structure and mechanism of na,k-atpase: functional sites and their interactions

14 Jan 2003 14:58 AR AR177-PH65-34.tex AR177-PH65-34.sgm LaTeX2e(2002/01/18)P1: IBC10.1146/annurev.physiol.65.092101.142558

Annu. Rev. Physiol. 2003. 65:817–49doi: 10.1146/annurev.physiol.65.092101.142558

Copyright c© 2003 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on January 9, 2003

STRUCTURE AND MECHANISM OF Na,K-ATPASE:Functional Sites and Their Interactions

Peter L. Jorgensen,1 Kjell O. Hakansson,1 andSteven J. D. Karlish21Biomembrane Center, August Krogh Institute, Copenhagen University,Universitetsparken 13, 2100 Copenhagen OE, Denmark; e-mail: [email protected];[email protected] of Biological Chemistry, Weizmann Institute of Sciences, Rehovot 76100,Israel; e-mail: [email protected]

Key Words Na-K-pump homology models, ATP binding, Na+ and K+ ionbinding, energy transduction mechanism

■ Abstract The cell membrane Na,K-ATPase is a member of the P-type family ofactive cation transport proteins. Recently the molecular structure of the related sar-coplasmic reticulum Ca-ATPase in an E1 conformation has been determined at 2.6Aresolution. Furthermore, theoretical models of the Ca-ATPase in E2 conformations areavailable. As a result of these developments, these structural data have allowed cons-truction of homology models that address the central questions of mechanism of activecation transport by all P-type cation pumps. This review relates recent evidence onfunctional sites of Na,K-ATPase for the substrate (ATP), the essential cofactor (Mg2+ions), and the transported cations (Na+and K+) to the molecular structure. The essentialelements of the Ca-ATPase structure, including 10 transmembrane helices and well-defined N, P, and A cytoplasmic domains, are common to all PII-type pumps such asNa,K-ATPase and H,K-ATPases. However, for Na,K-ATPase and H,K-ATPase, whichconsist of bothα- andβ-subunits, there may be some detailed differences in regions ofsubunit interactions. Mutagenesis, proteolytic cleavage, and transition metal-catalyzedoxidative cleavages are providing much evidence about residues involved in bindingof Na+, K+, ATP, and Mg2+ ions and changes accompanying E1-E2 or E1-P-E2-P con-formational transitions. We discuss this evidence in relation to N, P, and A cytoplasmicdomain interactions, and long-range interactions between the active site and the Na+and K+ sites in the transmembrane segments, for the different steps of the catalyticcycle.

INTRODUCTION AND SCOPE

The Na,K-pump is essential for all mammalian cells. At rest it consumes 20–30%of ATP production to actively transport Na+ out of and K+ into the cell. The Naand K gradients are required for maintaining membrane potentials, cell volume,

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14 Jan 2003 14:58 AR AR177-PH65-34.tex AR177-PH65-34.sgm LaTeX2e(2002/01/18)P1: IBC

818 JORGENSEN¥ HAKANSSON ¥ KARLISH

and secondary active transport of other solutes, e.g., the transcellular transportprocesses in intestine, glands, and kidney.

Na,K-ATPase is the largest protein complex in the family of P-type cationpumps. The minimum functional unit is a heterodimer of theα- andβ-subunits(1) and may also be coexpressed with small ion transport regulators of the FXYDfamily (2, 3). Individual genes of fourα-subunit isoforms and at least threeβ-subunit isoforms of Na,K-ATPase have been identified in mammals (4, 5). Theisoforms combine to form a number of Na,K-ATPase isozymes that are expressedin a tissue- and cell-specific manner. The central question in the comprehensivephysiology of active Na+ and K+ transport is the nature of the complex regulationof the expression of these isozymes and the variety of systems for posttranslationalmodification and short-term regulation.

Biochemical and spectroscopic data show that long-range E1-E2 conforma-tional transitions in theα-subunit mediate interactions between cytoplasmicdomains and the cation sites in the intramembrane domain (1, 6–8). These tran-sitions couple the scalar processes of ATP binding, phosphorylation, and de-phosphorylation to the vectorial extrusion of three Na+ ions and uptake of twoK+ ions.

High-resolution molecular structural information is essential for understand-ing these complex reactions and the physiological regulation of the system. TheNa,K-pump was first visualized as distinct particles of 30–50A diameter in pu-rified membrane preparations from mammalian kidney (9). Crystallization in themembrane after incubation in Mg2+ and vanadate (10) led to a three-dimensionalreconstruction at 20–25A resolution, with information on the mass distributionof the cytoplasmic protrusion of theα-subunit and the extracellular protrusionof the β-subunit (11). Recently, electron microscopy image reconstructions ofNa,K-ATPase at 9–11A resolution were reported (12, 13). Crystallization of sar-coplasmic reticulum Ca-ATPase in vanadate (14) led to 8–9A resolution struc-tures, which allowed tentative assignment of transmembrane helices (15). In 2000,Toyoshima and coworkers presented the 2.6A high-resolution structure based onX-ray diffraction of crystals grown in CaCl2 solution (16). In addition to this struc-ture at atomic resolution (1EUL), two structures of the E2 conformation at 6Aresolution are available, based on electron microscopy of Ca-ATPase stabilized bydecavanadate and thapsigargin, 1FQU (16) and 1KJU (17).

The focus of this review is the structure-function relationship of binding sitesfor Na+, K+, Mg2+, and ATP and the structural changes mediating interactionsof functional sites during the catalytic cycle. Ideally, resolution of these mech-anisms requires a high-resolution structure of each intermediate of the reactioncycle. In the absence of such information provisional concepts can be inferred bycomparing a high-resolution structure of the E1[2Ca] form (16) with the avail-able models of the E2-forms [1FQU and 1KJU (17)]. The structural reconstruc-tions of the E2-Mg-vanadate complexes of theα-subunit of Na,K-ATPase (12, 13)show considerable similarity to the E2 forms of Ca-ATPase (16, 17). Homol-ogy modeling of theα-subunit of Na,K-ATPase on the backbone of the

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Na,K-PUMP STRUCTURE AND FUNCTION 819

three-dimensional structures of Ca-ATPase therefore allows visualization of thedomain movements as large global changes of structure involving practically allamino acid residues. In light of these models, data on proteolytic (1, 8) or metal-catalyzed (6, 7) peptide bond cleavage, mutagenesis, and chemical modificationcan be interpreted to yield important information on the mechanism of energytransduction.

THE KINETIC MECHANISM OF Na,K-ATPase

Extensive studies into the cation transport mechanism and partial reactions, in-cluding definition of the properties of the phosphorylated intermediates (18), theoccluded Rb+ or K+ after dephosphorylation (19), and the conformational changesof the protein (1), provided the basis for the kinetic mechanism ofNa,K-ATPase (1, 20, 21). Known as the Albers-Post mechanism, it involves thesesteps (cyt refers to cytoplasmic, exc refers to extracellular with respect to Na+

and K+):

a. E2[2K]↔E12K + ATP↔ATP ·E1+ 2K+cyt

b. ATP·E1+ 3Na+cyt↔E1ATP3Na+

c. E1ATP3Na+↔E1-P[3Na]+ ADP

d. E1-P[3Na]↔E2-P[2Na]+ Na+exc

e. E2-P[2Na]+ 2K+exc↔E2-P[2K] + 2Na+exc

f. E2-P[2K]↔E2[2K] + Pi.

The steps of this ping-pong transport mechanism include (a) ATP acting with lowaffinity accelerates inward transport of two K+ ions coupled to the E2[2K]↔E1

conformational transition; (b) binding of three Na+ ions at cytoplasmic-orientedsites; (c) Na+cyt-dependent phosphorylation from ATP and occlusion of three Na+

ions; (d) outward transport of three Na+ ions coupled to the E1-P↔E2-P confor-mational transition; (e) binding of two K+ ions at extracellularly oriented sites; and( f ) K+exc-activated dephosphorylation and occlusion of two K+ ions. As pointedout by Jencks (22), an effective ion pump has strict cation specificity of the phos-phorylation and dephosphorylation reactions, (stepsc andf ), and tight couplingof the E1/E2 conformational changes to cation movements, (stepsa andd), as wellas features that minimize energy losses via nonproductive pathways. The latterinclude the cation occlusion in E1-P[3Na] and E2[2K] and change in specificityfrom ADP reactivity of E1-P to water reactivity of E2-P. Other P-type pumps, suchas sarcoplasmic reticulum Ca-ATPase or gastric H,K-ATPase have different cationspecificities of the phosphorylation-dephosphorylation steps and stochiometriesof cation movements, but the basic mechanisms are the same. An explanation ofthese mechanisms in terms of molecular structure is the question that needs to beaddressed.

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OVERALL STRUCTURE OF Ca- AND Na,K-ATPaseAND THE E1-E2 TRANSITIONS

The general characteristics of a P-type ATPase include a bundle of transmembranehelices making up the cation transport path and three domains on the cytoplasmicside of the membrane; the A (actuator), P (phosphorylation), and N (nucleotide-binding) domains. In Ca-ATPase and Na,K-ATPase, the A domain consists ofthe N-terminal portion of the polypeptide chain and an insertion between M2and M3, whereas the P and N domains are located between M4 and M5 (16)(Figure 1).

Rabbit sarcoplasmic reticulum Ca-ATPase is the only member of the P-typeATPase family for which an atomic resolution structure is available (16). Much ofthe Na,K-ATPase shows sufficient sequence similarity to allow construction of ahomology model, although there are exceptions, notably in the N and P domains.Key residues involved in ATP binding can be identified in both Ca-ATPase (23, 24)and Na,K-ATPase (25), but there are several large insertions and deletions in theN domain and in the first 70 residues of the P domain. The extracellular loops be-tween M1 and M2, and between M7 and M8, contain an insertion in Na,K-ATPase,and there is a deletion between M3 and M4. Nevertheless, at the extracellular face,a negatively charged surface area, where the Na+ ions exit and the K+ ions enter,is preserved in the two enzymes.

Very large movements of the cytoplasmic A, N, and P domains accompanythe E1-E2 transitions in Ca-ATPase, as visualized by comparing the 2.6A reso-lution atomic structure of Ca-ATPase in an E1[2Ca] conformation (1EUL) (16)with the E2-models (1FQU, 1KJU) (16, 17) (Figure 1). The P domain is rotatedwith reorientation of the catalytic site, including the important residues (Asp351,Thr625, and Asp703), on a plane along the Rossmann folds. Movement of the Ndomain comes mainly from rotation of the P domain (17). The A domain rotatesapproximately 90◦ about a vertical axis, thus docking the conserved 181-TGESsequence onto the P domain at the 625-TGD motif, the 701-TGDGVNDS motif,and the phosphorylated Asp351 in the catalytic site of the E2 form (16, 17).

The N Domain

The N domain contains the ATP-binding site and extends from the phosphorylationsite Asp369, with the 370-KTGTL sequence, to the C-terminal 586-DPPR hinge(Figure 2). This domain is inserted into the P domain and consists of a fairlytwisted antiparallel eight-strandedβ-sheet, with a basic fold known only from theCa-ATPase structure (16). The strand topology is the same as found in the eight-strandedβ-barrels of the lipocalins, which are carriers of hydrophobic compounds.(26, 27). Two long segments protrude from thisβ-sheet, one between the outermostβ-strands 3 and 4, and another between strands 6 and 7 (Figure 2). Each of theseprotrusions contains aβ-hairpin and two or three helices. The binding site forthe ATP adenine base is found in a hydrophobic pocket lined by strands 4, 5, 6,

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Figure 2 Topological representation of the N and P domains. The positions of someresidues around the ATP-binding site are shown.β-strands are shown as arrows but arenumbered only if they belong to any of the two centralβ-sheets;α-helices are shownas cylinders.

and 7 on one side and the N-terminal part of helix Asp443-Cys457 on the other.Na,K-ATPase has two large deletions relative to the Ca-ATPase structure, onebetween strands 1 and 2 and the other on the C-terminal side of helix Asp443-Cys457. In the tertiary structure, both deletions are far from the ATP-binding site.

The ATP-Binding Site

The ATP-binding site in the Ca-ATPase structure is detected by boundTNP-AMP in the N domain (16), but precise interactions between ATP and proteinhave not been characterized. The role of the N domain of Na,K-ATPase is alsoillustrated by binding of nucleotides to the isolated expressed loop between M4and M5 with the selectivity of native enzyme, although with much lower affinity(28). Several residues in the ATP-binding pocket are conserved in Na,K-ATPase

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and Ca-ATPase. Lys501 (Lys515 in Ca-ATPase) in the conserved KGA motif iscovalently labeled with fluorescein isothiocyanate (FITC) (29). Lys480 (Lys492)is the receptor for 8-azido ATP and pyridoxal 5′-diphospho-5′-adenosine (30, 31).ATP protects against insertion, and labeling abolishes ATPase activity. However,mutations of Lys515 in Ca-ATPase (32) and Lys480 in Na,K-ATPase (33) havemoderate effects on catalysis. Mutation of Lys480 appears to reduce the affinityof the Na,K-ATPase for both ATP and phosphate, but it is not essential for ATPbinding or hydrolysis (33). Although insertion of bulky groups in the bindingcavity may prevent binding of ATP, these basic residues may not be essential forcoordination of nucleotide. Hydrogen bonding of Lys501 with Glu446 may be ofimportance for the general structure of the ATP site. In Ca-ATPase, competitionagainst photolabeling of Lys492 (Lys480 in Na,K-ATPase) by [γ -32P]-TNP-8-azido-ATP has been exploited to analyze ATP binding (34). The results implicatePhe487 (Phe475 in Na,K-ATPase), Arg489, and Lys492 (Lys480) (34). Cross-linking of Lys492 and Lys515 also produces strong inhibition of ATPase activityand phosphorylation in Ca-ATPase (24).

In Na,K-ATPase, Arg544 (Arg560 in Ca-ATPase) is positioned at the mouth ofthe ATP-binding pocket near the interface with the P domain and initiates a WalkerB–like sequence (1, 25). Direct ATP binding or phosphorylation assays of mutantsshows that Arg544 contributes 7 to 8 kJ/mol to the change in Gibbs free energyof ATP binding (11Gb). ATP binding is unaffected in the Arg544Lys mutation,whereas the affinity for ADP is reduced, suggesting that Arg544 stabilizes theβ

or γ phosphate moieties of ATP and aligns theγ phosphate for interaction withthe carboxylate group of Asp369 (25).

Several investigators have proposed that Na,K-ATPase has two ATP sites lo-cated either on the sameα/β protomer, or on interacting protomers. A review ofthis controversial topic is presented in Reference 35 with many original references.The minimum asymmetric unit in membrane crystals and the minimal functionalunit in detergent solution are unequivocallyα/β protomers (36–38), and in ourview, the most recent findings favor a single ATP site mechanism. First andforemost, the Ca-ATPase structure (16) has only a single ATP-binding pocketand, insofar as they have been compared, ATP-binding residues of Na,K-ATPaseand Ca,ATPase are similar. The two-ATP site hypothesis is based extensively onbinding of bulky ATP derivatives (39) and modification by reagents such as ery-throsine isothiocyanate (Er-ITC) to FITC-modified Na,K-ATPase, which blockshigh-affinity ATP binding (40–42). Recently, pure duck salt gland Na,K-ATPasefully modified with FITC (6.5 nmol/mg protein) was shown to bind TNP-ADPand be modified by Er-ITC, although with much lower affinity than native enzyme(43). Thus the fluorescein label attached to Lys515 hinders binding of ATP andalso TNP-ADP and Er-ITC, but the latter are not absolutely blocked and can stillenter the ATP pocket and bind with a low affinity. Duck salt gland microsomalNa,K-ATPase is mostly monomeric (α/β). In pure Na,K-ATPase, higher oligomersare detectable, but the molecular activity is the same as for the monomer (α/β) inmicrosomes (44).

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The P Domain

The phosphorylation domain is homologous to the haloacid dehalogenase (HAD)family of enzymes for which the structure is known to atomic resolution in thecase of L-2-haloacid halogenase (45), phosphonoacetaldehyde hydrolase (46), andMethanococcus jannashiiphosphoserine phosphatase (47). As anticipated fromthe sequence similarities, these proteins share a common fold corresponding tothe phosphorylation domain in the Na,K-ATPase, although the substrate-bindingdomain insertions are different from each other and from the N domain of theATPases. The fold can be described as a six-stranded parallelβ-sheet made upof two Rossmann folds (βαβαβ), which in Ca-ATPase and Na,K-ATPase areflanked with one parallel and one antiparallelβ-strand (Figure 2). The N domainis inserted between the second and third strand. Three motifs are arranged next toeach other and outline the active site for binding of Mg2+ and phosphate. The firstof these motifs, 369-DKTGTL in Na,K-ATPase, contains the aspartic acid residue,which is phosphorylated during catalysis (48–50). The second motif is 610-TGDon strand 3, C terminal to the N domain. It associates with the phosphorylatedsegment because Asp627 in Ca-ATPase is hydrogen bonded to Lys352 and theAsp627Glu mutation blocks the Ca2+ transport at the E1-P-E2-P transition (32, 51).The third motif, 708-TGDGVND, terminates the sixthβ-strand of the P domainwhere Asp710 is required for binding of Mg2+ and phosphorylation (52).

L-2-haloacid halogenase and phosphonoacetaldehyde hydrolase act on rela-tively small substrates, and Mg2+ is not required for catalysis (45, 46). Similarto the phosphoserine phosphatase (47), they do not catalyze energy transfer fromphosphate bond ester cleavage or undergo sizeable domain-domain movements asin P-type ATPases. Their use as a model system for conformational changes of theATPases is therefore limited.

More can be learned from the binding of Mg2+ and phosphate in these en-zyme structures. InM. jannashiiphosphoserine phosphatase (47), the Mg2+ ion iscoordinated to residues homologous to Na,K-ATPase residues Asp710, the mainchain carbonyl oxygen of Thr371, the phosphorylation residue Asp369, and to aphosphate ion. The other phosphate oxygens interact with (a) Asn713, Lys691 andthe main chain NH group of Gly611, (b) Thr610 and the main chain NH groupsof Lys370, and (c) Thr371. Coordination of the phosphate group is most likely tobe similar to theγ phosphate of the ATP in P-type ATPases. The structure of theDKT motif is well adapted to bind both a negatively charged phosphate ion anda positively charged Mg2+ ion. Lys370 initiates a short, one-turn helix, and thedipole moment is utilized for binding phosphate by the main chain NH groups ofresidues Lys370 and Thr371. However, on one side of the helix, the peptide bondsof residues Thr371-Gly372 and Thr375-Gln376 are flipped, allowing the carbonyloxygen of Thr371 to interact with the Mg2+ ion. Thus two antiparallel dipolemoments in this unusual helix are utilized to dock both the positively chargedMg2+ and the negatively charged phosphate ion. Residues likely to interact withtheα andβ phosphate groups toward the binding site on the N domain include

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Lys370, Thr375, and Asp586. The structure of phosphonoacetaldehyde hydrolasecomplexed with tungstate is similar to the Mg-phosphate complex in the phos-phoserine phosphatase, although Lys691 and Asn713 are replaced by arginine andserine residues, respectively.

The A Domain

The A domain is the smallest cytoplasmic domain of theα-subunit ofNa,K-ATPase, consisting of the N-terminal segment (Gly1-Gln88) plus the loopbetween M2 and M3 (Lys205-Glu312). In Ca-ATPase, the cytoplasmic loop formsa distorted jelly roll structure, and there are two short helices in the 40 residuesof the N-terminal segment (16). In Na,K-ATPase, the N-terminal segment is 33residues longer and the bond at Lys30 is a well-defined tryptic cleavage site (53).The tryptic cleavage sequence of Lys30 and Arg438 implies that this part of theA domain interacts with the N domain in the E2[2K] form (1). The conserved212-TGES motif is located at an edge of the A domain (16). The critical impor-tance of this loop has been demonstrated in iron cleavage experiments, as it iscleaved in the E2-P form, but not in E1-P (54).

Exposure of proteolytic cleavage sites in the L2/3 loop in E1 forms and protec-tion in E2 forms are parallel in Na,K-ATPase (1, 55) and Ca-ATPase (1, 56, 57),suggesting that the conformational movements of the A domain are similar. Theimportance of the A domain movements and the L2/3 loop are underlined by thenature of its connections near the cytoplasmic end of M3 (Figure 3). In Ca-ATPase,a triangular hydrogen-bonded connection is made between L2/3, helix-1 of S4 andL6/7, involving Thr247 in 247-TPL of L2/3, Glu340 in 339-VETLG of the P1-helix in S4, and Arg822 in 819-RXPK of L6/7 (16). These segments are conservedin Na,K-ATPase 275-TPI, 357-VETLG, and Arg830 in 827-RXPK of L6/7 andare modeled in Figure 3, as in Ca-ATPase. The connection to P1-helix of S4 mayfunction as a lever mechanism to alter the position of M4. L6/7 runs as a collararound the P domain, and there are several conserved hydrogen bond connectionsbetween L6/7 and S5, i.e., between Asp813 in L6/7 and Asn755 in S5 and betweenthe main chain carbonyl oxygens of Arg819 and Arg751 in S5. Additional hydro-gen bonds and van der Waal interactions between L6/7 and M5 are described forCa-ATPase (16).

The Transmembrane Region

There are 10 transmembraneα-helices or 5 pairs of helices inserted from thecytoplasmic side (37, 57). With the exception of M9-M10, the returning helixis in close contact with the preceding one in Ca-ATPase, and the four M1-M2,M3-M4, M5-M6, and M7-M8 helical pairs are sequentially juxtaposed in themembrane. Three of the helical pairs contribute to cation binding via residues inM4, M5, M6, and M8 (55, 58–60) (Figure 4a and Figure 5). The M2, M4, and M5helices are extended to lengths of 41, 26,>27, and 60A, with several turns onthe cytoplasmic side where they connect with cytoplasmic domains. The M9-M10

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pair does not contribute directly to domain anchoring or cation binding, and its twohelices are not packed together but wrap around M8 (16). Determination of thehelical tilts in the bilayer is not straightforward because the membrane structureis not determined or defined in crystallographic structures. The inclination of thetransmembrane helices in Ca-ATPase has been estimated by electron microscopy(15) to range from less than 10◦ for M4 and M6 to∼30◦ for M5 and M9. Therelative orientations of helices varies from almost parallel to an angle close to 50◦

between M5 and M10.The transmembrane regions of P-type ATPases differ from those of known

ion channels by the absence of an obvious transport path in the form of an openwater-filled channel. Presumably, this reflects the difference between passive andactive transport mechanisms and the necessity of occluding the cations in the ionpumps. Probing the structure with the Hole software (61) in Figure 4c shows that achannel with dimensions suitable for passage of Na+ or K+ connects the proposedentry site for Na+ with site I and II near the center of the membrane region. In theextracellular half of the bilayer, the putative channel is constricted in a section thatappears to be too narrow for passage of a cation, as could be expected for an E1

conformation.

HELIX PACKING STABILITY AND α-β-SUBUNITINTERACTIONS

Because the Ca-ATPase structure or homology models are used extensively tointerpret data on Na,K-ATPase or H,K-ATPase, we need to know howα- andβ-subunits interact and whetherβ-subunits affect organization of theα-subunit. Asimilar question arises regarding theγ -subunit or other FXYD proteins. Trans-membrane helix arrangement has been predicted to be similar in Ca-ATPase andNa,K-ATPase (57, 62). However, as discussed below, there may be a significantdifference in the M7-M10 region whereα- andβ-subunits interact.

The α-Subunit

Several proximities and interactions betweenα- andβ-subunits have been detectedwith renal Na,K-ATPase, digested extensively with trypsin to shave off cytoplas-mic N, P, and A domains of theα-subunit, which leave well-defined membrane-embedded fragments (63–65). Theβ- andγ -subunits are largely intact. In these19-kDa membranes, cation occlusion and ouabain binding are preserved whereasthe ATP-binding sites are removed. This shows directly that cation sites are locatedwithin transmembrane segments and communicate with ATP sites via long-rangeinteractions. Cation occlusion is much less thermally stable in the 19-kDa mem-brane than in native Na,K-ATPase, although it is strongly protected by Rb+ or Na+

ions or ouabain. (66). Instability can be explained by loss of stabilizing interactionsof L6/7 after the cut at Arg830-Asn831 and removal of the P1 helix in S4. Thisfits well with a proposal that L6/7 of Ca-ATPase and its interacting stalk segments

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stabilize both phosphorylation (P) and Ca-binding (M) domains, based on effectsof mutations to charged residues (67). The M5/M6 fragment dissociates from di-gested Na,K-ATPase or H,K-ATPase upon warming at 37◦C, in the absence ofK(Rb+) ions (68, 69). This phenomenon indicates thermal lability of interactionsbetween M5/M6 and other segments, notably the M7-M10 segments (70), andsuggests that M5 and M6 are mobile segments in cation transport.

Cu-phenanthroline treatment of detergent-solubilized 19-kDa membranescauses S-S bridge formation between Mβ (Cys44) and a cysteine in M8 (Cys911or Cys930), an internal cross-link between M9 (Cys964) and M10 (Cys983) oftheα-subunit, and a cross-link of M1/M2 to M7/M10, which was not identified(71, 72). A homology model shows that Cys983 and Cys964, which form the in-ternal cross-link, lie at the ends of M9 and M10 but are not close to each other(Figure 5). The observation (66) that in 19-kDa membranes Cys983 in M10 re-acts with cysteine reagents only after dissociation of the M5/M6 fragment isnot self-explanatory because Cys983 already appears to be exposed (Figure 5).These discrepancies imply that M9 and M10 may not be organized identicallyin Ca-ATPase and Na,K-ATPase. In another studyo-pthalaldehyde produced twocross-links, one near the cytoplasmic entrance of M5 and M7, consistent withthe Ca-ATPase structure, and the other located between the extracellular en-trance of M8 and theβ-subunit, which fits other evidence on theα-β interaction.(73).

Cysteine modifications and mutations are beginning to provide valuable in-formation. Covalent modification of Cys964 and Cys911 in right-side-out renalvesicles shows that these residues lie at the extracellular boundaries of M9 andM8 and are conformationally mobile. Mobility of M9 is also demonstrated withBIPM-labeled Cys964 (74). Na,K-ATPase was expressed in insect cells, Cys964and Cys911 were mutated to non-cysteine residues, and residues in putative ex-tracellular loops were mutated to cysteine: Pro118 in L1/2; Thr309Cys in L3/4;Leu793Cys in L5/6; Leu876Cys in L7/8; and Met973Cys in L9/10. Each of themutants reacted with a membrane-impermeant cysteine reagent (MTSEA-biotin),confirming their location in extracellular loops (74). These results provide a strongindication for the model with 10 transmembrane segments (37) in the functionalenzyme. Val969Cys and Leu976Cys, located on either side of Met973, were not la-beled, suggesting that they lie at the boundaries of M9 and M10. On the homologymodel (Figure 5), Val969 and Leu976 are located in the center of the long L9/10,where cysteines should be exposed to MTSEA-biotin. Thus again, the unexpectedobservation raises the possibility of a different arrangement of M9 and M10 inNa,K-ATPase and Ca-ATPase. A functional cysteine-less Na,K-ATPase has nowbeen expressed in insect cells (75), providing an important tool for defining helixpacking, which uses cysteine-scanning mutagenesis as applied to transporters suchas lac permease (76).

Covalent labeling using photoactivated hydrophobic reagents detects lipid-exposed faces of transmembrane helices. Bothα- and β-subunits of renalNa,K-ATPase are labeled by INA (S-iodonaphtyl-L-azide) (77), TID

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[3-(trifluromethyl)-3-(m-iodophenyl)diazirine], and phosphatidylcholine-TID ana-logues (78, 79). Labeling of theα- but notβ-subunit is amplified in the E2[K]compared with the E1Na conformation. Recent work with Torpedo Na,K-ATPaseshows that TIDPC labels the hydrophobic segments M1, M3, M9, and M10 withno difference in E1 or E2 conformations. M2 and M4-M8 segments were not la-beled (80). This labeling pattern is expected for a helix arrangement such as that ofCa-ATPase, except for the lack of labeling of M2 and M7. Mβ has been hypothe-sized to conceal M7. TID labels the same fragments as TIDPC and also an M5-M6fragment; this labeling is enhanced in the E2 conformation. Thus the M5-M6 hair-pin is conformationally mobile, whereas there is little conformation-dependentchange of the peripheral segments M1, M3, M9, and M10.

Specific oxidative cleavage catalyzed by bound transition metals provides in-formation on proximity of cleaved polypeptide bonds (81). Fe2+ catalyzes specificcleavages of either Na,K-ATPase or H,K-ATPase (6, 82, 83) at two sites. Site Iis in the P and A domains (discussed below). Site II is at the membrane-waterinterface where Fe2+mediates cleavages near the entrance of M3 and M1, as doesalso a lipid-soluble complex of Cu2+ ions and bathophenanthroline (DPP) (54).A homology model confirms proximity of M3 and M1 at one point where thesequence 283-HFIH meets 81-EWVK to form a Fe2+ site II (85). Cleavages nearM3 and M1 are not affected by the E1/E2 conformations. This work indicates asimilar disposition of M3 and M1 in Ca,ATPase, Na,K-ATPase, and H,K-ATPase.

α-, β-, γ -Subunit Interactions

Several excellent reviews describe the role of theβ-subunit in stabilizing theα-subunit and facilitating its routing and insertion into the membrane, as well aseffects on functional properties, notably cation affinities (86–88). The strongestsubunit interactions exist between the extracellular loop L7/8 and theβ-subunit(86–88), but membrane and cytoplasmic sequences also play a part (86, 88). Recenttryptophan-scanning mutagenesis shows that two faces of Mβ interact with theα-subunit. Cys46 in theβ-subunit interacts with theα-subunit near M8 to which itcan be cross-linked (71, 72). Tyr40 and Tyr44 in theβ-subunit interact with otherα-subunit helices, which also affect K+ affinity (89).

Functional studies show that theγ - andα-subunits interact at more than oneposition (90), but the interaction sites ofα with γ or other FXYD proteins arelargely unknown. An interaction of theγ -subunit with a C-terminal portion ofthe α-subunit has been proposed based on thermal denaturation (90). A directcross-link ofγ to the M7-M10 fragment of 19-kDa membranes has been observedrecently (M. Fuzesi & S.J.D. Karlish, unpublished observation).

A Hypothesis Regarding Helix Packing

The helix packing of renal Na,K-ATPase proposed in the reconstruction basedon electron microscopy (13) assumes that the arrangement of theα-subunit andCa-ATPase are the same. Accordingly, Mβ is identified with a peripheral electron

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density (Figure 6A). This arrangement does not easily explain an interaction ofα with Mβ at more than one face (89). Also Mβ and M8 may be too far apartfor cross-linking (71). To resolve these inconsistencies, and those discussed abovein relation to L9/10, we propose an alternative arrangement in Figure 6B. Mβoccupies the position of M10 of Ca-ATPase, whereas M10 is displaced towardM9. Location of Mβ between M7 and M8 should allow Cys46 to approach M8,and Tyr40 and Tyr44 to approach M7. M10 (Cys983) should be close enough toM9 (Cys964) for S-S bridge formation. Finally,γ is close enough to M8 to permitcross-linking.

THE Na+- AND K+-ION-BINDING SITES IN THETRANSMEMBRANE REGION

The passage of cations through the low dielectric of the lipid bilayer is facilitatedby oxygen-containing side chains near the center of the bilayer and by interruptionof helices M4 and M6 in Ca-ATPase (16) and probably helix M5 in Na,K-ATPase(55). The helix dipoles of interrupted helices may impose a negative potential toattract the cations, and in the open loops, residues at positions -3 and -4 relative toprolines can expose main chain carbonyl oxygen groups for cation coordination(91).

Ca2+ ions may enter from the cytoplasmic side, between M2, M4, and M6,and be guided via a hydrophilic pathway to oxygen groups of the coordinatingresidues oriented toward the cytoplasm, in particular Glu309 and Asp800 (16).Electrostatic surface potential analysis, Figure 7a,b shows this region and thecorresponding region of Na,K-ATPase as red, negatively charged areas. On theextracellular side, a negatively charged grove, including Ile788 in Ca-ATPase,may serve as a cation outlet. The distance between these two putative entranceareas is not excessive and indicates that they are close to the membrane-waterinterface. The center of M4 and M6 are unwound loops with rows of exposedoxygen atoms, from Pro312-Glu309 in M4 and from Gly801-Asp800 in M6. Theside chains of Glu309, Asn768, Glu771, Asn796, Thr799, Asp800, and Glu908and several main chain carbonyl oxygens (Val304, Ala305, Ile307) contributesix coordinating groups for each of two Ca2+ ions (16). In theα-subunit ofNa,K-ATPase, the M5 helix may also be interrupted because the sequence 777-IPEITP is similar to the interrupted sequence 307-IPEGLP in M4 of Ca-ATPase(60). At the exit, there is a ring of oxygens with water molecules for rehy-dration, but a narrow water-filled access channel or ion well is not apparent(16).

In Na,K-ATPase, effects of several divalent or trivalent cations (Br-TITU),which act as Na-like competitive inhibitors of Na+ or Rb+ occlusion, led tothe concept of a cytoplasmic cation entry port, consisting of negatively chargedresidues in L6/7 that control access to the occlusion sites (92). Mutations of L6/7 ofCa-ATPase (Asp813, Asp815, Asp818) strongly reduce Ca+ affinity for activationof active Ca+ transport, suggesting a similar hypothesis (93). On the other hand,

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Figure 6 (A) Helix arrangement as in Hebert et al. (2002). (B)Alternative arrangement in the regions ofα-β- and α-γ -subunitinteraction.

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in the Ca-ATPase structure, Asp813, Asp815, and Asp818 appear too far apart tocreate a Ca+-binding site, implying that the mutations could have indirect effects(16). However, a peptide corresponding to L6/7 has been shown to form specific1:1 complexes with Ca+ and La+, and all three residues (Asp813, Asp815, andAsp818) are required (94). Thus L6/7 may have a dual role, mediating communi-cation and stabilizing the P and M domains as suggested by (16), and also actingas a port to control entry of Ca2+ ions (94).

Although the charges of Ca2+ and Na+ ions differ, their ionic radii and coor-dination properties are similar. In general, these hard cations coordinate to oxy-gen atoms of negatively charged or neutral groups. Two Na+-binding sites inNa,K-ATPase can therefore be assumed to be homologous to the two Ca2+ sitesfound in the Ca-ATPase structure. Sequence alignment and site-directed mutage-nesis are also in agreement with the role of residues Glu327, Asn776, Glu779,Asp804, and Asp808 in Na+ binding (55, 58–60). In site I, one of the Na+ ions iscoordinated to Asn776, Glu779, Thr807, and Gln923. In site II, the second Na+

ion coordinates to Glu327, Asp804, as well as to the carbonyl oxygen groups ofVal322, Ala323, and Val325, whereas the side chain of Asp808 coordinates to bothions (16, 32) (Figure 4a).

Location of a Third Na+ Site?

The model discussed above identifies two sites for binding of Na+, but the E1and E1-P forms are known to bind three Na+ ions, and a site for the third Na+

ion has not been identified. One possibility is that a third Na+ ion is bound nearthe middle of the membrane bilayer at the level of site I and II in Ca-ATPase.The coordinating groups might be located in M8 and M9, although mutagenesisdata have not revealed significant contributions to binding of Na+ from Glu953or Glu954 in M9 (95). Asp925 in M8 may contribute up to 5 kJ/mol to the freeenergy of Na+ binding (55, 96), but this residue is positioned in the cytoplasmichalf of the bilayer. In addition, the location of the third Na+ site in the middle of themembrane, near sites I and II, may not be compatible with the electrogenic bindingof the third Na+ ion with a dielectric coefficient of 0.25 in a neutral binding site orwith the electrogenic release with a dielectric coefficient of 0.7 at the extracellularsurface of the first Na+ ion from the E2-P form. For references to the literature onelectrogenecity of Na+- and K+-transport, see earlier reviews (97–99).

Another possibility for locating a third Na+ site is a cytoplasmic neutral site forbinding of a partially dehydrated Na+ ion. In a model of Na,K-ATPase built on thebackbone of the high-resolution structure of Ca-ATPase, a cavity appears betweenM4, M5, and M6 where Ser768 in Na,K-ATPase replaces Phe-760 in Ca-ATPase(Figure 4b) (K.O. Hakansson & P.L. Jorgensen, submitted for publication). A par-tially dehydrated Na+ ion can be docked into this site, 9–10A on the cytoplasmicside of the other two Na+ ions in sites I and II. The Na+ ion is coordinated bythe side chain and main chain oxygens of Ser768 and three water molecules andmakes contacts with Thr772, consistent with the selective effect on Na+ binding on

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mutation of Thr772 (60). Figure 4b illustrates how binding and partial dehydrationof a Na+ ion in a neutral site prior to binding in site I may explain both the electro-genic binding of the third Na+ (d = 0.25) (d is the dielectric distance explainedin References 97–99) and the electrogenic release of the first Na+ ion (d = 0.7)(97–99). Charge movements detected by electrochromic dyes are compatible witha Na+ site in the cytoplasmic membrane leaflet in Na,K-ATPase, whereas there isno indication of a cytoplasmic Ca2+ site in Ca-ATPase (100).

K+-Binding Sites

Structural changes in the cation-binding site during E1-E2 transition cause a switchin the selectivity from the Na+ ion (D = 1.9 A) to the somewhat larger K+ ion(D = 2.7A) (55). Most of the residues contributing to Na+ binding also bindK+ in the E2 form (55). The alkali metal ions do not move from one set of sitesto another; rather sites I and II are bifunctional and their coordination geometryand orientation are rearranged during the E1-E2 transitions. The E2 models of Ca-ATPase, 1FQU and 1KJU, show that the relative positions of M5 and M6 areroughly the same in E1 and E2, but that helices M4 and M8 move relative to thesetwo helices. In the case of Na,K-ATPase, this could mean that Glu327 and thecoordinating carbonyl oxygen groups of residues 322, 323, and 325, as well asGln923, move away, creating a more spacious cavity for binding of the largerK+ ion. The E2 structure of Na,K-ATPase, although not of sufficient resolution tovisualize individual helices, demonstrates that the E2 conformation is similar inthe two enzymes (12, 13).

Owing to the high K+ affinity, direct binding of two Rb+ or Tl+ ions perα-subunit can be measured at equilibrium in recombinant Na,K-ATPase (59, 60)after expression at high yield inSaccharomyces cerevisiae(101). Each of fourcarboxylate groups, Glu327 in M4, Glu779 in M5, Asp804, and Asp808 in M6,are essential for high-affinity binding Tl+ with Kd K 7–9µM in the wild-typeenzyme (58, 59). Significant contributions to K+ or Tl+ binding are provided byTyr771 (60), Ser775 (60, 102), and Asn776 (55, 60, 103). Parallel assays of K+

binding show that Tl+ and Rb+ are adequate analogues for K+ (55). Mutationsof Asp804 and Asp808, except Asp808Glu, are devoid of Na,K-ATPase activity(58, 104), whereas partial activities remain after mutations of Glu327 and Glu779(105, 106). In mutations of Glu327 or Glu779, the E2 form is destabilized andthe conformational equilibrium is poised in favor of the E1 form, with high ap-parent affinity for ATP and a greatly increased Na-ATPase activity in the absenceof K+(103, 106). Large changes in the currents generated by the recombinantNa,K-ATPase with Glu779Ala mutation (107) also implicate the 775-SNIPEITPsegment of M5 in Na+/K+ selectivity at the extracellular face.

Cation Specificity

The E1 and E2 conformations differ with respect to orientation and specificity ofcation sites for Na+ and K+. The adjustment of the cation sites from the E2 [2K]

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form with specificity for K+ over Na+ to the E1ATP form with specificity for Na+

over K+ is accompanied by changes of both the number and distances of coordi-nating groups from the bound cations. Twisting or tilting of the intramembranehelices may adapt the distances between the oxygen atoms of the coordinatinggroups of sites I and II (55, 60). In the E2 [2K] form, the dimensions are suitablefor coordinating two dehydrated K+ ions and orientation is toward the extracellu-lar phase. In the E1ATP form, the geometry is suitable for coordination of threedehydrated or partially dehydrated Na+ ions and the sites are oriented toward thecytoplasm.

Relative cation specificities of the two conformations can be estimated from ap-parent affinities at the cytoplasmic and extracellular surfaces. The apparent affini-ties for Na+ and K+ at the cytoplasmic surface have been estimated to be 0.6 and10 mM, respectively, corresponding to a 16-fold preference for Na+ over K+, atthe cytoplasmic surface. This requires that Na+ binding is stabilized by 6.4 kJ/molmore than binding of K+ (60). Specific contributions in the range of 4–7 kJ/molfrom Thr774, Thr776, Ser775, and Tyr771 in the cytoplasmic part of M5 ap-pear to be relevant for the selectivity for Na+ at the cytoplasmic surface (55, 60).At the extracellular surface, the affinities for Na+ and K+ amount to 600 and0.2 mM, respectively. The extracellular K/Na-affinity ratio of 3000-fold requiresthat K+binding is 18.4 kJ/mol more stable than binding of Na+. Glu327 contributes4–8 kJ/mol more to binding of Tl+ or K+ than to binding of Na+, but the contri-bution of several other residues appears to be required (55, 60).

ENERGY TRANSDUCTION, CYTOPLASMIC DOMAININTERACTIONS, AND THE CATALYTIC CYCLE

A characteristic feature of E1-E2 transitions is that the active site alters its sizeowing to domain association and dissociation and its inclination through tiltingor rotation of the P domain. The relationship between the N and P domains doesnot change much during the reaction, but association and dissociation of the Adomain is a prominent feature (16, 17). In the first step, ATP binding to the E2[2K]conformation displaces the A from the N and P domains and stabilizes the E1ATPconformation. In the E1 or E1-P conformations, the N domain remains docked ontothe P domain with the A domain displaced to one side. In the E2[2K] and E2-Pconformations, the A domain docks onto the P and N domains and interferes withthe tight N to P interactions. (8, 107a). The concepts, depicted in the schematic di-agrams in Figure 8, are applicable to both Na,K-ATPase and H,K-ATPase. Similarmodels for Ca-ATPase have been proposed recently (17, 108). Presumably, theyapply to all P-type ATPases.

N, P, and A Interactions in E1 and E2 Conformations

In light of the high-resolution structure, the exposure and protection of proteolyticcleavage sites in Na,K-ATPase and Ca-ATPase can now be interpreted in terms of

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Figure 8 Schematic diagrams of N, P, and A interactions and ATP-Mg2+- orMg2+-binding sites in the different states of the catalytic cycle.

cytoplasmic domain interactions. Association and dissociation of the A domainfrom P domain can be monitored by tryptic and chymotryptic cleavage (1) ortryptic, V8, and proteinase-K cleavages of Ca-ATPase (56, 57, 108, 109). Disso-ciation of the A domain from the P domain in E1[3Na] or E1-P[3Na] forms ofNa,K-ATPase exposes the bond at Leu266 (Cl) in the L2/3 loop to chymotrypsin(1, 110). Similarly, sites for tryptic, V8, and proteinase-K are exposed in the E1

[2Ca] form of Ca-ATPase (56, 57).

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Tryptic cleavage patterns reflect interaction between the A domain and theN domain in the E2[2K] or E2-P[2Na] forms. Trypsin cleaves theα-subunit atArg438 (T1) in the N domain and subsequently at Lys30 in the A domain. Lossof Na,K-ATPase activity is linear and associated with cleavage at Arg438. Thebiphasic inactivation of K phosphatase has a typical delay indicating that cleavageat Arg438 precedes cleavage at Lys30 (1, 111). Arg438 is exposed at the surface ofthe N domain close to a loop pointing toward the A domain. High-affinity bindingof ATP interrupts this interaction and exposes the T2 site (K30) to rapid trypticcleavage (111). The cleavage patterns are compatible with an interaction betweenthe 438-RAVAGDA loop in the N domain and the segment around Lys30 in theA domain in the E2 forms of Na,K-ATPase. The functional significance of theseconnections is apparent from the changes in catalytic properties because cleavage atLys30 strongly reduces the rate of the E1-P to E2-P conversion, stabilizing the E1

conformations three- to fourfold (112).Rotation of the A domain accompanying the E2 to E1 transition exposes Lys 30

(T2) in the N terminus to rapid cleavage, whereas Arg 262 (T3) in the exposed L2/3loop is cleaved 30-fold more slowly, resulting in biphasic inactivation of Na,K-ATPase in the E1 conformations (1). Synergistic effects on function of a truncationat M32 and a G233K mutation demonstrate the interplay of these segments in theA domain (113).

Changes in N, P, and A domain interactions also explain effects of K+ or Na+

ions on covalent labeling of Lys501 by various reagents (114) or cross-linkingof Lys501 to Lys480 (115). ATP or low concentrations of K+ ions protect theenzyme, whereas Na+ ions accelerate inactivation. Protection by ATP is expectedbecause Lys501 and Lys480 are contact residues. The cation effects are explainedmost simply by altered accessibility of Lys501 and Lys480 to the reagents. K+

stabilizes E2[2K] with a closed organization of the N, P, and A domains. Na+ ionsstabilize E1Na with the open N, P, and A domain organization.

The most direct evidence for P and A domain interactions has come from Fe2+-catalyzed cleavages of Na,K-ATPase or H,K-ATPase (116–118)α-subunits. In theE2[2K] form, Fe2+ bound at site I catalyzes splits in the conserved sequences ofthe A domain (at 214-ESE) and P domain (near 367-CSDK, near 608-MVTGD,and at 712-VNDS). The observations led to the prediction that the A and P do-mains interact near these sequences. In E1 conformations, cleavages at site I areabsent, although those at Fe site II remain. This can be explained by separationof the cytoplasmic domains so that Fe site I no longer exists. These inferencesare confirmed by the Ca-ATPase structure in the E1 form, or models in the E2conformation which indeed demonstrate proximity of A to P and also A to N do-main interactions (16, 17). Glu214 in the TGES sequence appears to be the linch-pin of the A to P domain interaction because it is hydrogen bonded to Asp369,Thr371, Lys370, Thr610, and Gly611. Suppression of different Fe2+-catalyzedcleavages within site I by Pi or Pi, Mg2+, ouabain, and vanadate/Mg2+ suggest thatPi interacts with residues in 608-MVTGD and 367-CSDK and Mg2+ with 212-TGES and 712-VNDS in the E2-P conformation (116). These predictions fit wellwith the structure of phosphoserine phosphatase (47) and phosphonoacetaldehyde

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hydrolase crystallized in the presence of Mg2+ and PO3−4 ions (46) or the phos-

phate analogues, BF−3 and WO2−4 (45), except that involvement of 212-TGES can-

not be tested for the HAD proteins, which lack an A domain and the homologousresidues.

Binding of ATP or MgATP to E1 (N↔P Interactions)and E2(K) (N↔A Interactions)

In the initial step of the Na,K-ATPase reaction cycle, binding of ATP to the E2[2K]conformation with low affinity (Km∼ 0.2–0.4 mM) accelerates the E2[2K]-E1(2K)transition, accompanied by release of K+ at the cytoplasmic surface. Mg2+ isnot required for this effect of ATP. ATP is bound with high affinity in the E1

conformation (Kd∼ 30–100 nM) (1, 7) stabilizing the E1ATP form by comparisonwith the E2[2K] form.

In Na,K-ATPase, binding of free ATP to E1 conformations in the absence ofMg2+ elicits strong electrostatic repulsion between theγ phosphate and the nega-tive charges at the surface of the P domain. Removal of the charge of the carboxylategroups in the Asp369Ala mutation increases the equilibrium-binding affinity forATP 30- to 40-fold (119). Substitution of Ala for Asp710 causes a more moderatetwo- to threefold increase of the ATP affinity (52). The apparent dissociation con-stant for wild-type Na,K-ATPase ofKapp

d = 39 nM is reduced toKappd = 13 nM

in Asp710Ala, and in the Asp369Ala mutation, theKappd is only 1.8 nM. The data

allow estimation of the free energy required to overcome the electrostatic interac-tion between theγ phosphate of ATP and the carboxylate groups to 2 kJ/mol forAsp710 and 7.9 kJ/mol for Asp369.

The strong electrostatic interaction of theγ phosphate of the tightly bound ATPwith the negative charges of Asp369 and Asp710 at the surface of the P domain inNa,K-ATPase shows that the N and P domains must be closed in the ATP-E1[3Na]form, even in the absence of Mg2+ ions (52, 119). As expected, coordination ofMg2+ in the negatively charged surface of the P domain reduces the electrostaticrepulsion of the phosphate groups and thus facilitates ATP binding. In presenceof Mg2+, the curve for ATP dependence of phosphorylation has a 0.5K value of12 nM ATP (25). This is at least threefold lower than theKd = 38 ± 5 nM forbinding of free ATP at equilibrium in the absence of Mg2+. An important functionof the negative charges of Asp369 and Asp710 could be to counterbalance the veryhigh intrinsic binding affinity of ATP for Na,K-ATPase. As proposed by Jencks(22), high-energy intermediates are likely to act as barriers to rapid turnover oftransport systems.

In order to explain ATP binding, a model of Ca-ATPase has been proposed inwhich rotation of the N domain about flexible hinges at Asn359 and Arg604 bringsN and P domains into alignment (17). The model also explains cross-linking ofLys492 (N domain) and Arg678 (P domain), which come into close proximity(120). This implies that the N to P domain interaction may occur even withoutATP or ATP-Mg, although binding of the nucleotides should stabilize closure. Thefact that Na,K-ATPase binds free ATP with a high affinity in the E1 state whereas

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Ca-ATPase binds only MgATP (121) may be explained if the N to P interaction ismuch stronger for Na,K-ATPase than for Ca-ATPase.

The complex of ATP or the non-phosphorylating analogues AMP-PNP withFe2+ act as affinity cleavage agents of Na,K-ATPase or H,K-ATPase (54, 83).Fe2+ substitutes for Mg2+ ions in catalyzing ATPase activity and phosphorylation(122). Thus the cleavages reveal points of contact of the Fe2+ or Mg2+ ions. InE1 conformations, ATP-Fe or AMPPNP-Fe catalyzes two cleavages at and near712-VNDS in the P domain and near 440-VAGDA in the N domain. The A domainis not in contact with bound ATP-Fe2+ and is not cleaved. The data imply thatMg2+ bound to ATP is in close proximity to both 708-TGDGVNDS in the Pdomain, presumably Asp710 and Asp714, and 440-VAGDASE in the N domain.This predicted N to P closure provides a testable constraint for model building.

Using an approach similar to that in Reference 17, N and P domains have beenbrought into proximity by tilting the N domain about 80◦.(107a). Asp703 (thehomologue of Asp710 in Na,K-ATPase) and Glu439 (the homologue of Asp443in 440-VAGDASE) can be brought within 4A of each other. With the ATP-Mg2+

docked, the model shows nucleotide interacting with Lys515, Lys492, Phe487,Arg560, and phosphates are close to Asp351, Thr353, Thr625, Lys684, Asp703,and Asp707, consistent with the known facts. The Mg2+ ion is close to bothAsp703 and Glu439. Thus cleavage and modeling fit well with the mutation datain identifying of Asp710 in Mg+ binding and point to Asp443 as an additionalcandidate.

The models of Ca-ATPase in the E2 conformation demonstrate N to A domaininteractions (16, 17). Specifically, Glu439 comes within 4A of Ser186 or 3A ofArg174 (A), and residues Glu486 and Arg489 (N) interact with Ser170-Thr171and Thr191-Glu192 (A). Recently, ATP-Fe2+ complex-mediated cleavages in theE2[2Rb] conformation have been detected near 212-TGES (A domain), near 440-VAGDA, and at a site between residues 460 and 490 (both in the N domain) (107a)that demonstrates directly the N to A domain interaction.

Comparison of the models for E2 (1KJU)- and E1-bound ATP can, at leastin part, explain the difference in affinity of ATP in E1 and E2 conformations.In E1, ATP is bound to residues in both N and P domains, whereas in the E2

conformation, the A domain interferes with the close N and P domain interaction.The low ATP affinity could be explained by the lack of accessibility to Asp351,Asp703, Asp707, Thr353, and Lys684 side chains within the P domain when theseresidues are completely overlaid by the A domain in the E2 conformation. In theE2 model, Glu183 appears to provide key contacts between the A to P domainsby H-bonding to Asp351, Lys352, Thr353, Thr625, and Gly626, several of which(Lys352, Thr353, Thr625) are predicted to be in direct contact with ATP in the E1

conformation. In addition, the A domain hinders access of ATP to Lys684. Thus inessence, stabilization of E1 by ATP can be understood as the result of competitionbetween the ATP and the A domain for contact residues in the P domain (107a). Inthis way, binding of ATP to E2[2K] with a low affinity induces the large outwardmovement of the A domain during the E2→ E1 transition and brings the P domainback into close proximity with the N domain (17).

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Mg2+ Binding and Formation of the MgE1-P[3Na] Complex

In the 1KJU model of the E2 conformation of sarcoplasmic reticulum Ca-ATPase(17), the transition from E2 to E1 involves a 53◦ rotation about an oblique axisof the P domain, which carries the N domain with it in addition to the movementof the A domain by 90◦ about a vertical axis. These motions can render the surface ofthe Rossmann folds with Asp369 and Asp710 more reactive toward binding of ATPand Mg2+ in the E1 conformation, but the transfer of theγ phosphate to Asp369occurs only after binding of three Na+ ions in the sites on the transmembranesegments.

Mg2+ is essential for all phosphoryl transfer reactions and for the E1-P to E2-Pconversion in Na,K-ATPase. To determine binding constants for Mg2+ binding itis important to stabilize either the E1-P or the E2-P conformations. In pig kidneyNa,K-ATPase, the E1-P-E2-P equilibrium is poised heavily in favor of the E2-P,and oligomycin is required to stabilize the E1-P form (1). The high affinity of theMgE2-P complex for ouabain provides a convenient assay of Mg2+ binding in theE2-P, but that conformation can be distinct from the MgE2-P[2Na] intermediate inthe catalytic cycle.

Analysis of mutations in the 708-TGDGVND loop shows that the Na,K-ATPaseactivity is severely reduced in mutations of Thr708, Asp710, Asn713, and Asp714(52). In plots of Na+-dependent phosphorylation versus free Mg2+ the apparentMg2+affinity, [Mg2+]0.5, for wild-type is 24± 5µM. A 27-fold reduction of Mg2+

affinity is observed for the Asp710Asn mutation, whereas phosphorylation is al-most abolished for the Asp710Ala mutation. Removal of the carboxamide groupof Asn713 causes a moderate 4-fold reduction of Mg2+ affinity (52). Coordinationof Mg2+ to Asp710 can establish a link via theγ phosphate to Asp369. The roleof Asp710 in this scheme is to stabilize Mg2+ in a position that facilitates nucle-ophilic attack of Asp369 to form the acyl phosphate intermediate (123), and Mg2+

shields the negative charges of the phosphate moiety and the two carboxylates.Another contribution to coordination of Mg2+ is the main chain carbonyl oxygenof Thr-371 in the 369-DKTGGT segment of the phosphorylated residue (Thr353 inCa-ATPase); however, the side chain hydroxyl of Thr371 (Thr-353) may partici-pate in interactions with nucleotide and phosphate (124).

Because ATP-Fe2+ serves as a substrate, the enzyme becomes phosphorylatedin the presence of ATP-Fe2+ and Na+ ions (83). In the presence of oligomycin,the E1-P form is stabilized. In this state, bound Fe2+ catalyzes exactly the samecleavages as in the E1 state (54). Thus the N to P interaction and Fe2+ or Mg2+

binding in 712-VNDS and near 440-VAGDA is assumed to be unchanged.

Transition from E1-P to E2-P (A to P Interaction)

During the transition to E2, the conserved 212-TGES loop engages with the surfaceof the P domain. Mutagenesis studies have not yet identified residues ligating Mg2+

in the E2-P conformation, but the Ca-ATPase structure suggests alternative ligatingloops in the A domain.

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Upon phosphorylation of Na,K-ATPase (ATP-Fe2+, Na+) or H,K-ATPase (ATP-Fe2+, Tris) to E2-P, Fe2+ is tightly bound and a major cleavage occurs near212-TGES in the A domain, whereas the cleavages near 712-VNDS are less promi-nent (54). This is indicative of the A to P domain interaction. In addition, the N to Pdomain interaction is relaxed because the cleavage near 440-VAGDA (N domain)is not seen in E2-P. The shift of Mg2+ coordination to the 212-TGES segment couldbe important for forming a bridge to stabilize interactions between the P and Adomains, and it may also have a crucial functional significance. The active sitecontains the Asp369 bound with phosphate, Mg2+ ions bound to the phosphate,and the 212-TGES sequence. A change in geometry of ligands around the phos-phate and the more hydrophobic environment produced by the domain closure maybe required for inducing reactivity of E2-P to water, compared with the reactivityof E1-P to ADP. Mg2+ ions interact less strongly with Asp710, although Asp710,Asp714, and Asn713 are still in close proximity.

Mutagenesis results imply that in E2-P the 708-TGDGVND loop is not requiredfor Mg2+ recognition but rather for stabilization of the transition state and for K+

stimulation of acyl-phosphate bond hydrolysis in the E2-P form. Asp710 maycontribute directly to coordination of pentacovalent vanadate in the transition state(52). Removal of the Asp710 also interferes with transmission of the structuralchange to the cation sites and ouabain-binding domain at the extracellular surface.The Asp710Ala mutation causes a threefold reduction of the affinity for bindingof Tl+ (52). In the forward reaction, this corresponds to a role of Asp710 intransmission of the K+ stimulation of phosphoenzyme hydrolysis from M5 to theP domain. Overall, the effects of mutations in the 708-TGDGVND loop on bindingof Mg2+ in E1-P and E2-P forms complement the results of Fe2+-catalyzed cleavage.

ENERGY TRANSDUCTION, LONG-RANGEINTERACTIONS BETWEEN CYTOPLASMICAND MEMBRANE DOMAINS

ATP binding, phosphorylation, and cytoplasmic domain movements associatedwith E1↔E2 transitions are tightly coupled to the vectorial processes in the in-tramembrane domain via long-range structural changes. The E2[2K] → E1(2K)transition is associated with an increase in binding energy of ATP, which drives K+

transport into the cell. The E1-P→ E2-P transition in the P domain is coupled toextrusion of Na+ from the cell. These long-range effects could be mediated directlyvia the P domain and S4 and S5 or by an indirect route via the large movementsof the A domain and the loops (L2/3 and L6/7) connecting to the transmembranesegments. The evidence for either path is not decisive, and it is likely that bothmechanisms could apply.

ATP-Driven K+ Uptake

The ATP-K+ antagonism is unique to Na,K-ATPase because this is the onlycation pump that possesses high-affinity binding sites for both ATP and K+. The

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structural adaptation following conversion of the E2[2K] → E1 transition drivenby high-affinity binding of ATP elicits an outward rotation of the A domain. Com-parison of E1 and E2 structures (Figure 1) suggests that there is a particularlylarge rearrangement of N domain structure in the region of the T1 cleavage site atthe 438-RAVAGDA loop, a known site of contact between the N and A domains(8). The large outward movement of the A domain alters the position of the L2/3loop and transmits the movement via the triangular hydrogen-bonded connectionto the P1-helix of S4 and the L6/7 loop. The significance of these connectionsfor energy transfer is apparent from the consequences of chymotryptic cleavageat Leu266 (1). This split eliminates the normal ATP-K antagonism, preventingATP-driven uptake of K+, normal displacement of occluded Rb+ by 1 mM ATP,and displacement of ATP binding by K+ ions, so that ATP is bound with highaffinity in both NaCl and KCl (110). These results show that there is only a verylimited contribution of direct energy transfer from the ATP-binding site in theN domain to the release of K+ ions from the binding sites to the cytoplasmicsurface.

E1-P to E2-P Transitions and Na+ Movements

It is also a question whether the long-range conformational transitions coupling theconversion of E1-P[3Na]→E2-P[2Na] to extrusion of Na+ are transmitted directlyor indirectly to the cation sites. With the architecture of the P domain in mind,direct transmission via S4 and S5 to M4 and M5 is clearly an option, but thereis considerable evidence for indirect transmission via the A domain and the L2/3loop and its connections to L6/7, M4, M5, and M6 (8). Chymotryptic cleavageat Leu266, which causes interruption of the L2/3 loop (Figure 3), blocks Na+

translocation and stabilizes the protein in the E1-P[3Na] form (110), thus allowingthe first demonstration of occlusion of Na+ (125). Similar to proteolytic splits,the mutations Gly263Ala and Arg264Ala (ratα1) stabilize the E1 conformation(126). In Ca-ATPase, a number of residues in L2/3 are crucial for the E1-P toE2-P transformation. They are located in the portion of L2/3 that connects theA domain to transmembrane segment M3 (127) or in S4, the stalk that links theP domain to M4 (128). Mutations in the P1-helix also prevent transition to theE2-P form (129), thus underlining the importance of the triangular connectionfrom L2/3 to the P1-helix and to L6/7. Additional hydrogen bonds and van derWaals interactions between L6/7 and M5 are described in the structure of Ca-ATPase (16). Structural changes can also be transmitted from the N-terminal partof the A domain through the helix loop connecting to M2. Both transmembranesegments M2 and S2, the helix loop connecting to the A domain, are in closecontact with M4 and its extension S4.

A direct transmission route is favored by two sets of information. First, a roleof the S4 segment is implied by observations that mutations of several conservedresidues of Ca-ATPase S4 (T316-356) (124), or an analogous segment ofS. cere-visiaeH-ATPase (129), stabilize E1 conformations. Second, mutations of Asp369or Asp710 to neutral residues stabilize E2 conformations, and double mutations

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(such as Asp369Ala, Asp710Asn) do not give greater effects (52, 119). Asp369and Asp710 need to be close in E1 in order to bind the ATP-Mg2+ complex. Onecan hypothesize that electrostatic repulsion between Asp369 and Asp710 stabi-lizes the E1 conformation, but when these are neutralized in E1-P-Mg2+ through Piand Mg2+ binding, interactions stabilizing E2-P predominate, notably those of the212-TGES loop in A with N and P domains. The resulting change in Mg2+ ligationand environment in E2-P makes it reactive with water, and the altered disposition ofAsp369 and Asp710 may trigger movement of S4 and S5 and the transmembranesegments.

Na+-Dependent Phosphorylation andK+-Activated Dephosphorylation

In contrast to the ion translocation reactions, long-range structural changes fromthe cation sites to the P domain appear to be communicated exclusively by a directmechanism. Activation of phosphorylation from ATP by binding of the third Na+

ion involves nucleophilic attack of Asp369 on theγ phosphate of ATP, whereasbinding of two K+ ions to E2-P[2K] stimulates nucleophilic attack by water on theAsp369–C–O–P bond and hydrolysis of phosphoenzyme. These cation-specificsignals are transmitted directly to the P domain without altering the associationwith the A or N domains.

The signal for K+ activation of E2-P hydrolysis appears to be transmitted di-rectly through M5 and S5 to the active site. This route of transmission has beenproposed after analysis of a series of mutations of residues in S5 of Ca-ATPase(130). The mutations interfere with transmission of conformational changes fromthe cation-binding sites in the membrane domain to the P domain controlling de-phosphorylation. Arg751 (Arg758 in Na,K-ATPase) in S5 is hydrogen bonded toL6/7 (Figure 3), and this residue is important for the structural integrity of the en-zyme. Through this and other connections in the network of hydrogen bonds andvan der Waals interactions of L6/7, the structural change can also be transmittedfrom K+ coordinating residues in M4 and M5 to the P domain without involvingthe A domain. Alanine substitution of Asp369, Asp710, or Asn713 eliminates theK+-stimulatedp-nitrophenylphosphate hydrolysis. The mutations have dramaticand specific effects, as removal of the carboxylate group of Asp710 eliminatesvanadate binding and removal of the carboxamide of Asn713 eliminates the phos-phate dependence of ouabain binding (52). The 708-TGDGVND loop is thereforerequired for stabilization of the transition state and for the dephosphorylationreaction.

A Na+-specific conformational change in the N domain has been detected byfluorescence (131). The analysis shows that the third Na+ ion binds to the neutralsite only after binding of two Na+ ions and triggers Na-dependent phosphorylationpresumably by inducing correct alignment of theγ phosphate of ATP with theAsp369. Effects of the Na+ ions in E1 could also be transmitted by the direct routevia M5 and S5.

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CONCLUSIONS AND PERSPECTIVES

This review demonstrates the power of homology models of theα-subunit of Na,K-ATPase, based on the high-resolution structure of Ca-ATPase (16), for analysis ofstructure-function relationships and molecular mechanisms of the conversion ofthe free energy of ATP hydrolysis to active transport of Na+ and K+ ions. Informa-tion from site-directed mutagenesis and cleavage experiments has been combinedwith molecular modeling, which allows us to discuss in detail the events in theactive sites, including binding of ATP and Mg2+ and the transfer of energy tothe cation sites for each reaction of the catalytic cycle. The progress in under-standing the structure and function of theα-subunit underlines the gaps in ourknowledge of the structure and subunit interactions of theβ- and γ -subunits,CHIF (corticosteroid-induced factor), and other members of the family of smallion transport regulators of the FXYD family, which are subunits of the Na,K-pump. Coexpression ofα1β1-subunits with theγ -subunit or CHIF regulates theactivity of the Na,K-pump (132) in the medullary thick ascending limb of Henleor inner medullary collecting duct. These nephron segments are important forthe regulation of water and salt excretion. High-resolution structural informationabout these Na,K-pump protein complexes is essential for solving the molecularmechanism of this regulation and is therefore a question of primary physiologicaland pharmacological significance.

ACKNOWLEDGMENTS

Work in the authors’ laboratories is supported by grants from the Carlsberg Foun-dation (KOH), the Danish Natural Science Research Council (PLJ and KOH), andthe Israel Science Foundation (SJK).

The Annual Review of Physiologyis online at http://physiol.annualreviews.org

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22 Jan 2003 21:37 AR AR177-34-COLOR.tex AR177-34-COLOR.SGM LaTeX2e(2002/01/18)P1: GDL

Figure 1 Models of E1 and E2 forms of the a-subunit of Na,K-ATPase based onthe high-resolution structure of Ca-ATPase (1EUL) (16) in the E1[2Ca] form and astructure model (1KJU) based on the 6A electron microscopy structure of the E2 formof Ca-ATPase stabilized by decavanadate and thapsigargin (17). Side chains in ball-and-stick are shown for Phe475, Lys501, and Arg 544 in the N-domain (green), forD369 and D710 in the P-domain (blue), and for Glu327, Glu779, Asp804, and Asp808in the cation-binding domain (gray). The position of the N-domain, which in the crystalstructure of Ca-ATPase might be displaced by crystal contacts (16), has been adjustedto allow spanning of an ATP molecule over the N- and P-domains (8). The resultingrelative positions of the N and P domains conform well with those observed in the E2form by electron microscopy (12, 13, 17).

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Figure 3 Direct or indirect transmission of structural changes from P-domain tocation sites? Homology model of the connections from the A-domain via L2/3 to M3,P1-helix, and L6/7 to M4, M5, and M6. Side chains of theα-subunit of Na,K-ATPaseare modeled on the backbone of Ca-ATPase in the E1[Ca] form (16). CHYM indicatesthe position of the peptide bond (Leu266), which is cleaved by chymotrypsin in theE1[3Na] form of theα-subunit of Na,K-ATPase (1). T2-TRYP (Arg198) and V8 PROT(Glu231) denote positions of bonds cleaved in the E1[2Ca] form of Ca-ATPase (56,57).

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Figure 4 (A) Ball-and-stick representation of Na+-binding sites I and II; homology modelof Na,K-pump side chains on the backbone of the high-resolution structure of SR Ca-ATPase(1EUL) (16). (B) Proposed mechanism of Na+ translocation. The entrance of the third Na+

from the cytoplasmic site and the release of the first ion on the extracellular side are elec-trogenic owing to a bucket brigade mechanism. Binding of the third Na+ will displace thesecond Na+ to site I. The E1-P-E2-P transition displaces the third Na+ ion from the entry siteto site I and expels the second Na+ ion (K.O. Hakansson & P.L. Jorgensen, submitted). (C)Probing for channels through the model of Na,K-ATPase structure with the HOLE softwarepackage (K.O. H˚akansson & P.L. Jorgensen, submitted).

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Figure 5 A homology model of Na,K-ATPase with transmembrane helices seen fromthe extracellular side. Note residues highlighted in extracellular loops, particularlyL9/10.

Figure 7 Grasp figure showing the electrostatic potential on the surface. (Left) Ca-ATPase. (Right) A Na,K-ATPase homology model (K.O. H˚akansson & P.L. Jorgensen,submitted). Positive charge is shown in blue and negative charge in red. Arrow pointsat the suggested site for Na+ entrance on the cytoplasmic side between M2, M4, andM6.

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January 17, 2003 11:23 Annual Reviews AR177-FM

Annual Review of Physiology,Volume 65, 2003

CONTENTS

Frontispiece—Jean D. Wilson xiv

PERSPECTIVES, Joseph F. Hoffman, Editor

A Double Life: Academic Physician and Androgen Physiologist,Jean D. Wilson 1

CARDIOVASCULAR PHYSIOLOGY, Jeffrey Robbins, Section Editor

Lipid Receptors in Cardiovascular Development, Nick Osborneand Didier Y.R. Stainier 23

Cardiac Hypertrophy: The Good, the Bad, and the Ugly, N. Freyand E.N. Olson 45

Stress-Activated Cytokines and the Heart: From Adaptation toMaladaptation, Douglas L. Mann 81

CELL PHYSIOLOGY, Paul De Weer, Section Editor

Cell Biology of Acid Secretion by the Parietal Cell, Xuebiao Yaoand John G. Forte 103

Permeation and Selectivity in Calcium Channels, William A. Satherand Edwin W. McCleskey 133

Processive and Nonprocessive Models of Kinesin Movement,Sharyn A. Endow and Douglas S. Barker 161

COMPARATIVE PHYSIOLOGY, George N. Somero, Section Editor

Origin and Consequences of Mitochondrial Variation in VertebrateMuscle, Christopher D. Moyes and David A. Hood 177

Functional Genomics and the Comparative Physiology of Hypoxia,Frank L. Powell 203

Application of Microarray Technology in Environmentaland Comparative Physiology, Andrew Y. Gracey andAndrew R. Cossins 231

ENDOCRINOLOGY, Bert W. O’Malley, Section Editor

Nuclear Receptors and the Control of Metabolism,Gordon A. Francis, Elisabeth Fayard, Frederic Picard, andJohan Auwerx 261

vii

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viii CONTENTS

Insulin Receptor Knockout Mice, Tadahiro Kitamura, C. Ronald Kahn,and Domenico Accili 313

The Physiology of Cellular Liporegulation, Roger H. Unger 333

GASTROINTESTINAL PHYSIOLOGY, John Williams, Section Editor

The Gastric Biology of Helicobacter pylori, George Sachs,David L. Weeks, Klaus Melchers, and David R. Scott 349

Physiology of Gastric Enterochromaffin-Like Cells, Christian Prinz,Robert Zanner, and Manfred Gratzl 371

Insights into the Regulation of Gastric Acid Secretion Through Analysis ofGenetically Engineered Mice, Linda C. Samuelson and Karen L. Hinkle 383

NEUROPHYSIOLOGY, Richard Aldrich, Section Editor

In Vivo NMR Studies of the Glutamate Neurotransmitter Fluxand Neuroenergetics: Implications for Brain Function,Douglas L. Rothman, Kevin L. Behar, Fahmeed Hyder,and Robert G. Shulman 401

Transducing Touch in Caenorhabditis elegans, Miriam B. Goodmanand Erich M. Schwarz 429

Hyperpolarization-Activated Cation Currents: From Moleculesto Physiological Function, Richard B. Robinson andSteven A. Siegelbaum 453

RENAL AND ELECTROLYTE PHYSIOLOGY, Steven C. Hebert, Section Editor

Macula Densa Cell Signaling, P. Darwin Bell, Jean Yves Lapointe,and Janos Peti-Peterdi 481

Paracrine Factors in Tubuloglomerular Feedback: Adenosine, ATP,and Nitric Oxide, Jurgen Schnermann and David Z. Levine 501

Regulation of Na/Pi Transporter in the Proximal Tubule,Heini Murer, Nati Hernando, Ian Forster, and Jurg Biber 531

Mammalian Urea Transporters, Jeff M. Sands 543

Terminal Differentiation of Intercalated Cells: The Role of Hensin,Qais Al-Awqati 567

RESPIRATORY PHYSIOLOGY, Carole R. Mendelson, Section Editor

Current Status of Gene Therapy for Inherited Lung Diseases,Ryan R. Driskell and John F. Engelhardt 585

The Role of Exogenous Surfactant in the Treatment of Acute LungInjury, James F. Lewis and Ruud Veldhuizen 613

Second Messenger Pathways in Pulmonary Host Defense,Martha M. Monick and Gary W. Hunninghake 643

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CONTENTS ix

Alveolar Type I Cells: Molecular Phenotype and Development,Mary C. Williams 669

SPECIAL TOPIC: LIPID RECEPTOR PROCESSES, Donald W. Hilgemann,Special Topic Editor

Getting Ready for the Decade of the Lipids, Donald W. Hilgemann 697

Aminophospholipid Asymmetry: A Matter of Life and Death,Krishnakumar Balasubramanian and Alan J. Schroit 701

Regulation of TRP Channels Via Lipid Second Messengers,Roger C. Hardie 735

Phosphoinositide Regulation of the Actin Cytoskeleton,Helen L. Yin and Paul A. Janmey 761

Dynamics of Phosphoinositides in Membrane Retrieval andInsertion, Michael P. Czech 791

SPECIAL TOPIC: MEMBRANE PROTEIN STRUCTURE, H. Ronald Kaback,Special Topic Editor

Structure and Mechanism of Na,K-ATPase: Functional Sitesand Their Interactions, Peter L. Jorgensen, Kjell O. Hakansson,and Steven J. Karlish 817

G Protein-Coupled Receptor Rhodopsin: A Prospectus,Slawomir Filipek, Ronald E. Stenkamp, David C. Teller, andKrzysztof Palczewski 851

INDEXES

Subject Index 881Cumulative Index of Contributing Authors, Volumes 61–65 921Cumulative Index of Chapter Titles, Volumes 61–65 925

ERRATA

An online log of corrections to Annual Review of Physiology chaptersmay be found at http://physiol.annualreviews.org/errata.shtml

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structure and mechanism of na,k-atpase: functional sites and their interactions

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