Kunkel, T. A. (1985) Proc. Nat. Acad. Sci. USA 82, 488 – 492. 22. Price, E. M., and Lingrel, J. B. (1988) Biochemistry 27, 8400 – 8408. 23. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745–2752. 24. Lane, L. K., Copenhaver, J. H., Jr., Lindenmayer, G. E., and Schwartz, A. (1973) J. Biol. Chem. 248, 7197–7200. 25. Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925–16930. 26. Bradford, M. M. (1976) Anal. Biochem. 72, 248 –254. 27. Malik, B., Jamieson, G. A., Jr., and Ball, W., Jr. (1993) Protein Sci. 2, 2103–2111. 28. Blostein, R. (1988) Methods Enzymol. 156, 171–178. 29. Munzer, J. S., Daly, S. E., Jewell-Motz, E. A., Lingrel, J. B., and Blostein, R. (1994) J. Biol. Chem. 269, 16668 –16676. 30. Feng, J., and Lingrel, J. B. (1994) Biochemistry 33, 4218 – 4224. 31. Vilsen, B. (1995) FEBS Lett. 363, 179 –183. 32. Peluffo, R. D., Argu¨ello, J. M., Lingrel, J. B., and Berlin, J. R. (1998) Biophys. J. 74, A171. 33. Arato-Oshima, T., Matsui, H., Wkizaka, A., and Homareda, H. (1996) J. Biol. Chem. 271, 25604 –25610. 34. Mardh, S., and Post, R. L. (1977) J. Biol. Chem. 252, 633– 638. 35. Heginbotham, L., and MacKinnon, R. (1992) Neuron 8, 483– 491. 36. Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69 –76.