K+-PUMPS EXPRESSED INXENOPUSOOCYTES

Cell Biology International 1996, Vol. 20, No. 1, 67–72

STRUCTURE–FUNCTION RELATIONSHIPS OF NA + /K + -PUMPS EXPRESSED IN XENOPUS OOCYTES WOLFGANG SCHWARZ and LARISA A. VASILETS* Max-Planck-Institut für Biophysik, D-60596 Frankfurt/M, Germany

During the last years we have examined structure–function relationships in the Na + /K + -ATPase with respect to interactions of the external cations with the pump molecule. We have analysed in voltage-clamp experiments the influence of extracellular Na + and K + on the current generated by Na + /K + -pumps expressed in Xenopus oocytes. Our results demonstrated that external Na + and K + have to pass an access channel in the electrical field of the membrane to reach their binding sites. This external access, therefore, is voltage-dependent and is affected by lysine residues within the cytoplasmic N-terminus, by glutamic acid residues in intramembraneous domains, the ouabain sensitivity and phosphorylation by protein kinases. ? 1996 Academic Press Limited

K: sodium pump; current–voltage dependence; cation binding mutation; regulation

INTRODUCTION +

+

external +

+

The Na /K -ATPase or Na /K -pump was discovered by Skou in 1957 as a Na + - and K + dependent ATPase. It maintains the electrochemical gradient for Na + and K + across the cell membrane with low [Na + ] and high [K + ] in the cytoplasm compared to the external environment. This pump is present in nearly all animal cells, and its expression and regulation forms the basis for a large variety of cellular conditions and functions (see e.g. Vasilets and Schwarz, 1993). These include, for example, the resting potential of the cell membrane, excitability of nerve and muscle cells, Na + -driven secondary active transport as well as regulation of cell volume or of muscle contraction. For the description of the transport of Na + out of the cell and of K + into the cell Albers and Post have developed a reaction scheme (Albers, 1967; Post et al., 1969) assuming that the molecule can exist in two conformations, E1 and E2, with inwardly and outwardly oriented binding sites for the transported cations, respectively. Phosphorylation–dephosphorylation steps drive the pump through cyclic transitions between the Na + -binding E1 and K + -binding E2 conformation (see Fig. 1). Since three Na + -ions are transported *Permanent address: Institute of Chemical Physics in Chernogolovka, Russian Academy of Sciences, Chernogolovka, Moscow region 142432, Russia. 1065–6995/96/010067+06 $12.00/0

aNa+

2K+

Na Na K

Na

K

aNa E2P

E2P

2KE2P

Pi

(3Na)E1P ADP

(2K)E2

ATP

3NaE1ATP

E1ATP 3Na+

2KE1ATP +

2K internal

Fig. 1. Modified Albers–Post reaction scheme illustrating external cation interaction with the pump molecule in its E2P form through a narrow access channel. The external cation interactions can be described by voltage-dependent binding rates k=k*o[cation]ne "zEF/RT and voltage-independent unbinding (Gadsby et al., 1993; Vasilets et al., 1993). n represents a Hill coefficient, and z an effective valence of the charge moved during the corresponding binding step.

out of the cell and only two K + -ions into the cell per ATP molecule that is hydrolysed, the pump is electrogenic. The generated current is a measure for pump activity and can be analysed by voltage-clamp techniques provided the number of ? 1996 Academic Press Limited

68

Cell Biology International, Vol. 20, No. 1, 1996

Fig. 2. Amino acid sequence of the á- and â-subunit of the Na + /K + -ATPase of Torpedo electroplax (Kawakami et al., 1985) and their possible orientation in the membrane. Amino acids that have been identified to be functionally relevant are indicated, as well as trypsin (T1-T3, 19 kDa) and chymotrypsin (C3) cleavage sites (Jørgensen and Andersen, 1988; Kalish et al., 1990). Based on Fig. 3 from Vasilets and Schwarz (1993).

pump molecules under voltage control is high enough. For our investigations of structure–function relationships of the Na + /K + -pump we use as a model system the oocytes of Xenopus, and examine in these cells by two-microelectrode voltage-clamp techniques the endogenous Xenopus pump, as well as wild-type and mutated pumps from Torpedo electroplax expressed in the oocytes. The Na + /K + -ATPase has been cloned and sequenced from a large variety of animal species and tissues (for review see Vasilets and Schwarz, 1993; Lingrel et al., 1994). The pump molecule is a heterodimer and is composed of an á-subunit of about 112 kDa and a smaller glycosylated â-subunit of about 55 kDa. Three, possibly four (Shamraj and Lingrel, 1994) isoforms of the á- and three of the â-subunit have been described. They are expressed to different extents in the various tissues, and functional differences between the isoforms may have physiological relevance. The amino acid sequence and its possible orientation in the membrane is illustrated in Fig. 2 for the pump of Torpedo californica.

VOLTAGE DEPENDENCE OF PUMP ACTIVITY Since the transport of the Na + and K + is electrogenic, pump activity has to depend on membrane potential. We demonstrated that the reaction cycle of the pump can be modulated by at least two voltage-dependent steps (Lafaire and Schwarz, 1986; Vasilets et al., 1991; Vasilets and Schwarz, 1992; Vasilets et al., 1993). These steps are represented by extracellular binding of K + and Na + as illustrated in Fig. 1. For the pump in Xenopus oocytes this interpretation is based on the finding that the voltage dependence of pump activity changes dramatically if the extracellular cation composition is altered (Fig. 3). The complex dependencies on [Na + ] and [K + ] and on membrane potential can be described by exponentially voltage-dependent K1/2-values for pump inhibition by external Na + (apparent KI) and for pump activation by external K + (apparent Km). At physiological [K + ] and [Na + ] stimulation by K + becomes rate-determining at positive potentials and inhibition by Na + at negative potentials,

Cell Biology International, Vol. 20, No. 1, 1996

69

Eqn [1]

1.00 0.75 0.50 0 –50 –100 –150

0.25 0.00 75 50 [Na + ] (mM

25 )

0

–200

M po em ten br tia ane l (m V)

t Normal pump curren

(a)

Km =Km(0)ezKEF/mRT

1.00 0.75 0.50

0.00 1.0 [K +] (mM )

0.1

–200

Me ten mbr tia ane l (m V)

0 –50 –100 –150

0.25

po

t Normal pump curren

(b)

with voltage-dependent apparent Km values for pump stimulation by K + and K1/2 values for pump inhibition by Na + . m and n represent corresponding Hill coefficients. The Km value for pump stimulation can be determined in the absence of external Na + and has in this description an exponential potential dependence (Rakowski et al., 1991; Vasilets and Schwarz, 1992):

Fig. 3. Voltage and concentration dependence of normalized and endogenous pump current in Xenopus oocytes for (a) different Na + concentrations (100, 75, 50, 25, 5, 1 m) in presence of 5 m K + externally, and (b) different K + concentrations (5, 2.5, 0.5, 0.25, 0.1, 0.05 m) in absence of external Na + . The mesh lines represent concentration dependencies for different membrane potentials and potential dependencies for different concentrations. The symbols with dropped lines represent K1/2 values for pump inhibition by external Na + in (a) and pump stimulation by external K + in (b).

leading to a maximum in the current-voltage dependence at about 0 mV (Lafaire and Schwarz, 1986). Since charge translocations in steps following K + occlusion or preceding Na + occlusion have been excluded (Goldshleger et al., 1987), a straightforward explanation for these dependencies is a voltage-dependent access of the cations to their occlusion sites through a narrow highfield access channel (Läuger, 1991). We have performed detailed analyses of the dependencies of pump current on membrane potential, and on external [Na + ] and [K + ] by experiments of the type shown in Fig. 3. If the binding of K + and Na + are the dominating voltage-dependent steps in the reaction cycle, the potential and concentration dependencies can be fitted on the basis of the diagram of Fig. 1 by (Vasilets et al., 1993):

Eqn [2]

zK represents an effective valence for charge that is moved in the electrical field during steps associated with the K + binding. In terms of the access channel it is a measure for the apparent dielectric length of the channel. The voltage dependence of the apparent Km was determined for the endogenous Xenopus pump and for the Torpedo pump expressed in the oocytes. In contrast to the voltage dependence of the Km value for the Xenopus pump, the voltage dependence for the Torpedo pump has to be described by the sum of two exponentials: Km =K1(0)ez1EF/RT+K2(0)ez2EF/RT

Eqn [3]

This has been interpreted by a sequential binding of two K + ions (Vasilets and Schwarz, 1992), the component with the higher effective valence (z2) representing the binding of the first K + ion, the component with the lower effective valence (z1) the binding of the second ion that senses less of the electrical field (for values see Fig. 4). In terms of the access channel, this could mean that the Xenopus pump has a less pronounced highfield access channel than the Torpedo pump with a dielectric geometry where the K + ions have to move in single file to reach their binding sites. The K1/2 value for pump inhibition by Na + shows a more complex dependency demonstrating voltage-dependent competition between Na + and K + in their interaction with the E2P form (see Vasilets et al., 1993). Assuming this competition, a voltage-dependent apparent KI can be calculated from the K1/2 value that is also described by an exponential corresponding to eqn [2] and is related to K1/2 by (see Vasilets et al., 1993):

70

Cell Biology International, Vol. 20, No. 1, 1996

Fig. 4. Cartoon of Na + /K + -pump molecules with external access channel for K + . The parameters are effective valences and apparent Km-value for stimulation of the Torpedo pumps by K + . Data are partially taken from Vasilets and Schwarz (1993) and Vasilets et al. (1994). Amino acids marked with a dark dot have been mutated, those with a white dot are considered as further channel-forming residues.

Eqn [4]

The nominator in eqn [4] illustrates the dependence of KI on [K + ] and that for the K1/2 value additional voltage dependence is introduced by the voltage dependence of Km. EFFECTS OF PUMP MODIFICATIONS ON VOLTAGE DEPENDENCE Effects of N-terminal mutations on external cation interaction Structurally, there is a high degree of homology in the amino acid sequence of the á-subunits of different animal species and isoforms. Major differences are predominantly located in the N-terminal region, which includes sites that determine sensitivity for pump-specific inhibitors, the cardiac glycosides like ouabain, and a lysine-rich cluster (see Fig. 2). A K-cluster is found in all á-subunits, but the number of lysine residues varies considerably. Therefore, the question arises whether the cytoplasmic, N-terminal K-cluster can account for functional isoform and species differences. To approach this

question, we examined mutants that were truncated at different positions within the K-cluster. Removal of 28 amino acids including Lys28 leads to a pronounced increase in the effective valencies z1 and z2 (see Fig. 4). Leaving Lys28 has a much less pronounced effect with concentration and voltage dependencies very similar to the wild type. Isoform differences are not only restricted to differences in the amino acid sequence of the cytoplasmic N-terminus, also characteristic for an isoform is its sensitivity for cardiac glycosides. Since endogenous cardiac glycosides or ouabain-like substances have been discovered (Blaustein, 1994), the differences in ouabain sensitivity may play an important role in physiological pump regulation. The ouabain-sensitive Torpedo pump can be converted to a ouabainresistant one by mutation of Gln-118 to Arg and Asn-129 to Asp (see Fig. 2). This results in slightly reduced effective valencies but a clear increase in apparent affinity for external K + (see Fig. 4). Therefore, not only the ouabain sensitivity by itself, but also the different external cation interactions may have functional tissue-specific significance. Effects of regulatory phosphorylation on external cation interaction Since the sodium pump is responsible for maintenance of a variety of cellular functions, its activity

Cell Biology International, Vol. 20, No. 1, 1996

should be reliably regulated with respect to different physiological conditions and particularly during intracellular signalling. We have demonstrated that transport activity of the Na + /K + -pump is, indeed, modulated by activation of protein kinases (Vasilets and Schwarz, 1992). Activation of protein kinase C (PKC) by microinjection of diC8 into the oocytes or application of phorbol ester leads to inhibition of transport activity of the endogenous and of expressed pumps; activation of protein kinase A (PKA) by cAMP injections, in contrast, stimulates. On the other hand, in experiments with purified and microsomal preparations of the Na + / K + -ATPase as well as with phosphorylation of the pump in crude homogenates of Xenopus oocytes it was demonstrated that the á-subunit is target protein for both protein kinases (Chibalin et al., 1991, 1992). Finally, Ser-942 was identified as unique site for phosphorylation by PKA (Aperia et al., 1994; Beguin et al., 1994) and Thr-347 and also Ser-375 located between C3 and T1 cleavage sites were suggested as candidates for PKC phosphorylation (Chibalin et al., 1993). Interestingly, phosphorylation of these residues by protein kinases leads to pronounced alterations in the binding characteristics for external cations (Vasilets and Schwarz, 1992). The changes of effective valences z1,2 and of Km-values are presented in Fig. 4. In terms of interpretation of the data by an access channel, these alterations may reflect a reduction of dielectric length of the channel by phosphorylation by PKA, and lengthening by phosphorylation by PKC. In view of the cytoplasmic location of these phosphorylation sites, modification of externally oriented cation binding sites by PKA and PKC suggests allosteric interactions between these domains within the pump protein. Effects of mutation of glutamic acid residues The access channel of the Na + /K + -pump has characteristics similar to K + -selective channels (see Vasilets and Schwarz, 1993) where ions have to pass in single-file fashion a series of energy barriers and binding sites. Therefore, for the access channel one might also expect binding sites in the intramembraneous domains. Based on chemical modifications the glutamic acid residues Glu334, Glu959 and Glu960 have been discussed as possible candidates for the binding site (Goldshleger et al., 1992). On the other hand, it has been shown by several other groups (Feng and Lingrel, 1995; Vilsen, 1995) and ourselves (Vasilets et al., 1994) that mutations of these amino acids lead to fully functional en-

71

zymes, but interactions of the pump with external cations may be altered. Changes in the apparent dielectric length of the access channel and in the apparent affinity depend on whether a ouabainsensitive or ouabain-resistant pump is examined, and they are more pronounced in ouabain-sensitive forms (see parameters in Fig. 4). This indicates that the intramembraneous glutamic acid residues are not essential for transport but that they, nevertheless, may be involved in forming a sequence of intermediate binding sites within the access channel, although allosteric effects cannot yet be excluded. In conclusion, our results demonstrate complex allosteric interactions between access channel, ouabain-binding site, N-terminus and sites for regulatory phosphorylation. This can form a basis for additional mutations to achieve further insights into structure–function relationships in the Na + /K + pump molecule. ACKNOWLEDGEMENTS We thank Drs M. Kawamura, T. Ohta and K. Takeda for providing cDNAs for the Torpedo pumps. The work was supported by Deutsche Forschungs-gemeinschft (SFB 169 and Schw 446/ 2-1) and by International Science Foundation (Grant NJD000). REFERENCES A RW, 1967. Biochemical aspects of active transport. Ann Rev Biochem 36: 727–756. A A, F J, F G, et al., 1994. Hormonal regulation of the Na + ,K + -ATPase activity. In: B E, S W (Eds), The Sodium Pump, pp. 662–669. Darmstadt: Steinkopff Verl. B P, B AT, C AV, V LA, R BC, J F, G K, 1994. Phosphorylation of the Na,K-ATPase by protein kinases: structure–functional relationship. In: B E, S W (Eds), The Sodium Pump: Structure, mechanism, hormonal control and its role in disease, pp. 682–684. Darmstadt: Steinkopff Verlag. B MP, 1994. Endogenous ouabain—physiologicalactivity and pathophysiological implications. Clin Invest 72: 706–707. C AV, L OD, P SP, V LA, 1991. Phosphorylation of Na,K-ATPase by protein kinase C and cAMP-dependent protein kinase. Biol Membr 8: 1440–1441. C AV, L OD, P SP, V LA, 1993. Phosphorylation of the Na,K-ATPase by Ca,phospholipiddependent and cAMP-dependent protein kinases: Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase. J Bioenerg Biomembr 25: 61–66. C AV, V LA, H H, P D, G K, 1992. Phosphorylation of Na,K-ATPase á

72

subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C. J Biol Chem 267: 22378–22384. F JN, L JB, 1995. Functional consequences of substitutions of the carboxyl residue glutamate-779 of the na,k-atpase. Cell Mol Biol Res 41: 29–37. G DC, R RF, D W P, 1993. Extracellular access to the Na,K pump—pathway similar to ion channel. Science 260: 100–103. G R, K SJD, R A, S WD, 1987. The effect of membrane potential on the mammalian sodium-potassium pump reconstituted into phospholipid vesicles. J Physiol 387: 331–355. G R, T DM, M J, S WD, K SJD, 1992. Chemical modification of Glu-953 of the alphachain of Na + ,K + -ATPase associated with inactivation of cation occlusion. Proc Natl Acad Sci USA 89: 6911–6915. Jø PL, A JP, 1988. Structural basis for E1-E2 conformational transitions in Na,K-pump and Ca-pump proteins. J Memb Biol 103: 95–120. K SJD, G R, S WD, 1990. A 19-kDa C-terminal tryptic fragment of the alpha-chain of Na/KATPase is essential for occlusion and transport of cations. Proc Natl Acad Sci USA 87: 4566–4570. K K, N S, N M, et al., 1985. Primary structure of the á-subunit of Torpedo californica (Na + +K + )ATPase deduced from cDNA sequence. Nature 316: 733–736. L AV, S W, 1986. Voltage dependence of the rheogenic Na + /K + ATPase in the membrane of oocytes of Xenopus laevis. J Membr Biol 91: 43–51. L¨  P, 1991. Kinetic basis of voltage dependence of the Na,K-pump. In: K JH, D W P (Eds), The Sodium Pump: Structure, mechanism, and regulation, pp. 303–315. New York: Rockefeller University Press. L JB, VH J, O W, J E, S P, 1994. Na,K-ATPase—Structure–function studies. Renal Physiol Biochem 17: 198–200.

Cell Biology International, Vol. 20, No. 1, 1996

P RL, K S, T T, O B, S AK, 1969. Flexibility of an active centre in sodium-plus-potassium adenosine triphosphatase. J Gen Physiol 54: 306s–326s. R RF, V LA, LT J, S W, 1991. A negative slope in the current-voltage relationship of the Na + /K + pump in Xenopus oocytes produced by reduction of external [K + ]. J Memb Biol 121: 177–187. S OI, L JB, 1994. A putative fourth Na + ,K + ATPase alpha-subunit gene is expressed in testis. Proc Natl Acad Sci USA 91: 12952–12956. S JC, 1957. The influence of some cations on an adenosine triphosphatase from peripherial nerves. Biochim Biophys Acta 23: 394–401. V LA, K M, T K, O T, S W, 1994. The role of putative intramembraneous glutamic acid residues of the á-subunit of the sodium pump in external cation binding. In: B E, S W (Eds), The Sodium Pump, pp. 557–560. Darmstadt: Steinkopff Verlag. V LA, O T, N S, K M, S W, 1993. Voltage-dependent inhibition of the sodium pump by external sodium: Species differences and possible role of the N-terminus of the á-subunit. Eur Biophys J 21: 433–443. V LA, O H, O T, N S, K M, S W, 1991. Stimulation of the Na + /K + pump by external [K + ] is regulated by voltage-dependent gating. J Biol Chem 266: 16285–16288. V LA, S W, 1992. Regulation of endogenous and expressed Na + /K + pumps in Xenopus oocytes by membrane potential and stimulation of protein kinases. J Membr Biol 125: 119–132. V LA, S W, 1993. Structure–function relationships of cation binding in the Na + /K + -ATPase. Biochim Biophys Acta 1154: 201–222. V B, 1995. Mutant glu781->ala of the rat-kidney Na + ,K + -ATPase displays low cation affinity and catalyzes atp hydrolysis at a high-rate in the absence of potassiumions. Biochemistry 34: 1455–1463.