inside-out oriented (i-s-o) and two right-side-oriented ... both sides of thé membrane so that Rb ions are thé sole regulator ... Swiss National Science Foundation.
The Na + ,K + -Pump, Part A: Molecular Aspects, pages 461-468 © 1988 Alan R. Liss, Inc.
SYMMETRIC ACTIVE TRANSPORT IN CHOLATE-DIALYSED LIPOSOMES CONTAINING RANDOMLY ORIENTED SODIUM PUMPS
B.M. Armer, M. Moosmayer and H.G. Rey Department of Pharmacology Geneva University Médical Center (CMU) CH-1211 Geneva 4, Switzerland
INTRODUCTION Despite thé récent détermination of thé atnino acid séquence of thé * and fi subunits of thé Na,K-ATPase protein in several laboratories (Shull et al., 1988), thé tight link between thé extracellular receptor and thé transmembrane active transport catalyzed by intracellular ATP is still poorly understood. A sided model-system containing purified Na,K-ATPase molécules performing active transport, is a prerequisite to connect spécifie structural modifications to altérations of thé active transport and of thé transport-receptor interaction. We hâve now designed such a model-system based on thé previous extensive analysis of numerous cholate-dialysed Na,K-ATPase-liposome préparations by électron microscopy that determined thé liposome size distribution, thé average entrapped volume per liposome, thé number of Na,K-ATPase molécules per liposome and their orientation (Anner et al., 1984; Anner and Moosmayer, 1985). On thé average, four co-reconstituted pumps, i.e. two inside-out oriented (i-s-o) and two right-side-oriented (r-s-o) pumps perform active transport per liposome of 100 nm diameter, by adding first internai ATP and external RbCl and then, when sufficient internai RbCl has accumulated to drive thé i-s-o pumps, external ouabain (to block thé r-s-o pumpf;) ,: (to activate thé i-s-o pumps).
462 / Anner, Moosmayer, and Rey Exploiting this bifunctional model, we noticed, first, that thé transmembrane Rb-gradient did not influence thé pump-rate, and, second, we confirmed thé pump orientation in freeze fractured liposomes.
RESULTS The equal întramembrane particle density on concave and convex faces of freeze-fractured liposomes (Table 1) indicates a symmetric orientation of thé reconstituted Na,K-ATPase, provîded that thé fracture plane goes asymmetrically around thé pump.
TABLE 1 . Symmetric distribution of intramerabrane partiales in cholate-dialysed freeze-fractured liposomes
IMP density (concave surface) = 1.022 ± 0.052* IMP density (convex
surface)
*Value is mean ± S.D.; thé number of intramembrane partiales of 250 concave and 250 convex liposome faces of 17 différent préparations were counted.
A putative mechanism by which thé cholate-dialysis procédure changes thé original asymmetric orientation of thé enzyme to a symmetric one is outlined in Fig. 1. Another possibility might be that artificial «2R2 dimers are formed by thé enzyme purification procédure, where thé intracellular part of one «n monomer associâtes with thé extracellular part of another «n (Zampighi et al., 1986), leading to units which perform active transport in both directions if thé uM purified Na,K-ATPase protein (assuming a mol. wt- of 300 000 dalton for thé «2^2 unit) in 23 mM sodium cholate, 50 mM Na2ATP, 5 mM MgCl2, 30 mM histidine, 1 mM Tris-EDTA, pH 7.10, 12 mM dioleoylphosphatidylcholine and accelerated removal of thé cholate by dialysis at 0°C in thé présence of cholestyramine resin (Rey et al., 1987).
average volume of a single liposome yields thé average number of liposomes per volume, which is equal to thé liposome density illustrated in Fig. 2. As a conséquence, thé number of substrate molécules entrapped per liposome as well PS thé number of external molécules and ions in thé surrounding solution can be calculated during both resting state and active transport. In theory, at identical bilatéral pump-ligand concentrations and with a symmetric pump orientation, thé active transport should proceed at equal rate in both directions. However, thé Rb-extrusion rate was consistently faster than thé uptake rate, even at saturating RbCl concentrations (Rey et al., 1987). Finally, we discovered that ATP concentrations above 5 mM lowered thé pump turnover (Fig. 4).
464 / Anner, Moosmayer, and Rey
Figure 2. Calculated density of thé standardized Na,K-ATPase-liposome suspension prepared as described in Legend of Fig. 1. Values are nm. The ratio of thé entrapped space to thé external space is 1%. Thus, 1 ml suspension contains 2.6 x 10 " liposomes representing an external surface of 8 230 cm2,
Fig. 3 shows thé principle of thé bifunctional liposomes. Excess Na and Mg ions and ATP are présent on both sides of thé membrane so that Rb ions are thé sole regulator of thé pump rate.
Symmetnc Active Transport in Na-Pump Liposomes / 465 Figure 3. After cholate-dialys îs and removal of thé external ATP (see Legend of Fig. 1), an average 100 nm liposome contains about 10 000 ATP (50 mM) molécules, 40 000 Na ions (200 mM), and 1000 Mg ions (5 mM). The r-s-o pump population (1) is activated by thé addition of 5 to 2 000 xiM external RbCl. When thé liposomes hâve accumulated thé desired concentration of Rb ions (usually an inside-out Rb gradient ranging between 1 and 70), 100 xiM ouabain are added externally, together with 10 to 50 mM ATP to block thé r-s-o and to activate thé i-s-o pumps (2) which then extrude thé accumulated Rb-ions.
100
ao 60
20
O1 0-01
01
025
0-5
10
2-5
5-0 7-5 10
25
50
ATP CONCENTRATION ri at 10 mM ATP.
Fig. 5 shows thfe typical triangular curves obtained by successive activation of r-s-o and î-s-o pumps. The 50 mM ATP-containing Na,K-ATPase-liposomes were incubated at 25°C in thé présence of external RbCl to drive Rb accumulation via thé r-s-o pumps (Fig. 5). The linear phase was determined by measuring thé Q6Rb-uptake at min intervais; external ATP was added to extrude thé captured 66Rb, via
466 / Anner, Moosmayer, and Rey
200--
2
TIME AFTER
3
0
0
5
1
1-5
86 Rb-ADDITION(min)
Figure 5. Successive active Rb-transport by co-reconstituted r-s-o and i-s-o Na,K-ATPase. At time 0, 10juM (A) or 1 mM (B) external 86RbCl are added to aliquots of thé ATPcontaining liposomes to s t a r t thé turnover of thé r-s-o pumps; 10 mM ( O ) or 50 mM ( D , A) external ATP and 100 AiM ouabain are added to activate thé i-s-o oriented pumps (•, • , ) and to inhibit thé r-s-o oriented pumps ( o > O » A ) . In thé absence of external ATP, thé r-s-o pumps continue to accumulate Rb ions ( O ) . By adding 10 mM external ATP instead of 50 mM, thé i-s-o pumps are less i n h i b i t e d ( i n s e t ) . The internai RbCl concentration is calculated frem thé 10 jal water space entrapped by 1 ml s t a n d a r d i z e d liposome préparations, i.e., 8&Rb-entrapment above 1 % indicates inside-out Rb-gradients. Each expérimental point represents 1 - 2 measu>ements in a typical préparation.
Symmetric Active Transport in Na-Pump Liposomes / 467 thé i-s-o pumps. The ratio of thé 86Rb-extrusion-rate to thé uptake-rate is deterrained by dividing thé descending by thé ascending slope, at both suboptimal and saturating (external and internai) RbCl concentrations.
CONCLUSIONS AND PERSPECTIVES Co-reconstituted i-s-o and r-s-o oriented Na,K-ATPase molécules in 1iposomes are activated successively by asymmetric addition of ATP to thé internai and external liposome copartment. In thé présence of mirror-symmetrical conditions, i.e. identical and saturating bilatéral pump-ligand concentrations, thé transport rates of thé two pump populations are about equal, indicating that thé random distribution of intramembrane partiales, seen in freeze-fractured préparations, corresponds to thé actual pump orientation. This observation provides thé first functional confirmation of thé random distribution of intramembrane particles in Na,K-ATPase-liposomes.
The model, in which thé extra- and intracellular sides of actively transporting Na,K-ATPase molécules are successively exposed at thé exterior surface of liposomes is a helpful tool for studying thé structure-function relationship of Na,K-ATPase.
ACKNOWLEDGMENTS The work was supported by grant no. 3.536-0.83 of thé Swiss National Science Foundation. We thank Dr. Paolo Meda for crîtically reading thé text and Mr. Fred Pillonel for careful artwork.
REFERENCES Anner BM, Moosmayer,M (1985) Right-side-out pumping Na,K-ATPase-liposomes: a new tool to study thé enzyme's receptor function. Biochem. Biophys. Res. Commun. 129: 102-108. Anner BM, Robertson JD, Ting-Beall HP (1984) Characterization of (Na + K)-ATPase-liposomes. I. Effect of enzyme concentration and modification on
468 / Anner, Moosmayer, and Rey 1iposome size, intramembrane partiales formation and Na,K-transport. Biochim. Biophys. Acta 773: 253-261. Rey HG, Moosmayer M, Anner BM (1987) Characterization of (Na + K)-ATPase-liposomes. III. Controlled activât ion and inhibition of symmetrîc pumps by timed asymmetric ATP, RbCl, and cardiac glycoside addition. Biochim. Biophys. Acta 900: 27-37. Shull GE, Young J, Greeb J, Lingrel JB (1988) Amino acid séquence of thé a and g subunits of thé Na,K-ATPase. This volume.. Zampighi G, Simon SA, Kyte J, Kreman M (1986) Onedîmensional crystals of,(Na + K)-ATPase dimers. Biochim. Biophys. Acta 854: 45-57.
