MINIREVIEW The sodium pump Its molecular properties and mechanics of ion transport Georgios Scheiner-Bobis From the Institut fu ¨ r Biochemie und Endokrinologie, Fachbereich Veterina ¨ rmedizin, Justus-Liebig-Universita ¨ t Giessen, Germany The sodium pump (Na + /K + -ATPase; sodium- and potas- sium-activated adenosine 5¢-triphosphatase; EC 3.6.1.37) has been under investigation for more than four decades. During this time, the knowledge about the structure and properties of the enzyme has increased to such an extent that specialized groups have formed within this field that focus on specific aspects of the active ion transport catalyzed by this enzyme. Taking this into account, this review, while some- what speculative, is an attempt to summarize the informa- tion regarding the enzymology of the sodium pump with the hope of providing to interested readers from outside the field a concentrated overview and to readers from related fields a guide in their search for gathering specific information concerning the structure, function, and enzymology of this enzyme. Keywords: ATPase; P-type; ouabain; palytoxin; ion transport. THE SODIUM PUMP: A BRIEF RETROSPECTIVE Today there is a vast amount of information concerning ion transport through biological membranes and primary structures, crystals, mutants, and chimeras of ion trans- porters. It is difficult to imagine that the impressive progress achieved thus far was originally generated by a few researchers who had the ability to observe simple phenom- ena connected with ion distribution, to question their origin, and to assemble experimental evidence in ways that did not allow any other conclusion but that there must a mechanism that enables ions to be actively transported against their electrochemical gradients. This mechanism, termed a Ôsodium pumpÕ by Dean in 1941, originates from the observation that sodium ions within muscle fibers can exchange with radioactive sodium added to their environ- ment. Nevertheless, although a large amount of data and interpretation of it followed Dean’s proposal, it was not until 1954 that Gardos discovered that ion pumping in red blood cell ghosts was supported by ATP, which in turn became hydrolyzed. (Due to space limitations, some of the early, seminal work is not included in the reference list; instead, an up-to-date selection of papers from a variety of groups from which both the current progress in the field can be assessed and in which earlier, landmark discoveries are fully referenced is provided.) These observations, together with the finding that 18 sodium ions were transported for each molecule of oxygen consumed (4.5 Na + per electron or, in other words, 3 Na + per ATP) and the fact that ouabain had already been shown to inhibit sodium fluxes on frog skin, contributed to the overall acceptance of Skou’s conclusion from 1957, which identified in crab nerve membrane preparations the sodium pump as an ATPase that was activated by Na + and K + and inhibited by ouabain [1]. Undoubtedly, however, all of these findings helped to lay the cornerstone in the research field of ion transport, which currently includes a vast number of primarily and secondarily active transporters or ion channels. Among them, the Na + /K + -ATPase takes its place within the family of the so-called P-type ATPases, enzymes that become autophosphorylated by the gamma phosphate group of the ATP molecule that they hydrolyze. The Na + / K + -ATPase was the first discovered ion transporter, and indeed the first-discovered P-type ATPase. It is still, however, not well understood; after many years of investigation, the sodium pump is still at the center of researchers’ attention. Na + /K + -ATPASE: SUBUNIT COMPOSITION Every living cell is negatively charged in comparison with its environment. Thus, in principle, the cell/environment pair constitutes a battery. Just as a battery can be used to perform work, a cell uses this electrochemical gradient to obtain nutrients, ionic or nonionic, from its environment and to extrude metabolites and ions from its interior. In this fashion, the composition of the intracellular milieu remains constant while allowing for adaptation to a changing environment to occur. Correspondence to G. Scheiner-Bobis, Institut fu ¨ r Biochemie und Endokrinologie, Fachbereich Veterina ¨ rmedizin, Justus-Liebig-Universita ¨ t Giessen, Frankfurter Str. 100, D-35392 Giessen, Germany. Fax: + 49 641 9938189, Tel.: + 49 641 9938180, E-mail: Scheiner-Bobis@vetmed.uni-giessen.de Abbreviations:Na + /K + -ATPase, sodium- and potassium-activated adenosine 5¢-triphosphatase; FSBA, 5¢-p-fluorosulfonylbenzoyl- adenosine; ClR-ATP, c-[4-(N-2-chloroethyl-N-methylamino)]benzyl- amide ATP; FITC, 5¢-isothiocyanate. Enzyme: sodium- and potassium-activated adenosine 5¢-triphosphatase (EC 3.6.1.37). (Received 15 October 2001, revised 11 December 2001, accepted 28 January 2002) Eur. J. Biochem. 269, 2424–2433 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02909.x The sodium pump, also known as the Na + /K + -ATPase, is responsible for establishing and maintaining this electro- chemical gradient in animal cells. This enzyme is a component of the plasma membrane and transports Na + and K + using ATP hydrolysis. For every molecule of ATP hydrolyzed, three Na + ions from the intracellular space and two K + ions from the external medium are exchanged. Thus, the sodium pump contributes substantially to the maintenance of the membrane potential of the cell, provides the basis for neuronal communication, and contributes to the osmotic regulation of the cell volume. In addition, the electrochemical Na + gradient is the driving force behind secondary transport systems. The Na + /K + -ATPase belongs to the P-type ATPases, a family of enzymes that become phosphorylated during transport by the c-phosphate group of ATP at an aspartic acid localized within the highly conserved sequence DKTGS/T [2]. This family, which contains more than 50 members, includes membrane-bound enzymes responsible for the transport of heavy metal ions (P 1 -type ATPases), other metal ions (P 2 -type ATPases), and the K + -selective Kdp-ATPase of Escherichia coli (P 3 -type ATPase). Within the group of the P 2 -type ATPases, the Na + / K + -ATPase, together with the colonic or gastric H + / K + -ATPases, constitute a subgroup of oligomeric enzymes consisting of a and b subunits. A third peptide referred to as the c subunit appears in some tissues to be involved in regulating the activity of the sodium pump and its interactions with Na + or K + ions. A number of isoforms of the a and b subunits has been isolated from various tissues of numerous species, and it has been repeatedly demonstrated that the function of Na + / K + -ATPase requires the presence of both subunits. The a subunit, which is referred to as the catalytic subunit, has a relative molecular mass of 100–113 kDa, depending on the presence of different isoforms: a1, a2, a3, or a4. It crosses the membrane 10 times, forming trans- membrane domains M1 to M10; both N- and C-termini are localized on the cytosolic side [3]. Various studies have shown that both ATP binding and ion occlusion occurs in this subunit. The b subunit is highly glycosylated and has a relative molecular mass of about 60 kDa. The mass of the protein moiety of this subunit is 36–38 kDa, depending on the isoforms b1, b2, or b3. The bsubunit crosses the membrane only once, and the N-terminus is localized on the intracel- lular side of the membrane. The respective roles of these proteins is still not entirely clear. More recent results have shown that the b subunit makes direct contact with the a subunit [4], thereby stabilizing the a subunit and assisting in its transport from the endoplasmic reticulum to the plasma membrane [5]. In addition, numerous experiments have shown that the b subunit is important for ATP hydrolysis, ion transport, and the binding of inhibitors such as ouabain. The third subunit of Na + /K + -ATPase, the csubunit of 7–11 kDa, was first identified as a component involved in the binding of [ 3 H]ouabain. The c subunit specifically associates with the sodium pump [6], possibly via interac- tions with the C-terminal domain of the a subunit [7]. The c subunit belongs to type I membrane proteins and is related to phospholemman and to the human Mat8 protein, a type I membrane protein associated with mammary tumors. The availability of the cDNA coding for the peptide permitted analysis of the role of the c subunit in the function of the enzyme. Consistent with the fact that c expression is not seen in all tissues where a or b expression is otherwise easily identified, the presence of the c peptide is not essential for obtaining Na + / K + -ATPase activity in heterologous expressions systems of the enzyme [8]. Nevertheless, c subunit expression in HEK cells apparently modifies the affinity of the enzyme for ATP, and its expression in different segments of the nephron is associated with modulation of the affinity of Na + /K + -ATPase for Na + or K + ions [9,10]. These data, together with the fact that several peptides similar to the c subunit have already been determined to interact with and influence the sodium pump [11] confirm that the ion pumping activity can be finely modulated by type I membrane peptides and also offers the possibility of addressing physiologically relevant questions in connec- tion with the regulation of the expression of this type of protein. THE CATALYTIC MECHANISM OF THE Na + /K + -ATPASE The Na + /K + -ATPase has two conformational states, E 1 and E 2 . These states are not only characterized by differ- ences in their interactions with Na + ,K + , ATP, or ouabain, they also have been clearly defined by tryptic cleavage experiments. In the first step of the reaction sequence, Na + and ATP bind with very high affinity (K d values of 0.19–0.26 m M and 0.1–0.2 l M , respectively) to the E 1 conformation of the enzyme (Fig. 1, step 1), during which phosphorylation at an aspartate residue occurs via the transfer of the c-phosphate of ATP (Fig. 1, step 2) [12,13]. Magnesium is very important for this reaction. Thereafter, three Na + ions are occluded while the enzyme remains in a phosphorylated condition. After the E 2 -P3Na + conformation is attained, the enzyme loses its affinity for Na + (K 0.5 ¼ 14 m M )and the affinity for K + is increased (K d  0.1 m M ). Thus, three Na + ions are released to the extracellular medium (Fig. 1, step 3) and K + ions are taken up (Fig. 1, step 4). The binding of K + to the enzyme induces a spontaneous dephosphorylation of the E 2 -P conformation. The dephosphorylation of E 2 -P leads to the occlusion of two K + ions, leading to E 2 (2K + ) (Fig. 1, step 5) [12,13]. Intracellular ATP increases the extent of the release of K + from the E 2 (2K + ) conformation (Fig. 1, step 6) and thereby also the return of the E 2 (2K + ) conformation to the E 1 ATPNa conformation. The affinity of the E 2 (2K + ) conformation for ATP, with a K 0.5 value of 0.45 m M ,is very low [12,13]. Through the juxtapositioning of these three reaction sequences, the full catalytic cycle of Na + /K + -ATPase is obtained (Fig. 1). All P-type ATPases function in a similar way: they all hydrolyze ATP and occlude ions during the translocation process within the membrane-inserted segment of the protein. Through this process, the ionophore of every ion-transporting ATPase is accessible from only one side of themembraneatanygiventime. The sequential model presented above, however, often referred to as the Albers–Post scheme [13] does not take into Ó FEBS 2002 Sodium pump structure and properties (Eur. J. Biochem. 269) 2425 consideration that the sodium pump might exist as a diprotomer of cooperating (ab) 2 subunits and thus contain two binding sites for ATP. The concentration–effect curve for ATP hydrolysis is biphasic, which can be explained by an extrapolation of the single-site model shown in Fig. 1. Each ab protomer has a single ATP binding site that changes from high affinity to low affinity with changes in conformation. This model is strongly supported by experiments showing that the stoichiometry of binding for either ATP, phosphate, or ouabain is 1 per a subunit, and that solubilized enzyme retains its catalytic activity [14]. Results obtained with highly purified enzyme from duck salt gland lend credence to this hypothesis [15]. A second model, which was originally put forward by Repke, postulates that the biphasic nature of the ATP concentration curve is due to the presence of two catalytic a subunits that work cooperatively [16]. Each catalytic subunit goes through the same conformational changes that are described in the single-site model but in such a way that they are shifted 180° from each other. Thus, in this model the high affinity and low affinity ATP binding sites occur simultaneously, and there is also simultaneous transportofNa + out of the cell and K + into the cell. Several experimental results support this model. In a third model proposed by Plesner, the cooperativity of the a subunits described by Repke occurs only in the presence of Na + and K + [17]. The partial reactions of the Na + /K + -ATPase are catalyzed by the ab protomeric enzyme, as is the case with Na + -ATPase or K + -stimulated phosphatase. The models of Repke and Plesner differ from the single- site model in that they predict the presence of two binding sites on each functional enzyme entity. The results of many investigations support the existence of two binding sites on one (ab) 2 diprotomer. Kinetic studies have shown that the single-site model is not sufficient to explain the coupling of ATP hydrolysis to ion transport [18]. Moreover, crystallo- graphic studies have demonstrated that Na + /K + -ATPase crystallizes in a way that allows ab protomers to be in close contact with each other [19]. Finally, radiation inactivation has shown in several cases that the target size is consistent with that of an (ab) 2 diprotomeric structure. These data, however, are not compelling proof of the simultaneous existence of two ATP binding sites and therefore do not definitively establish the (ab) 2 diprotomer as the basic functional unit of Na + /K + -ATPase. Alternative proposals suggest the existence of (ab) 4 tetrameric enzymes [20] or enzymes with two ATP binding sites per a subunit [21]. THE K + -STIMULATED PHOSPHATASE ACTIVITY A special characteristic of the Na + /K + -ATPase is its ability to hydrolyze phosphoesters and phosphoanhydrides in the presence of K + ions [22]. This so-called K + -stimulated phosphatase activity is ouabain-sensitive. The physiological relevance of this reaction is unknown. Fig. 1. Reaction cycle of Na + /K + -ATPase. Na + /K + -ATPase binds Na + and ATP in the E 1 conformational state (step 1) and is phosphorylated at an aspartate residue by the c-phosphate of ATP. This leads to the occlusion of three Na + ions (step 2) and then to their release to the extracellular side (step 3). This new conformational state (E 2 -P) binds K + with high affinity (step 4). Binding of K + leads to dephosphorylation of the enzyme andtotheocclusionoftwoK + cations (step 5). K + is then released to the cytosol after ATP binds to the enzyme with low affinity (step 6). The dashed box highlights the electrogenic steps of the catalytic cycle. 2426 G. Scheiner-Bobis (Eur. J. Biochem. 269) Ó FEBS 2002 THE ATP BINDING DOMAIN The cytosolic protein structure between membrane domains M4 and M5 (L4/5) is of great importance for the function of the enzyme, because a series of amino acids within this region have been identified to be either essential for or highly involved in ATP hydrolysis and enzyme function. (The prefix L stands for loop, a transmembrane domain- connecting peptide. L2/3, L4/5, L6/7, and L8/9 are localized on the cytosolic side, and L1/2, L3/4, L5/6, L7/8, and L 9/10 are accessible from the extracellular side.) First, the ATP phosphorylation site is localized within this loop as a part of the sequence DKTGT/S that is highly conserved among all P-type ATPases. In addition, all ATP analogs used thus far label peptide structures within this loop, and the recently published Ca 2+ -ATPase crystal structure was shown to contain TNP-AMP bound within this L4/5 peptide. Therefore, it is justified to refer to this part of the enzyme as the ATP binding domain. By using the protein-reactive ATP analogs 2-azido-ATP and 8-azido-ATP, it was possible to label and identify Gly502 and Lys480, respectively, as possible recognition sites for the adenosine moiety of ATP [23,24]. (Hereafter, the amino-acid sequence numbers refer to that of the a1 isoform of the sheep.) The fact that Lys480 is also labeled by both pyridoxal 5¢-diphospho-5¢-adenosine and pyridoxal 5¢-phosphate suggests that this amino acid might be involved additionally in the recognition of phosphate groups, as proposed by Hinz & Kirley [25]. Thus, in this point of view, the labeling of Lys480 by 8-azido-ATP [23] does not necessarily indicate that this amino acid directly interacts with the adenine moiety of the ATP molecule, but that it is merely within reach of the highly reactive azido group of 8-azido-ATP. In the crystal structure of the Ca 2+ -ATPase, Lys492, the equivalent of Lys480 of the sodium pump a1 subunit, seems to interact with the phosphate group of TNP-AMP [26]. Site-directed muta- genesis experiments have confirmed the importance of Lys480 for ATP hydrolysis and enzyme function [27]. Various other ATP analogs such as 5¢-p-fluoro- sulfonylbenzoyl-adenosine (FSBA) or c-[4-(N-2-chloro- ethyl-N-methylamino)]benzylamide ATP (ClR-ATP) were successfully used for identifying amino acids within the L4/5 peptide. Nevertheless, although these substances resemble nucleotide triphosphates and their interaction with the enzyme can be prevented by ATP, they are not substrates of the sodium pump. Thus, it was still uncertain whether Cys656 and Lys719, the FSBA labeling sites [28], and Asp710, the ClR-ATP labeling site [29], were truly constituents of the ATP binding site. In contrast to these ATP-like substances, fluorescein 5¢-isothiocyanate (FITC), a protein-reactive probe, was shown to modify Lys501 of the sodium pump a1 subunit [30]. Although there is no apparent similarity between FITC and ATP, the fact that ATP prevents modification of Lys501 by FITC led to the conclusion that Lys501 is localized within the adenosine- recognizing moiety of the a1 subunit. This proposal has been supported by findings concerning the conformation of Mg 2+ -complexed ATP analyzed by 1 H-NMR and ultra- violet spectrophotometric methods. According to these reports, the a-phosphate group of the ATP molecule is in close proximity to the C8 atom of the adenine moiety. Therefore, if ATP is assumed to retain a similar conformation when bound within the ATP binding site, one can imagine that the C8-azido group of 8-azido-ATP labels Lys480, which originally interacts with the a-phosphate group of ATP. Taking into account that the distance between Lys501 and Lys480, as determined by labeling experiments with dihydro-4,4¢-diisothiocyanostilbene-2,2¢- disulfonate, is approximately 1.4 nm [31], it is conceivable that the azido group of 8-azido-ATP labels Lys480 while the azido group of 2-azido-ATP labels Gly502. The recently resolved crystal structure of Ca 2+ -ATPase demonstrates that all ATP analogs used so far label functional areas of the a subunit. The azido derivatives of ATP, pyridoxal 5¢-diphospho-5¢-adenosine and pyridoxal 5¢-phosphate, or FITC label near the adenosine binding pocket, as demonstrated for the binding of TNP-AMP within the crystal structure of Ca 2+ -ATPase. This area is referred to as the N (nucleotide binding) domain of the L4/5 peptide. Other ATP analogs such as FSBA or ClR-ATP label the enzyme in the vicinity of the phosphorylation site, within a substructure of the L4/5 peptide referred to as the P (phosphorylation) domain. This area of the protein, consti- tuting a Rossman fold, was first identified as being conserved among various hydrolases by comparison of the primary sequences of P-type ATPases with the primary sequence of the L -2-haloacid dehalogenase from Pseudo- monas sp. and was thought to directly participate in the phosphorylation/dephosphorylation of Asp369 via the terminal phosphate of ATP. More recent studies, however, have suggested that this area of the protein is a Mg 2+ binding site [32]. The distance between the adenosine binding area of the N domain and the phosphorylation site in the P domain is rather large (2.5 nm) to be bridged by the ATP molecule. Thus, some conformational transition must occur prior to ATP hydrolysis, which results in the two domains approaching each other. A third subdomain formed by the L2/3 peptide might be involved in these conforma- tional changes. This area of the protein is referred to as the actuator domain (A domain). No functional analysis has yet been published, however, that supports this proposal. Nevertheless, the A domain undoubtedly contributes to the conformational transitions associated with ATP hydrolysis, ion transport, and dephosphorylation of the phosphoenzyme formed by the transfer of the c phosphate group of ATP. In experiments involving ascorbate/ H 2 O 2 -catalyzed peptide cleavage in the presence of ATP-Fe 2+ , it was demonstrated that the peptide TGESE(212–216) from the A domain moves towards the phosphorylation site in the P domain, supporting the dephosphorylation of the enzyme during the E 2 -P fi E 2 (K + )-transition [33]. Because this peptide (TGES/A) is highly conserved among all known P-type ATPases, transport catalyzed by these other enzymes is likely to take place by similar mechanisms. MEMBRANE-SPANNING DOMAINS AND THEIR INVOLVEMENT IN THE CATION TRANSLOCATION PROCESS Investigations using isolated Na + /K + -ATPase have shown that after tryptic removal of the hydrophilic part of the enzyme, the remaining C-terminal, membrane-spanning segment (so-called Ô19-kDa membranesÕ) is still able to Ó FEBS 2002 Sodium pump structure and properties (Eur. J. Biochem. 269) 2427 occlude Na + or the K + analog Rb + [34], indicating that the ionophore, as expected, must consist of membrane- spanning domains. Negatively charged amino acids within this structure are viewed as possible interfaces between the protein and ions being transported. However, analysis of mutants has not always demonstrated that substitution of acidic amino acids within the membrane-spanning domains has a marked effect on enzyme activity. Substitution at Glu327 (within the fourth membrane-spanning domain, denoted M4), Asp926 (M8), Glu953, or Glu954 (both M9) does not lead to significant changes in the affinity of the mutant enzyme for Na + or K + or affect its electrical properties [35–37]. Mutation of Glu953 or Glu954 also has no effect on the interaction of the enzyme with palytoxin (G. Scheiner-Bobis, unpublished observations). Mutation of Glu779 from the sixth membrane-spanning domain has a number of effects, depending on the nature of the substitution. A Glu779Ala mutant has an ATPase activity that is independent of K + (a Na + -ATPase) [38]; here, it may be that Na + mimics the binding of K + at extracellular sites. Nevertheless, mutation of this Glu779 to Gln, Asp, or Lys leads to only moderate changes in the K 0.5 for the cation activation of Na + /K + -ATPase. For this reason, and because the Glu779fiLys mutants have a slightly higher affinity for Na + , a direct role for Glu779 in the cation binding process is fairly unlikely. Rather, it may be assumed that Glu779 is a part of the overall structure that participates in the formation of an ion coordination complex involved in cation selectivity and activation of the sodium pump. Of all acidic amino acids examined thus far, only nonconservative mutation of Asp804 and Asp808 leads to a nonfunctional enzyme. It is possible that these mutations have a deleterious effect on K + recognition at the extracellular face of the enzyme [39]. The interaction with the conservative mutation Asp808fiGlu. The conclusion drawn from these studies is that Asp804 and Asp808 from the sixth membrane-spanning domain of the a1 subunit are involved in cation coordination [39]. The data reported thus far, however, give the impression that the mutations have an effect only on K + and not on Na + recognition. The examination of various acidic residues from the transmembrane domains of the sodium pump has not brought us closer to the goal of identifying amino acids that are essential for ion transport. In general, it would appear as if it weren’t the individual negatively charged amino acids of the membrane-spanning domains that were directly involved in ion transfer, but larger peptide structures that contain these amino acids. This conclusion, as unsatisfac- tory as it may be, agrees well with investigations of a considerable number of mutants of the Ca 2+ -ATPase that clearly demonstrate that numerous amino acids within the transmembrane domains M4, M5, M6, and M8 are important for the function of the enzyme, independent of whether they are charged or not [40]. If no single acidic residue from the transmembrane domains is essential for ion transport, then which structures are important? It is known that cations are transferred along the backbone of carbonyl groups by ion/dipole interactions from studies of the ionophores valinomycin and gramicidin [40a]. This general preference for ion/dipole instead of ion/ ion interactions has also been noted for soluble enzymes that bind monovalent cations. Should ion translocation by the sodium pump also occur by ion/dipole interactions, one would assume that cations interact with carbonyl or hydroxyl groups and not just with carboxyl groups. In analogy to the Ca 2+ -ATPase, these amino acids would be in the membrane-spanning domains M4, M5, M6, and M8 of the a subunit of the sodium pump. In fact, the crystal structure of Ca 2+ -ATPase, which was recently reported with a resolution of 2.6 A ˆ , shows two binding sites for Ca 2+ within the transmembrane region (Fig. 2). One calcium ion is bound within a pocket formed by Asn768 and Glu771 (M5), Thr799 and Asp800 (M6), and Glu908 (M8) [26]. These results agree well with previous conclusions drawn from mutation experiments [40]. A second Ca 2+ binds via interaction with the carbonyl groups of Val304, Ala305, and Ile307 (M4) and through the side-chain oxygen atoms of Asn796 and Asp800 (M6) and Glu309 (M4) [26]. A similar situation could be assumed for the coordination of cations within the membrane-spanning domains of the Na + /K + -ATPase, because several structures are, as dem- onstrated in extensive and thorough theoretical work, very similar to those of the Ca 2+ -ATPase. This constellation would also explain why single mutations within this region do not lead to a complete loss of transport, because the cations are coordinated simultaneously by several amino acids. If this is the case, then only the mutation of several amino acids concomitantly would lead to a marked change in ion transport properties. Fig. 2. Cation coordination sites of Ca 2+ -ATPase. The view is a cross- section of the protein from the lumen of the sarcoplasmic reticulum. Areas of the protein not involved in Ca 2+ coordination have been eliminated. Two Ca 2+ ions shown in green are coordinated by Val304, Ala305, Ile307 and Glu309 (M4), Asn768 and Glu771 (M5), Thr799 and Asp800 (M6), and Glu908 (M8). The side chain carboxyl group of Asp800 participates in the coordination of both Ca 2+ ions. The cor- responding amino acids of the sodium pump a1 subunit of the sheep are given in parentheses. Atoms of interest: oxygen, red; nitrogen, blue; calcium, green. 2428 G. Scheiner-Bobis (Eur. J. Biochem. 269) Ó FEBS 2002 COUPLING OF ATP HYDROLYSIS TO ION TRANSPORT Despite the appreciable amount of knowledge about the ATP-recognition area of the protein or its ion coordination sites, the molecular mechanisms that couple ATP hydrolysis to the opening of the ionophore for the translocation of ions against their electrochemical gradient are not well under- stood. Comparison with some other known ion transporters might be helpful in understanding the translocation process, or at least in gaining some room for speculation. The Kdp-ATPase of bacteria is a particularly interesting K + -transporting ATPase made up of three protein components: KdpA, KdpB, and KdpC. KdpA is inserted into the membrane and is similar in sequence to the hydrophobic portion of other P-type ATPases. KdpB is hydrophilic and analogous to the hydrophilic, ATP-binding L4/5 domains of other P-type ATPases. Finally, the KdpC protein is equivalent to the b subunit of K + -transporting P-type ATPases [41]. Furthermore, the KdpA component has similarities to K + channels [42]. Taking into account these observations, one could speculate that during evolu- tion an ion channel has, together with the help of an ATP hydrolase, been selected to move ions against their electro- chemical gradients. In the further development of P-type ATPases, ATP hydrolases and ion channels became phys- ically fused. InthecaseofNa + /K + -ATPase, by taking into consid- eration Armstrong’s proposal regarding the selectivity of ion channel ionophores for Na + or K + [43], such trans- formations in the ion binding structure could explain how one single structure could coordinate Na + in one instance and K + in another. In the E 1 conformation, Na + is bound by ion/dipole interactions to carbonyl groups of the M4, M5, M6, and M8 domains. This applies for a sodium ion in an aqueous milieu. Because K + is larger (r ¼ 1.33 A ˚ )than Na + (r ¼ 0.95 A ˚ ), K + would not fit into the Na + binding site. Phosphorylation of Na + /K + -ATPase causes a conformational change that brings about an alteration in the Na + binding site, allowing Na + to exit toward the extracellular side. One can assume that this conformational change occurs concomitantly with an expansion of the cation binding site (E 2 conformation of Na + /K + -ATPase), so that now the larger K + can be accommodated. The ion/ dipole interactions in this case are also those of K + in an aqueous environment. This newly expanded binding site does not bind Na + well because Na + cannot be adequately coordinated by the carbonyl groups. In this state, an exchange of the water molecules surrounding Na + for carbonyl groups would be thermodynamically unfavorable. For the rigid pore opening of K + channels, Armstrong [43] calculated that an energy expenditure of approximately 10 kcalÆmol )1 would be required to remove two water molecules 0.38 A ˚ (difference in ionic radii between Na + and K + )fromNa + . This results in a preference for selecting K + over Na + of 10 6 : 1. The mechanism of ion selectivity proposed by Armstrong guarantees that despite an enormous excess of Na + in the extracellular medium, the binding of K + is preferred. Thus, the Eisenman hypothesis, which dictates that smaller ions pass more easily through a pore than larger ones, does not apply for all ion channels or pores. It is conceivable that after the release of Na + , the selectivity for K + at the extracellular side of Na + /K + -ATPase is maintained by such a rigid pore opening, which may be formed by the L7/8 peptide of the a subunit as well as the b subunit. THEROLEOFTHEa / b SUBUNIT INTERACTIONS FOR ION TRANSPORT The a and b subunits of the sodium pump must interact with each other in order to accomplish ion transport. In several reports from the laboratory of Fambrough and colleagues, it was shown that 26 amino acids from within the L7/8 peptide loop of the a subunit interact with extracellular parts of the bsubunit [4]. Such interactions appear not only to stabilize the a/b heterodimer but also to have functional relevance, as ATP hydrolysis, ouabain binding, and paly- toxin-induced K + efflux occur only in the presence of both subunits and are markedly influenced by mutations in this region of the enzyme. Moreover, the bsubunit appears to influence the confor- mation and ion sensitivity of the sodium pump. If the b subunit of the sodium pump is replaced by that of the H + / K + -ATPase, Na + -independent specific ouabain binding can still be measured in the presence of Mg 2+ and ATP [44]. Apparently, the b subunit of the H + /K + -ATPase confers a conformational change on the a subunit that enhances the binding of ouabain. Besides verifying that the interaction between a and b subunits involves the L7/8 region, our own investigations using an NGH26 chimera have additionally shown that the binding of specific inhibitors is mediated through this interaction. Thus, an NGH26/HKb heterodimer recognizes not only palytoxin and ouabain but also the gastric H + /K + -ATPase-specific inhibitor SCH 28080 [45]. Taken together, these results point to the function of the b subunit as being more than just a vehicle for the transport of the a subunit from the ER to the plasma membrane [46]. This hypothesis is supported by the fact that there are three or possibly even four isoforms of the b subunit. Besides the b1 isoform, which is the most widely distributed isoform, there is the b2 isoform that is found in excitable tissues (muscle and nervous tissue), the b3intestes,adrenal,and brain, and the bm in skeletal and heart muscle. In view of the variety of isoforms that have been identified, it is not unreasonable to speculate that this multiplicity has a physiological relevance. Interestingly, the b2 isoform was known for some time in glial cells as Ôadhesion molecule on gliaÕ [47]. This lends further support to the idea that the b2 isoform has a function besides that of stabilizing the a subunit. For example, in tissue sections from cerebellum, Fab fragments of monoclonal antibodies against adhesion molecule on glia inhibit the migration of granulocytes. In the cochlea, the expression of b2 is specifically associated with the striata vulgaris, a tissue that forms the barrier between endolymph and extracellular fluid. The endolymph contains a high concentration of K + andalmostnoNa + . It is also strongly electropositive, and K + must be transported against this potential (+80 mV). Thus, it appears that b2 expression is associated with structures that have a high K + -transporting capability. Finally, a dual function for the b2 isoform is also suggested by the fact that it is expressed in tissues that contain no b1 isoform, including pineal gland, photorecep- tor cells, and astrocytes, and also in tissues in the CNS Ó FEBS 2002 Sodium pump structure and properties (Eur. J. Biochem. 269) 2429 (glia, choroid plexus, arachnoid membrane) that have specialized ion-translocating characteristics. Nevertheless, although these observations suggest that the b2 subunit influences ion transport via the sodium pump, data that confirm this function are still lacking. An extracellularly localized peptide composed of 34 amino acids of the b1 subunit (Val93-Asp126) interacts with the 26 amino-acid peptide of the a1 subunit already mentioned [48]. The corresponding fragment of the b2 subunit (Val96-Arg129) has only 29% identity with the Val93-Asp126 fragment of the b1, and 47% homology. Whether these differences in the primary structure of these two regions are responsible for any differences in enzyme characteristics has yet to be investigated. Nevertheless, the overall impression is that the 26 amino- acid peptide and possibly the entire L7/8 region are somehow involved in ion conduction by the pump. Our own results show that mutations of Asp884 and Asp885 from within the L7/8 peptide to Arg considerably affect the interactions of the enzyme with Na + , while, if anything, the affinity for K + increases [49]. Notably, an SYG motif is present within the 26-amino-acid peptide that somewhat resembles the GYG motif of the P-loop of K + channels. There, this tyrosine is essential for ion translocation. Although it is not clear yet whether the corresponding tyrosine of the asubunit is also involved in K + conduction, it is certainly interesting to note that all but one of the K + -transporting P-type ATPases, which always have a and b subunits, have this tyrosine residue conserved (in Hydra, it is a phenylalanine). A further point worth mentioning is that naturally occurring mutation of the highly conserved GYG sequence of the pore opening of K + channels to SYG (which is the sequence in the Na + /K + -ATPase) leads to a reduction in K + selectivity and an increase in Na + permeability [50]. Although there are currently no data directly indicating a role for the SYG(894–896) sequence of the Na + /K + -ATPase in ion transport, Cu 2+ -catalyzed cleavage of the L7/8 loop (possibly near His875) results in the loss of Rb + occlusion [51] usually obtained with the 19-kDa-membrane preparations of the a subunit. This, together with the likelihood that the b subunit may play a roleincationocclusion[52],makestheL7/8areaandthe 26 amino-acid peptide within this region attractive for further investigation. Besides this peptide, aromatic amino acids from the transmembrane domain of the b subunit might be import- ant for a/b subunit interactions and might influence the properties of the enzyme. In the membrane-spanning domains of the b1, b2, and b3 subunits of the sodium pump, there is a relatively high number of amino acids with aromatic side chains (phenylalanine, tyrosine, tryptophan) whose position is conserved in almost all isoforms. In a more recent study it was confirmed that Tyr40 and Tyr44 of the membrane-spanning domain of the b1 subunit influence the transport kinetics of the Na + /K + -ATPase and its affinity towards K + [53]. However, the mechanism by which the tyrosine residues might influence interactions of theenzymewithK + are not yet understood. SPECIFIC INHIBITORS Possibly due to its key function in cellular physiology and indeed the entire organism, the sodium pump has been a target of a vast number of toxins produced by both plants and animals. Thus, its ion pumping activity is specifically inhibited by a series of naturally occurring steroids, termed cardiac steroids or cardiac glycosides, such as ouabain and digitalis. Other substances, like palytoxin from marine corals of the genus Palythoa or sanguinarine from the plant Sanguinaria canadensis, are also specific inhibitors of the sodium pump. Unlike the cardioactive steroids, which inhibit ion flow through the pump, palytoxin and possibly also sanguinarine convert the enzyme into an open channel that allows ions to flow down their concentration gradient. In all cases, however, the toxin/receptor interactions result in loss of the membrane potential, a fatal situation for the cell or organism. Cardioactive steroids bind reversibly to the extracellular side of the Na + /K + -ATPase and inhibit ATP hydrolysis and thus ion transport. The Na + /K + -ATPase is the only enzyme known to interact with this class of substances. Cardioactive steroids, especially water-soluble ouabain (g-strophanthine), have often been used to identify Na + /K + -ATPase and to study ion transport mechanisms involved in this system. Under optimal conditions, 1 mole of Na + /K + -ATPase binds 1 mol of ouabain. Optimal binding occurs when the incubation medium contains one of the following groups of ligands: (a) Mg 2+ ,Na + ,andATPor (b) Mg 2+ and P i . Because both conditions can induce the E 2 -P conformation of the enzyme, this is the conformation to which the cardioactive steroids bind, resulting in the formation of a stable phosphoenzyme/cardioactive steroid complex, termed [E 2 –P*Æouabain]. The presence of the ions to be transported influences the dissociation constant of the enzyme–ouabain complex of the Na + /K + -ATPase: K + lowers the affinity of the enzyme for cardioactive steroids at their high affinity, extracellular binding site. The presence of extracellular Na + competitively inhibits this effect of K + , and high concentrations of Na + enhance cardioactive steroid binding. This probably occurs via interaction with sites from which Na + is released to the extracellular medium. On the other hand, with purified enzyme in the presence of Mg 2+ and P i , low concentrations of Na + have the effect of lowering the affinity of Na + /K + -ATPase for cardioactive steroids when K + is present. Inhibition of the sodium pump by cardiac steroids is clinically relevant. Application of these substances, especi- ally of digitalis and its congeners, helps to increase muscular contractility of the failing heart, possibly by indirectly inducing an elevation in the Ca 2+ concentration in the myocardium. The wide use of digitalis for many centuries in medicine, the great therapeutic impact of these substances, and the need for a regulatory substance that increases heart tonus without influencing its beating frequency led more than 50 years ago to the proposal that endogenous factors must exist that either have a similar structure or act in a similar way to the cardiac steroids currently in use for clinical purposes. The discovery of various isoforms of the sodium pump that are specifically expressed in discrete tissues indirectly supports this concept of an endogenous digitalis- like factor, especially because in some cases distinctive differences were found in the interaction of the various pump isoforms with cardiac steroids and transported cations. Recently, various research groups have succeeded in both isolating endogenous circulating factors that interact with the sodium pump and inhibit 86 Rb + uptake (Rb + is a 2430 G. Scheiner-Bobis (Eur. J. Biochem. 269) Ó FEBS 2002 surrogate for K + ) and also in identifying several of them as ouabain or its congeners [54]. In addition, evidence was provided in several investigations that the concentration of so-called endogenous ouabain increases in plasma upon excessive work and is present at higher levels in the serum of hypertensive patients [54]. All these data indicate that ouabain might be directly or indirectly involved in the regulation of vascular tone and possibly also in the pathogenesis of hypertension. Never- theless, the mechanisms that might be relevant have not yet been elucidated, and ouabain or cardiac glycosides do not appear in the list of vasoactive endogenous substances that includes such agents as endothelin and nitric oxide. Recent experiments demonstrating mitogen-activated protein kin- ase activation in rat cardiomyocytes by low concentrations of ouabain [55,56], however, indicate that investigating signal cascades induced by the glycoside might be helpful in understanding its potential physiological relevance and its possible involvement in vascular tone regulation or in the pathogenesis of hypertension. The Na + /K + -ATPase is a target of other substances besides the cardiac glycosides. Palytoxin, produced by corals of the genus Palythoa, is the most potent toxin of animal origin. The LD 50 for rodents is 10–250 ngÆkg )1 [57]. Previous investigations demonstrated that palytoxin opens ion channels in vertebrate cells with a conductance of approximately 10 pS. These channels remain open for some time and allow K + ions to flow out of the cytosol. This is probably the reason for the high toxicity of palytoxin, as the outflow of K + and the resulting collapse of the membrane potential lead to a general loss of basic cell functions. Furthermore, depolarization is a key event that affects numerous secondary systems. Thus, the concentration of Ca 2+ becomes elevated in several organs through the opening of Ca 2+ channels and leads to the production of inositol trisphosphate [57], the activation of phospholi- pase A 2 and metabolism of arachidonic acid, and numerous other physiological responses that all stem from the increased Na + influx and the ensuing increase in the concentration of cytosolic Ca 2+ that accompany the initial K + outflow [57]. The actual binding site for palytoxin has been the subject of controversy for some time, despite the fact that the Na + / K + -ATPase was known to be inhibited by the toxin. This issue was resolved by expressing Na + /K + -ATPase hetero- logously in yeast [58]. Untransformed yeast cells are insensitive to palytoxin, whereas cells transformed with both subunits of the Na + /K + -ATPase show a marked efflux of K + in response to the toxin. This fact, and the observation that this palytoxin-induced K + efflux is inhib- ited by ouabain and other cardiotonic steroids, confirmed that the sodium pump is the target of palytoxin. In vitro expression experiments have lent further support to this theory by showing that the palytoxin-induced channel is directly associated with the presence of the Na + /K + - ATPase [59]. Through its binding to the Na + /K + -ATPase, the toxin appears to convert the enzyme into a permanently open conformation that allows K + to flow down its concentration gradient out of the cell. This channel is possibly the permanently open state of the natural iono- phore of the sodium pump. Palytoxin binds predominantly to the E 1 -P conformation of the pump. This observation results from experiments demonstrating that ATP and Na + , which first induce the E 1 -P conformation, enhance the binding of 125 I-labeled palytoxin. Mg 2+ and P i , which support the direct formation of the E 2 -P conformation, decrease binding [57]. ATP hydrolysis or enzyme autophosphorylation, however, are not necessary for the formation of the palytoxin-induced channel because palytoxin produces K + efflux in yeast cells expressing an Asp369Ala mutant of the a1 subunit that is enzymatically inactive. Palytoxin is apparently not the only molecule that converts the sodium pump into an ion channel. Sanguin- arine, one of a number of alkaloids developed by the plant Sanguinaria canadensis in the course of evolution to protect itself from herbivores, was described about 25 years ago as an inhibitor of the sodium pump. Nevertheless, the interactions between sanguinarine and the pump were not pursued because at that time experiments that would yield conclusive results were not possible. Using the yeast expression system for the sodium pump, we recently showed that sanguinarine induces the formation of a ouabain- or proscillaridin A-sensitive channel in the sodium pump that allows K + ions to flow out of the cell cytosol [60]. Sanguinarine also appears to bind primarily to the E 1 -P conformation of the enzyme and to inhibit the binding of [ 3 H]ouabain, although, as with palytoxin, phosphorylation is not absolutely required. The experiments with palytoxin and sanguinarine show that under the appropriate conditions an ion channel can be created within an ion pump. This ion channel, which is possibly the ionophore of the pump arrested into a permanently open state, is regulated under normal, physio- logical conditions so that at any given time it is open to only one side of the membrane. Interestingly, the electrogenic step in the catalytic cycle of the sodium pump is associated with the E 1 -P conformation of the enzyme. Viewed from this standpoint, the reaction cycle of the sodium pump (Fig. 1) takes on a new aspect: in the first part of the reaction up to the occlusion of Na + , the pump can be seen as a ligand-inactivated ion channel where P i is the ligand that blocks the backflow of Na + out of the occlusion pocket. In the last part of the reaction sequence, the release of K + into the intracellular medium, the enzyme can be viewed as a ligand-activated ion channel where ATP is the ligand whose binding opens the occlusion pocket and allows the release of K + to the cytosol. PROSPECTS FOR FUTURE RESEARCH Although much has been learned about the mechanics of the transport of ions against their electrochemical gradients by ATPases or the role of these enzymes as targets of either endogenous or foreign toxins, the picture is still not complete. The resolution of the crystal structure of Ca 2+ - ATPase has appeared at a time when it was being suggested that additional efforts might only result in semantic refinements rather than the gain of new information. This structure has provided new hope that the mechanisms of this enzyme can be unveiled by addressing new questions in new projects, and with the expectation of gaining new perspectives. Thus, although they are long-known enzymes, ATPases remain a fresh target for researchers and may soon be discovered anew. Ó FEBS 2002 Sodium pump structure and properties (Eur. J. Biochem. 269) 2431 ACKNOWLEDGEMENTS The author has been supported through DFG, grants Sche 307/5-1 and 307/5-2. He wishes to thank Drs W. Schoner and R. A. Farley for many constructive discussions. REFERENCES 1. Skou, J.C. (1957) The influence of some cations on adenosine- triphosphatase from peripheral nerves. Biochim. Biophys. Acta 23, 394–401. 2. Lutsenko, S. & Kaplan, J.H. (1995) Organization of P-type ATPases: significance of structural diversity. Biochemistry 34, 15607–15613. 3. Antolovic,R.,Bruller,H.J.,Bunk,S.,Linder,D.&Schoner,W. (1991) Epitope mapping by amino-acid-sequence-specific anti- bodies reveals that both ends of the a subunit of Na + /K + - ATPase are located on the cytoplasmic side of the membrane. Eur. J. Biochem. 199, 195–202. 4. Lemas, M.V., Hamrick, M., Takeyasu, K. & Fambrough, D.M. (1994) 26 Amino acids of an extracellular domain of the Na, K-ATPase a-subunit are sufficient for assembly with the Na,K-ATPase b-subunit. J. Biol. Chem. 269, 8255–8259. 5. Geering, K., Meyer, D.I., Paccolat, M.P., Kraehenbuhl, J.P. & Rossier, B.C. (1985) Membrane insertion of a-andb-subunits of Na + ,K + -ATPase. J. Biol. Chem. 260, 5154–5160. 6. Beguin,P.,Wang,X.,Firsov,D.,Puoti,A.,Claeys,D.,Horis- berger, J.D. & Geering, K. (1997) The c subunit is a specific component of the Na,K-ATPase and modulates its transport function. EMBO J. 16, 4250–4260. 7. Donnet, C., Arystarkhova, E. & Sweadner, K.J. (2001) Thermal denaturation of the Na,K-ATPase provides evidence for a–a oligomeric interaction and c subunit association with the C-ter- minal domain. J. Biol. Chem. 276, 7357–7365. 8. Scheiner-Bobis, G. & Farley, F.A. (1994) Subunit requirements for the expression of functional sodium pumps in yeast cells. Biochim. Biophys. Acta 1193, 226–234. 9. Therien, A.G., Karlish, S.J. & Blostein, R. (1999) Expression and functional role of the c subunit of the Na,K-ATPase in mam- malian cells. J. Biol. Chem. 274, 12252–12256. 10. Arystarkhova, E., Wetzel, R.K., Asinovski, N.K. & Sweadner, K.J. (1999) The c subunit modulates Na + and K + affinity of the renal Na,K-ATPase. J. Biol. Chem. 274, 33183–33185. 11. Mahmmoud,Y.A.,Vorum,H.&Cornelius,F.(2000)Identifi- cation of a phospholemman-like protein from shark rectal glands. Evidence for indirect regulation of Na,K-ATPase by protein kinase C via a novel member of the FXYDY family. J. Biol. Chem. 275, 35969–35977. 12. Skou, J.C. (1988) Overview: the Na,K-pump. Methods Enzymol. 156, 1–25. 13. Glynn, I.M. (1993) Annual review prize lecture. ÔAll hands to the sodium pumpÕ. J. Physiol. 462, 1–30. 14. Vilsen, B., Andersen, J.P., Petersen, J. & Jorgensen, P.L. (1987) Occlusion of 22 Na + and 86 Rb + in membrane-bound and soluble protomeric ab-subunits of Na,K-ATPase. J. Biol. Chem. 262, 10511–10517. 15. Martin, D.W., Marecek, J., Scarlata, S. & Sachs, J.R. (2000) ab protomers of Na + ,K + -ATPase from microsomes of duck salt gland are mostly monomeric: formation of higher oligomers does not modify molecular activity. Proc. Natl Acad. Sci. USA 97, 3195–3200. 16. Repke, K.R. & Schoen, R. (1973) Flip-flop model of (NaK)- ATPase function. ActaBiol.Med.Ger.31, K19–K30. 17. Plesner, I.W., Plesner, L., Norby, J.G. & Klodos, I. (1981) The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na + +K + )-ATPase from ox brain. III. A minimal model. Biochim. Biophys. Acta 643, 483–494. 18. Askari, A. & Huang, W. (1982) Na + ,K + -ATPase: evidence for the binding of ATP to the phosphoenzyme. Biochem. Biophys. Res. Commun. 104, 1447–1453. 19. Skriver,E.,Maunsbach,A.B.,Hebert,H.,Scheiner-Bobis,G.& Schoner, W. (1989) Two-dimensional crystalline arrays of Na, K-ATPase with new subunit interactions induced by cobalt-tet- rammine-ATP. J. Ultrastruct. Mol. Struct. Res. 102, 189–195. 20. Taniguchi, K., Kaya, S., Abe, K. & Mardh, S. (2001) The oli- gomeric nature of Na/K-transport ATPase. J. Biochem. 129, 335–342. 21. Ward, D.G. & Cavieres, J.D. (1993) Solubilized ab Na,K- ATPase remains protomeric during turnover yet shows apparent negative cooperativity towards ATP. Proc. Natl Acad. Sci. USA 90, 5332–5336. 22. Bader, H. & Sen, A.K. (1966) (K + )-Dependent acyl phosphatase aspartofthe(Na + +K + )-dependent ATPase of cell mem- branes. Biochim. Biophys. Acta 118, 116–123. 23. Tran, C.M., Scheiner-Bobis, G., Schoner, W. & Farley, R.A. (1994) Identification of an amino acid in the ATP binding site of Na + /K + -ATPase after photochemical labeling with 8-azido- ATP. Biochemistry 33, 4140–4147. 24. Tran, C.M., Huston, E.E. & Farley, R.A. (1994) Photochemical labeling and inhibition of Na,K-ATPase by 2-azido-ATP. Identification of an amino acid located within the ATP binding site. J. Biol. Chem. 269, 6558–6565. 25. Hinz, H.R. & Kirley, T.L. (1990) Lysine 480 is an essential residue in the putative ATP site of lamb kidney (Na,K)-ATPase. Identification of the pyridoxal 5¢-diphospho-5¢-adenosine and pyridoxal phosphate reactive residue. J. Biol. Chem. 265, 10260– 10265. 26. Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A ˚ resolution. Nature 405, 647–655. 27. Scheiner-Bobis, G. & Schreiber, S. (1999) Glutamic acid 472 and lysine 480 of the sodium pump a1 subunit are essential for activity. Their conservation in pyrophosphatases suggests their involvement in recognition of ATP phosphates. Biochemistry 38, 9198–9208. 28. Ohta, T., Nagano, K. & Yoshida, M. (1986) The active site structure of Na + /K + -transporting ATPase: location of the 5¢-(p-fluorosulfonyl)benzoyladenosine binding site and soluble peptides released by trypsin. Proc.NatlAcad.Sci.USA83, 2071–2075. 29. Ovchinnikov, Y.A., Dzhandzugazyan, K.N., Lutsenko, S.V., Mustayef, A.A. & Modyanov, N.N. (1987) Affinity modification of E1-form of Na + ,K + -ATPase revealed Asp-710 in the cata- lytic site. FEBS Lett. 217, 111–116. 30. Farley, R.A., Tran, C.M., Carilli, C.T., Hawke, D. & Shively, J.E. (1984) The amino acid sequence of a fluorescein-labeled peptidefromtheactivesiteof(Na,K)-ATPase.J. Biol. Chem. 259, 9532–9535. 31. Gatto, C., Lutsenko, S. & Kaplan, J.H. (1997) Chemical modification with dihydro-4,4¢-diisothiocyanostilbene-2,2¢- disulfonate reveals the distance between K480 and K501 in the ATP-binding domain of the Na,K-ATPase. Arch. Biochem. Biophys. 340, 90–100. 32. Jorgensen, P.L. & Pedersen, P.A. (2001) Structure–function relationships of Na + ,K + ,ATP,orMg 2+ binding and energy transduction in Na,K-ATPase. Biochim. Biophys. Acta 1505, 57–74. 33. Patchornik, G., Goldshleger, R. & Karlish, S.J. (2000) The complex ATP-Fe 2+ serves as a specific affinity cleavage reagent in ATP-Mg 2+ sites of Na,K-ATPase: altered ligation of Fe 2+ (Mg 2+ ) ions accompanies the E1 fi E2 conformational change. Proc. Natl Acad. Sci. USA 97, 11954–11959. 34. Shainskaya, A. & Karlish, S.J. (1994) Evidence that the cation occlusion domain of Na/K-ATPase consists of a complex of 2432 G. Scheiner-Bobis (Eur. J. Biochem. 269) Ó FEBS 2002 membrane-spanning segments. Analysis of limit membrane- embedded tryptic fragments. J. Biol. Chem. 269, 10780–10789. 35. Jewell-Motz, E.A. & Lingrel, J.B. (1993) Site-directed mutagen- esis of the Na,K-ATPase: consequences of substitutions of negatively-charged amino acids localized in the transmembrane domains. Biochemistry 32, 13523–13530. 36. Vilsen, B. (1993) Glutamate 329 located in the fourth trans- membrane segment of the a-subunit of the rat kidney Na + , K + -ATPase is not an essential residue for active transport of sodium and potassium ions. Biochemistry 32, 13340–13349. 37. Van Huysse, J.W., Jewell, E.A. & Lingrel, J.B. (1993) Site- directed mutagenesis of a predicted cation binding site of Na, K-ATPase. Biochemistry 32, 819–826. 38. Vilsen, B. (1995) Mutant Glu781 fi Ala of the rat kidney Na + ,K + -ATPase displays low cation affinity and catalyses ATP hydrolysis at a high rate in the absence of potassium ions. Bio- chemistry 34, 1455–1463. 39. Kuntzweiler,T.A.,Arguello,J.M.&Lingrel,J.B.(1996)Asp804 and Asp808 in the transmembrane domain of the Na,K-ATPase alpha subunit are cation coordinating residues. J. Biol. Chem. 271, 29682–29687. 40. Rice, W.J. & MacLennan, D.H. (1996) Scanning mutagenesis reveals a similar pattern of mutation sensitivity in transmem- brane sequences M4, M5, and M6, but not in M8, of the Ca 2+ - ATPase of sarcoplasmic reticulum (SERCA1a). J. Biol. Chem. 271, 31412–31419. 40a. Eisenmann, G. & Dani, J.A. (1987) An introduction to molecular architecture and permeability of ion channels. Annu. Rev. Bio- phys. Biomol. Struct. 16, 247–263. 41.Altendorf,K.,Siebers,A.&Epstein,W.(1992)TheKDP ATPase of Escherichia coli. Ann. NY Acad. Sci. 671, 228–243. 42. Durell, S.R., Bakker, E.P. & Guy, H.R. (2000) Does the KdpA subunit from the high affinity K + -translocating P-type KDP- ATPase have a structure similar to that of K + channels? Biophys. J. 78, 188–199. 43. Armstrong, C. (1998) The vision of the pore. Science 280, 56–57. 44. Eakle, K.A., Lyu, R M. & Farley, R.A. (1995) The influence of b subunit structure on the interaction of Na + /K + -ATPase complexes with Na + . A chimeric b subunit reduces the Na + dependence of phosphoenzyme formation from ATP. J. Biol. Chem. 270, 13937–13947. 45. Farley,R.A.,Schreiber,S.,Wang,S G.&Scheiner-Bobis,G. (2001) A hybrid between Na + ,K + -ATPase and H + ,K + - ATPase is sensitive to palytoxin, ouabain, and SCH 28080. J. Biol. Chem. 276, 2608–2615. 46. Geering, K. (1991) Posttranslational modifications and intra- cellular transport of sodium pumps: importance of subunit assembly. Soc. General Physiol. Series 46, 31–43. 47. Schmalzing, G., Kroner, S., Schachner, M. & Gloor, S. (1992) The adhesion molecule on glia (AMOG/b2) and a1 subunits assemble to functional sodium pumps in Xenopus oocytes. J. Biol. Chem. 267, 20212–20216. 48. Colonna, T.E., Huynh, L. & Fambrough, D.M. (1997) Subunit interactions in the Na,K-ATPase explored with the yeast two- hybrid system. J. Biol. Chem. 272, 12366–12372. 49. Schneider, H. & Scheiner-Bobis, G. (1997) Involvement of the M7/M8 extracellular loop of the sodium pump a subunit in ion transport. Structural and functional homology to P-loops of ion channels. J. Biol. Chem. 272, 16158–16165. 50. Silverman,S.K.,Kofuji,P.,Dougherty,D.A.,Davidson,N.& Lester, H.A. (1996) A regenerative link in the ionic fluxes through the weaver potassium channel underlies the pathophy- siology of the mutation. Proc. Natl Acad. Sci. USA 93, 15429– 15434. 51. Shimon, M.B., Goldshleger, R. & Karlish, S.J. (1998) Specific Cu 2+ -catalyzed oxidative cleavage of Na,K-ATPase at the extracellular surface. J. Biol. Chem. 273, 34190–34195. 52. Lutsenko, S. & Kaplan, J.H. (1993) An essential role for the extracellular domain of the Na,K-ATPase b-subunit in cation occlusion. Biochemistry 32, 6737–6743. 53. Hasler, U., Crambert, G., Horisberger, J.D. & Geering, K. (2001) Structural and functional features of the transmembrane domain of the Na,K-ATPase b subunit revealed by tryptophan scanning. J. Biol. Chem. 276, 16356–16364. 54. Schoner, W. (2002) Endogenous cardiac glycosides, a new class of steroid hormones. Eur. J. Biochem. 269, 2440–2448. 55. Haas, M., Askari, A. & Xie, Z. (2000) Involvement of Src and epidermal growth factor receptor in the signal-transducing function of Na + /K + -ATPase. J. Biol. Chem. 275, 27832–27837. 56. Xie, Z. & Askari, A. (2002) Na + /K + -ATPase as a signal transducer. Eur. J. Biochem. 269, 2434–2439. 57. Habermann, E. (1989) Palytoxin acts through Na + /K + -ATPase. Toxicon 27, 1171–1187. 58. Scheiner-Bobis, G., Meyer zu Heringdorf, D., Christ, M. & Habermann, E. (1994) Palytoxin induces K + efflux from yeast cells expressing the mammalian sodium pump. Mol. Pharmacol. 45, 1132–1136. 59. Hirsh, J.K. & Wu, C.H. (1997) Palytoxin-induced single-channel currents from the sodium pump synthesized by in vitro expres- sion. Toxicon 35, 169–176. 60. Scheiner-Bobis, G. (2000) Sanguinarine induces K + outflow from yeast cells expressing mammalian sodium pumps. Naunyn- Schmiedeberg’s Arch. Pharmacol. 363, 203–208. Ó FEBS 2002 Sodium pump structure and properties (Eur. J. Biochem. 269) 2433 . enzyme. Moreover, the bsubunit appears to influence the confor- mation and ion sensitivity of the sodium pump. If the b subunit of the sodium pump is replaced by that of. interactions and might influence the properties of the enzyme. In the membrane-spanning domains of the b1, b2, and b3 subunits of the sodium pump, there is