Ligand-sensitive Interactions among the Transmembrane Helices of Na+/K+-ATPase*

(Received for publication, November 11, 1996, and in revised form, December 16, 1996)

Noune A. Sarvazyan , Alexander Ivanov , Nikolai N. Modyanov and Amir Askari

From the Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699-0008

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES


ABSTRACT

An extensively trypsin-digested Na+/K+-ATPase, which retains the ability to bind Na+, K+, and ouabain, consists of four fragments of the alpha -subunit that contain all 10 transmembrane alpha  domains, and the beta -subunit, a fraction of which is cleaved at Arg142-Gly143. In previous studies, we solubilized this preparation with a detergent and mapped the relative positions of several transmembrane helices of the subunits by chemical cross-linking. To determine if these detected helix-helix proximities were representative of those existing in the bilayer prior to solubilization, we have now done similar studies on the membrane-bound preparation of the same digested enzyme. After oxidative sulfhydryl cross-linking catalyzed by Cu2+-phenanthroline, two prominent products were identified by their mobilities and the analyses of their N termini. One was a dimer of a 11-kDa alpha -fragment containing the H1-H2 helices and a 22-kDa alpha -fragment containing the H7-H10 helices. This dimer seemed to be the same as that obtained in the solubilized preparation. The other product was a trimer of the above two alpha -fragments and that fraction of beta  whose extracellular domain was cleaved at Arg142-Gly143. This product was different from a similar one of the solubilized preparation in that the latter contained the predominant fraction of beta  without the extracellular cleavage. The cross-linking reactions of the membrane preparation, but not those of the solubilized one, were hindered specifically by Na+, K+, and ouabain. These findings indicate that (a) the H1-H2 transmembrane helices of alpha  are adjacent to some of its H7-H10 helices both in solubilized and membrane-bound states, (b) the alignment of the residues of the single transmembrane helix of beta  with the interacting H1-H2 and H7-H10 helices of alpha  is altered by detergent solubilization and by structural changes in the extracellular domain of beta , and (c) the three-dimensional packing of the interacting transmembrane helices of alpha  and beta  are regulated by the specific ligands of the enzyme.


INTRODUCTION

The coupled active transport of Na+ and K+ across the plasma membrane of most eucaryotic cells is carried out by Na+/K+-ATPase. The enzyme consists of two subunits, alpha  and beta , both of which are essential to normal function (1). That some transmembrane domains of the subunits must be involved in cation transport is self-evident, and specific roles of several such domains of the alpha -subunit in ion binding and transport have been explored (1-6). There is one transmembrane domain in the beta -subunit, and the 10-span model of alpha  is favored by most evidence (1).

To define the roles of the intramembrane domains in ion binding and transport, it is clearly necessary to go beyond two-dimensional schemes of folding and obtain information about the three-dimensional packing of the transmembrane domains of Na+/K+-ATPase. There is a paucity of such information, however, because it has been difficult to apply high resolution structural methods to hydrophobic helix-bundle proteins such as Na+/K+-ATPase. Molecular modeling studies have been helpful (7-9). Limited experimental data on the helix packing of Na+/K+-ATPase have resulted from our recent chemical cross-linking studies on enzyme preparations that were subjected to controlled proteolysis and solubilized in a detergent (10). These findings suggested contact between the intramembrane H1,H2 helix pair and H8-H10 intramembrane helices of alpha , and between these N-terminal and C-terminal helices of alpha  and the single intramembrane helix of beta  (10). The use of detergent-solubilized membrane proteins in chemical cross-linking studies is both necessary and problematic. On the one hand, solubilized preparations must be used to rule out the possibility of cross-linking due to random collisions of proteins and peptides that are highly concentrated within the membrane phase (11-13). On the other hand, once the existence of a stable noncovalent complex is established by the formation of a cross-linked product in a solubilized preparation, it is necessary to ensure that the detected protein-protein interaction is not entirely an artifact of solubilization (11). The purpose of the present experiments was to determine if the helix-helix proximities that we had detected in solubilized preparations (10) were indeed representatives of the interactions within the native membrane.


EXPERIMENTAL PROCEDURES

The purified membrane-bound Na+/K+-ATPase of canine kidney medulla, with specific activity of 1000-1600 µmol of ATP hydrolyzed/mg of protein/h, was prepared and assayed as described previously (14). The enzyme was exposed to trypsin (Sigma, Type III-S, bovine pancreas) in the presence of Rb+ and EDTA at pH 8.5 (10, 15) to remove large portions of the cytoplasmic domains of the alpha -subunit, and obtain a preparation containing an essentially intact beta -subunit and several fragments of the alpha -subunit (10, 16, 17). This preparation is often called the "19-kDa membranes" because its cation occlusion capacity was originally thought to be due to a C-terminal fragment of alpha with the apparent molecular mass of 19 kDa (15). It is now evident, however, that several, if not all, peptides of this preparation are involved in its functions (5, 16, 17). The capacity of this digested preparation to occlude Rb+ was verified before use by previously described procedures (15, 18).

For cross-linking, the digested enzyme was suspended (2 mg/ml) in a solution containing 10 mM Tris-HCl (pH 7.4), 0.25 mM CuSO4, and 1.25 mM o-phenanthroline, and incubated at 24 °C for 15 min. These conditions were chosen after preliminary experiments using the approach described previously (13). The reaction was terminated by the addition of EDTA to a final concentration of 30 mM, followed by SDS to a final concentration of 2%. Samples were subjected to electrophoresis on Tricine1/SDS-polyacrylamide gels (10). Unless indicated otherwise, sulfhydryl reagents were omitted from the buffers used for electrophoresis to preserve the cross-linked products. The stained peptide bands were quantified by a scanning densitometer (Bio-Rad). N-terminal analyses of the peptide bands were done as described previously (10).


RESULTS

Our previous conclusions regarding the intramembrane helix-helix interactions of Na+/K+-ATPase were based on the identification of cross-linked products obtained from enzyme preparations that were cleaved proteolytically, solubilized in digitonin, and oxidized in the presence of Cu2+ (10). The present experiments were initiated with the specific aims of determining (a) if the same or similar cross-linked products could be obtained in membrane-bound preparations that were not detergent-solubilized prior to cross-linking, and (b) if the functional specificities of the protein-protein interactions could be established by the sensitivities of the cross-linking reactions to ligands specific to Na+/K+-ATPase. Based on promising results of preliminary experiments, we focused attention on the cross-linking reactions of the membrane moiety of the trypsin-digested enzyme in the presence of Cu2+-phenanthroline complex.

The trypsin-digested preparation used here consists of four well characterized fragments of the alpha -subunit (10, 16, 19) that contain the 10 transmembrane helices of the alpha -subunits, and an essentially intact beta -subunit, a fraction of which is cleaved at Arg142-Gly143 (16, 19). The schematic structure of this preparation is shown in Fig. 1, and the resolution of its constituent peptides on Tricine/SDS gels is shown in Fig. 2 (lane 1).


Fig. 1. Schematic representation of the alpha -subunit fragments and an essentially intact beta -subunit in the extensively trypsin-digested Na+/K+-ATPase. The indicated N termini have been determined experimentally, but most of the indicated C termini of the fragments have been estimated based on apparent molecular masses (10, 16, 17). The circled cysteines are those most likely to be involved in cross-linking reactions of the transmembrane helices of alpha  and beta . See "Results" for other details.
[View Larger Version of this Image (33K GIF file)]



Fig. 2. Ligand-sensitive cross-linking reactions of the membrane-bound preparation of the trypsin-digested Na+/K+-ATPase in the presence of Cu2+-phenanthroline. The extensively digested preparation was prepared, cross-linked, and resolved on 10% Tricine gels as described under "Experimental Procedures." Lane 1, control prior to cross-linking. Lane 2, after cross-linking. The other lanes are samples that were cross-linked in the presence of 100 mM NaCl (lane 3), 5 mM KCl (lane 4), and 10 µM ouabain plus 7.5 mM MgCl2 (lane 5).
[View Larger Version of this Image (74K GIF file)]


When the membrane-bound preparation was first exposed to Cu2+-phenanthroline and then resolved on Tricine/SDS gels, several of the original bands were reduced in intensity, and several new bands appeared (Fig. 2, lane 2), indicating the cross-linkings of some peptides. The most prominent, and the most consistently observed, cross-linked products were the two designated as bands a and b in Fig. 2. These bands were excised from gels such as those of lane 2 of Fig. 2, electroeluted, and subjected to N-terminal analysis. Also analyzed as controls were the materials eluted from the positions corresponding to those of bands a and b from the gels on which the uncross-linked samples were resolved (Fig. 2, lane 1). These analyses (Table I), in conjunction with the apparent molecular mass of band a, showed that band a was the cross-linked dimer of the 22-kDa C-terminal fragment of alpha  containing the H7-H10 transmembrane helices (Fig. 1), and the 11-kDa N-terminal fragment of alpha  containing the H1,H2 helix pair (Fig. 1).

Table I.

N-terminal sequences of isolated cross-linked products

Products designated as a (11,12-dimer) and b (11,22,beta -trimer) in Fig. 2 (lane 2) were sequenced as indicated under "Experimental Procedures." The sequences were followed for 11 cycles and were identical with the indicated fragments of alpha - and beta -subunits of canine kidney Na,K-ATPase. Xaa, amino acid residues that were not identified upon sequencing and correspond to Trp11 and Cys148 of beta -subunit. The yield of each sequence was estimated from the average elevation of residues in cycles 2-11.
Cross-linked product Sequence Polypeptide chain region Yield

pmol
11,22  68Asp-Gly-Pro-Asn-Ala-Leu-Thr-Pro-Pro-Pro-Thr-  alpha -Subunit, H1-H2 19.8
831Asn-Pro-Lys-Thr-Asp-Lys-Leu-Val-Asn-Glu-Arg-  alpha -Subunit, H7-H10 18.9
11,22,beta  68Asp-Gly-Pro-Asn-Ala-Leu-Thr-Pro-Pro-Pro-Thr-  alpha -Subunit, H1-H2 6.0
831Asn-Pro-Lys-Thr-Asp-Lys-Leu-Val-Asn-Glu-Arg-  alpha -Subunit, H7-H10 6.5
  5Ala-Lys-Glu-Glu-Gly-Ser-Xaa-Lys-Lys-Phe-Ile-  beta -Subunit, N-terminal half 6.2
143Gly-Glu-Arg-Lys-Val-Xaa-Arg-Phe-Lys-Leu-Glu-  beta -Subunit, C-terminal half 5.6

Four sequences shown in Table I were identified upon N-terminal sequencing of band b, indicating that this cross-linked product contains the following four peptides in equimolar stoichiometry: the 11-kDa N-terminal fragment of alpha , the 22-kDa C-terminal fragment of alpha , a fragment of beta  beginning at Ala5, and a fragment of beta  beginning at Gly143. That the trypsin-digested enzyme contains beta  beginning at Ala5, and that a fraction of this truncated beta  is also cleaved at Arg142-Gly143 has been demonstrated (10, 16, 17, 19). Since the latter cleavage site (Fig. 1) is between Cys125 and Cys148, which are connected by a disulfide bridge (1), and since the bridge is expected to remain intact under oxidative cross-linking reaction conditions, we conclude that band b is the cross-linked trimer of 11-kDa and 22-kDa fragments of alpha -subunit and a beta -subunit that begins at Ala5, is cleaved at Arg142-Gly143, but is otherwise intact. Because we have not been able to detect beta  that is cross-linked either with the 11-kDa fragment alone or with the 22-kDa fragment alone, we suspect that formation of a disulfide bond between the two alpha -fragments stabilizes a conformation of the alpha -subunit helix-bundle, which is the mandatory first step to the subsequent direct contact of the Cys44 of beta  with an intramembrane cysteine of the two alpha -fragments.

The above identifications of 11-kDa and 22-kDa fragments of alpha  in the two most prominent cross-linked products are consistent with the reduced intensities of 11-kDa and 22-kDa bands after exposure to Cu2+-phenanthroline (Fig. 2, lanes 1 and 2). Closer examination of these gels shows, however, that other peptides also may have participated in cross-linking reactions. To date, we have been unable to characterize other discrete cross-linked products.

The specificities of the cross-linking reactions were tested in experiments, a representative of which are shown in Fig. 2. Formations of the cross-linked products, and the disappearances of 11-kDa and 22-kDa alpha  fragments were prevented by Na+ and K+ (Fig. 2, lanes 3 and 4), but not by the same concentrations of choline and N-methylglucamine (data not shown). Incubation in the presence of ouabain and Mg2+ also prevented cross-linking (Fig. 1, lane 5), but Mg2+ alone was not effective (data not shown). When the protective effects of different concentrations of Na+ and K+ were quantitated, K0.5 values were in the range of 10-20 mM for Na+ and less than 0.5 mM for K+ (Figs. 3 and 4). The relative values of these constants are in general agreement with the relative K0.5 values for occlusions of Na+ and Rb+ by this digested preparation (15), and clearly indicate the specificities of the cation effects on the cross-linking reactions. The concentration of ouabain for half-maximal protection of cross-linking was reduced when Mg2+ was present (Fig. 5), indicating that the well established interactive effects of ouabain and Mg2+ on the native enzyme (14) continue to exist within the peptide fragments of the digested enzyme.


Fig. 3. Protective effects of varying Na+ concentrations on the cross-linking reactions of the 22-kDa alpha -fragment of Na+/K+-ATPase. Experiments were done as in Fig. 2, and the stained bands were quantitated by densitometry.
[View Larger Version of this Image (17K GIF file)]



Fig. 4. Protective effects of varying K+ concentrations on the cross-linking reactions of the 22-kDa alpha -fragment of Na+/K+-ATPase. Experiments were done as in Fig. 2, and the stained bands were quantitated by densitometry.
[View Larger Version of this Image (18K GIF file)]



Fig. 5. Protective effects of varying ouabain concentrations on the cross-linking reactions of the 22-kDa alpha -fragment of Na+/K+-ATPase. Experiments were done as in Fig. 2 in the presence of ouabain alone or ouabain plus MgCl2 (inset). The stained bands were quantitated by densitometry.
[View Larger Version of this Image (16K GIF file)]


When the digitonin-solubilized preparation of the digested enzyme was cross-linked in the presence of Cu2+ as described previously (10) or in the presence of Cu2+-phenanthroline as in experiments of Fig. 2, no Na+, K+, or ouabain effects on the cross-linking reactions of the solubilized preparation were noted (data not shown).


DISCUSSION

The trypsin-digested preparation of the membrane-bound Na+/K+-ATPase used here (Fig. 1) has no ATPase activity, but retains the capacity to occlude Na+ and Rb+ and to carry out a slow Rb+/Rb+ exchange after reconstitution (15). When this preparation is solubilized in digitonin and oxidatively cross-linked, a major product is a dimer of the 11-kDa N-terminal fragment of alpha  and the 22-kDa C-terminal fragment of alpha  (10). We now show (Fig. 2 and Table I) that such a dimer is also a major cross-linked product of the membrane-bound version of this preparation. It has long been recognized that the demonstration of similar cross-linked complexes of proteins in native membranes and in solubilized preparations of the membranes is a good indication of the existence of specific protein associations within the native membranes (11, 13). The present findings and our previous data (10), therefore, establish the proximities of the N-terminal and C-terminal helices of alpha  in the functional membrane. Although the details of the contact domains remain to be mapped, as discussed previously (10), the estimated locations of the sulfhydryl groups of these helices (Fig. 1) suggest that the most likely interacting helices are the H1,H10 pair and the H2,H8 pair.

The other prominent cross-linked product of the membrane preparation identified here is a trimer of an essentially intact beta  and the above two fragments of alpha  (Fig. 2 and Table I). This is also similar to such a trimer that we obtained previously from the detergent-solubilized preparation (10). The two trimers, however, differ significantly in respect to the nature of beta  that participates in cross-linking. In the solubilized preparation the predominant reactant is the beta  that begins at Ala5, but is otherwise intact (10), while the predominant reactant in the membrane preparation is the same N-terminally truncated beta  that is also cleaved at Arg142-Gly143 between two half-cystines (Table I). Since under the conditions we have used to prepare the digested enzyme (i.e. at pH 8.5, the Arg142-Gly143 cleavage occurs in a small fraction of total beta  content (16)), we conclude that without this cleavage beta  does not cross-link with the alpha -fragments in the membrane-bound preparation. Evidently, some spatial flexibility of the beta -subunit chain, induced either by cleavage between the half-cystine residues or by detergent solubilization, is required for close juxtaposition of the single transmembrane cysteine of beta  with one of the nearby transmembrane cysteines of alpha  to allow the zero-length cross-linking of the two sulfhydryl groups. The five intramembrane sulfhydryl groups available for the formation of the two disulfides of the cross-linked trimer are Cys44 of beta  and cysteines 104, 138, 930, and 983 of alpha  (Fig. 1).

That the area of contact between the transmembrane helix of beta  and one or more transmembrane helices of alpha  is altered significantly by a cleavage in the extracellular domain of beta  is pertinent to a number of studies that have provided considerable information on the effects of various structural modifications of the extracellular domains and the transmembrane domain of beta  on the assembly of the alpha ,beta -complex (20-23), and on the functions of the enzyme (17, 21-24). We suggest that, in the interpretation of these findings, the often neglected possibility should be considered that a structural change of an extramembrane domain of beta  may be transmitted to its transmembrane helix, and then to the interacting helices of alpha .

A large number of studies on ligand-induced conformational transitions of Na+/K+-ATPase in relation to its function have been conducted over the years (25). Few of these, however, have addressed such structural transitions of the transmembrane domains of the enzyme. Experiments on hydrophobic labeling, followed by proteolysis, have suggested the existence of different Na+- and K+-dependent conformations of the transmembrane domains (8, 26); a fluorescent label attached to a specific transmembrane domain of the alpha -subunit of the intact enzyme has been shown to respond differently to various ligand-induced states (27); and structural changes of some transmembrane helices, either by chemical modification or by site-directed mutagenesis, have been shown to alter specific ligand interactions with the enzyme (2-6). To our knowledge, these and related previous studies, however, have not provided experimental evidence for ligand-induced interactions among the intramembrane domains. The sensitivities of the cross-linking reactions to Na+, K+, and ouabain demonstrated in the present studies (Figs. 2, 3, 4, 5) clearly indicate that the highly specific interactions of these ligands with the extensively digested enzyme regulate the three-dimensional packing of the transmembrane helices. Specifically, our data show that the conformational states induced by the indicated ligands affect the alignments of the residues of H1,H2 helix pair relative to those of the H8-H10 helices, and the proximities of these helices with the residues of one transmembrane helix of beta . This does not exclude the involvement of other transmembrane domains in ligand-induced conformational changes of the helix-bundle. The use of cysteine-scanning mutagenesis, other cross-linking reagents, and other functionally competent fragments of the enzyme may reveal additional ligand-sensitive transmembrane helix-helix interactions. Such studies are in progress.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant HL-36573 from the NHLBI, United States Public Health Service, Department of Health and Human Services.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: Dept. of Pharmacology, Medical College of Ohio, P. O. Box 10008, Toledo, OH 43699-0008. Tel.: 419-381-4182; Fax: 419-381-2871.
1   The abbreviation used is: Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

Acknowledgment

We thank Dr. J. Pohl, Emory University Microsequencing Facility, for amino acid analysis.


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