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J Biol Chem, Vol. 275, Issue 3, 1976-1986, January 21, 2000

Transport and Pharmacological Properties of Nine Different Human Na,K-ATPase Isozymes^*

Gilles Crambert^§¶, Udo Hasler, Ahmed T. Beggah, Chuliang Yu, Nikolai N. Modyanov, Jean-Daniel Horisberger, Lionel Lelièvre^§, and Käthi Geering^**

From the  Institut de Pharmacologie et de Toxicologie de l'Université, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland, the ^§ Laboratoire de Pharmacologie des Transports Ioniques Membranaires, Université Paris VII, 2 place Jussieu, 75251 Paris Cedex 05, France, and the  Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614-5804

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Na,K-ATPase plays a crucial role in cellular ion homeostasis and is the pharmacological receptor for digitalis in man. Nine different human Na,K-ATPase isozymes, composed of 3  and  isoforms, were expressed in Xenopus oocytes and were analyzed for their transport and pharmacological properties. According to ouabain binding and K⁺-activated pump current measurements, all human isozymes are functional but differ in their turnover rates depending on the  isoform. On the other hand, variations in external K⁺ activation are determined by a cooperative interaction mechanism between  and  isoforms with 2-2 complexes having the lowest apparent K⁺ affinity.  Isoforms influence the apparent internal Na⁺ affinity in the order 1 > 2 > 3 and the voltage dependence in the order 2 > 1 > 3. All human Na,K-ATPase isozymes have a similar, high affinity for ouabain. However, 2- isozymes exhibit more rapid ouabain association as well as dissociation rate constants than 1- and 3- isozymes. Finally, isoform-specific differences exist in the K⁺/ouabain antagonism which may protect 1 but not 2 or 3 from digitalis inhibition at physiological K⁺ levels. In conclusion, our study reveals several new functional characteristics of human Na,K-ATPase isozymes which help to better understand their role in ion homeostasis in different tissues and in digitalis action and toxicity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Na,K-ATPase (Na,K-pump) belongs to the P-type ATPase family of cation transporters which are characterized by intermediate phosphorylation during the catalytic cycle. The Na,K-ATPase is an ubiquitous plasma membrane enzyme which transports 2 K⁺ ions into and 3 Na⁺ ions out of the cell by using the energy of the hydrolysis of 1 molecule of ATP. This enzyme plays a crucial role in cell homeostasis since it maintains Na⁺ and K⁺ gradients between the intra- and extracellular milieu which are necessary for the maintenance of the cell volume. Furthermore, the Na⁺ gradient created by the Na,K-ATPase provides the energy for the transport activity of many secondary transporters which provide the cell with nutrients or regulate intracellular concentrations of ions which are implicated in specialized cellular functions such as muscle contraction, transmission of nerve impulses, or Na⁺ reabsorption in the kidney. Moreover, the Na,K-ATPase is the pharmacological receptor for cardiac glycosides which are widely used in the treatment of heart failure because of their positive inotropic effect and is possibly also the physiological receptor for endogenous ouabain compounds. In view of its important "housekeeping" and specialized functions, it is expected that any dysfunction or dysregulation of the Na,K-ATPase may have important pathophysiological consequences (for review, see Ref. 1).

The minimal functional unit of Na,K-ATPase is composed of an  and  subunit. The  subunit has 10 membrane-spanning domains and exposes the N and C terminus to the cytoplasmic side while the  subunit is a type II glycoprotein with a single transmembrane segment, a short cytoplasmic tail, and a large ectodomain. The  subunit carries the functional properties of the Na,K-ATPase, namely it binds and transports the cations, hydrolyzes ATP, and is intermediately phosphorylated. Furthermore, the  subunit bears the binding site for cardiac glycosides (for review, see Ref. 2). The  subunit is necessary for the structural and functional maturation of the  subunit and also influences the K⁺ and Na⁺ activation kinetics of mature pumps (for references, see Ref. 3).

One of the remaining questions concerning the structure-function relationship in Na,K-ATPase is the functional role of existing  and  isoforms. Indeed, 4  and 3  isoforms have been identified which exhibit 85 and 45% of sequence identity, respectively, and which show a tissue-specific distribution and a developmentally regulated pattern of expression (for review, see Ref. 4). Biochemical evidence (5) and transfection studies (for review, see Ref. 4) suggest that  and  isoforms can assemble in different combinations and potentially form functional pumps. So far, analysis of functional differences among isozymes has mainly been performed with rat Na,K-ATPase. These studies have led to several hypotheses on the specific role of different isozymes in different tissues. 1 Isoforms are ubiquitous and may assume a housekeeping function in all cells. In the adult rat, 2 isoforms are expressed predominantly in brain, skeletal muscle, and heart, whereas 3 isoforms are primarily expressed in brain. These  isoforms are thought to form auxiliary pumps working in particular physiological situations. For instance, compared with 1 and 2 isoforms, 3 isoforms have a lower affinity for Na⁺ and may only be active after an increase in intracellular Na⁺ concentrations due to a series of action potentials (for review, see Ref. 4). On the basis of the large differences in ouabain sensitivities between 1 isoforms (ouabain-resistant) and 2 or 3 isoforms (ouabain-sensitive) of rat or dog, it was also speculated that "inotropic" and "toxic" isoforms determine digitalis action (6, 7).

Similar to the 1 isoform, the 1 isoform is expressed ubiquitously. In rat, the 2 isoform is mainly expressed in muscle and brain where it could have a complementary role as an adhesion molecule (8), whereas the 3 isoform is found in a variety of rat tissues. Some evidence exists that  isoforms may differentially influence the enzymatic and transport properties of Na,K-ATPase isozymes (for review, see Ref. 4) but the effects appear to be less pronounced than those of  isoforms.

Despite the physiological and pathophysiological importance of Na,K-ATPase and its role in digitalis action in the treatment of heart failure, little is known about the physiological and pharmacological properties of human Na,K-ATPase isozymes. It may indeed be expected that the extrapolations from data obtained with rat Na,K-ATPase isozymes on the pharmacological properties, e.g. therapeutic and toxic targets of digitalis, may not hold true for humans. In contrast to rat, the 3  and the 3  isoforms are present in the human heart, which raises the possibility that in this tissue, 9 different - complexes may exist with different transport and/or pharmacological properties. Furthermore, in the human heart, only one or two high affinity digitalis-binding sites were identified suggesting that, in contrast to rat Na,K-ATPase isozymes, human isozymes do not significantly differ in their digitalis sensitivity (for review, see Ref. 9).

To better understand the physiological and pharmacological relevance of the existence of different Na,K-ATPase isozymes in general, and in humans in particular, we expressed human 1, 2, or 3 cRNAs together with 1, 2, or 3 cRNAs in Xenopus oocytes and investigated several transport characteristics (turnover, Na⁺ and K⁺ affinities, voltage dependence) and pharmacological properties (K_d, k₊₁, k₁ of ouabain binding and K⁺/ouabain antagonism) of the 9 possible human Na,K-ATPase isozymes. The functional comparison in the same experimental system of the various - complexes revealed several new characteristics of Na,K-ATPase isozymes which are discussed with respect to their physiological and pharmacological relevance.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cloning of Human 2, 3, and 2 Isoforms of the Na,K-ATPase and cRNA Preparations-- Based on the genomic or cDNA sequences available (see below), we cloned 2, 3, and 2 cDNAs from a human cardiac uncloned cDNA library (Marathon-ready cDNA, CLONTECH) using long distance PCR¹ (LD-PCR) technology (Advantage cDNA PCR kit, CLONTECH).

The 2 isoform (Ref. 10, GenBank^TM accession number J05096) was cloned, using a sense oligonucleotide covering the sequence coding for Met¹ up to Ala¹² and tailed with a sequence containing 10 nucleotides including an EcoRI restriction site. The antisense oligonucleotide covered the sequence coding for Pro¹⁰¹⁰ up to the stop codon and was tailed with a sequence coding for a HpaI restriction site (blunt end). The LD-PCR was performed using a touchdown protocol with a first denaturing step (1 min at 94 °C), followed by two pre-amplification steps (5 cycles at 94 °C for 30 s and 72 °C for 5 min 30 s and 5 cycles at 94 °C for 30 s and 70 °C for 5 min 30 s) and the amplification step (25 cycles at 94 °C for 30 s and 68 °C for 5 min 30 s). Finally, a last elongation step was performed at 68 °C for 8 min. The 2 identity of the PCR product was confirmed by SmaI digestion. The 2 PCR product was then subcloned into a pSD5 vector (containing the Xenopus 1 cDNA), using EcoRI and PmaCI (blunt end) sites. The nucleotide sequences of clones were analyzed by dideoxy sequencing and, in the chosen clone, 2 mutations introduced by LD-PCR were identified and were corrected according to the published genomic sequence using PCR techniques (11). These clones were resequenced to verify their identity.

The 3 isoform (Ref. 12, GenBank^TM accession number X12910) was cloned by using a sense oligonucleotide covering the sequence coding for Met¹ up to Ser¹² and containing a tail consisting of 10 nucleotides of the 5'-untranslated region of a N-terminal truncated Xenopus 1 cDNA (Xe 5'-UT (13)) and an antisense oligonucleotide covering the region coding for Gly⁹⁸⁶ up to the stop codon. This oligonucleotide was tailed with a sequence coding for EcoRI and EcoRV (blunt end) restriction sites. LD-PCR was performed as for 2 (see above). The 3 identity of the PCR product was confirmed by PvuII digestion. We added the Xe 5'-UT sequence by performing an additional PCR between the human 3 PCR product (containing part of the Xe 5'-UT) and the entire Xe 5'-UT (containing a NheI site). This PCR leads to a cDNA of the full-length human 3 preceded by the entire Xe 5'-UT. This product was subcloned into a pSD5 vector using NheI and PmaCI (blunt end) sites. The addition of the Xe 5'-UT was previously shown to improve expression of foreign proteins in oocytes (14), but in our case, the expression of 3 was still low. We, therefore, subcloned the cDNA into the pNKS2 vector (kindly provided by G. Schmalzing). The human 3 cDNA in the pSD5 vector was removed by EcoRI digestion and blunted at both ends. It was then ligated with the pNKS2 vector previously digested with NcoI (fill-in) and SalI (blunt). The nucleotide sequences of clones were analyzed by dideoxy sequencing. In the chosen clone, 2 mutations were identified and were corrected according to the published genomic sequence using PCR techniques (11). These clones were resequenced to verify their identity.

The 2 isoform (Ref. 15, GenBank^TM accession number P14415) was cloned by using a sense oligonucleotide encompassing a sequence coding for Met¹ up to Glu⁶ and containing a NcoI restriction site within the Met¹ codon. The antisense oligonucleotide covered a region coding for Leu²⁹⁵ up to the stop codon and was tailed with a sequence coding for a XbaI restriction site. The LD-PCR was similar to that used for 2 and 3 except that the amplification times were decreased to 2 min 30 s. PCR products obtained were checked by digestion with PvuII and subcloned into the pSD5 vector between NcoI and XbaI restriction sites. The nucleotide sequences of 2 clones were analyzed by dideoxy sequencing and were found to differ from the sequence of Martin-Vassalo et al. (15) by a Leu replacement of Pro⁵¹. This was not considered as a mutation since Ruiz et al. (16) have reported the same sequence for human 2 from fetal and adult retinal pigment epithelia (GenBank U45945).

The full-length human 3 cDNA was identified in the IMAGE Consortium clone number 133072 obtained from Research Genetics (Huntsville, AL) by restriction mapping and sequencing as described (17). To generate the pSD5 vector covering the entire 3 coding region preceded by Xe 5'-UT, the AlwnI-EcoRV fragment containing the coding region (without 36 nt at the 5' end) and 15 nt of the 3'-UT, and a synthetic oligonucleotide linker which was designed to encode the first 12 amino acids and fit NcoI and AlwnI sites (sense strand of 37 nt and antisense strand of 30 nt), were ligated with the vector preliminary digested with NcoI and SmaI.

The full-length cDNA encoding human 1 isoform (18) was reconstructed from two overlapping clones, pSNa100 and pSN54, which were kindly provided by K. Kawakami. The NcoI-EcoRV fragment of 1 cDNA containing the entire coding part and 397 nt of 3'-UT was subcloned into the pSD5 vector containing Xe 5'-UT between NcoI and SmaI restriction sites. In order to improve the level of expression, the 1 cDNA was transferred into the pNKS2 vector.

The human 1 cDNA (19) (a gift from K. Kawakami) containing the entire coding part and 70 nt of 3'-UT was subcloned into the pSD5 vector containing Xe 5'-UT. cRNA coding for human  and  isoforms was obtained by in vitro translation (20).

Expression of the - Complexes of Human Na,K-ATPase in Xenopus laevis Oocytes and Detection by Nondenaturating Immunoprecipitation of  Subunits-- Oocytes were obtained from X. laevis females, as described (21), and were injected with 10 ng of human  isoform cRNAs alone or together with 1 ng of  isoform cRNA. Oocytes were incubated for 24 or 72 h in modified Barth's medium containing 0.5-0.7 mCi/ml of [³⁵S]methionine (Hartman Analytic). In some experiments, oocytes were subjected to a 48-h chase period in the presence of 10 mM cold methionine. Digitonin extracts were prepared after the pulse and chase periods as described and  subunits were immunoprecipitated under nondenaturating conditions, as described (21), with a polyclonal anti-Bufo Na,K-ATPase 1 antibody. Reliable quantification of  subunits associated with  subunits is difficult due to the complex-type glycosylation of  subunits which can result in diffuse migration on gels of  subunits containing multiple glycosylation sites. Thus, to determine the ratio between  and  isoforms in Na,K-ATPase isozyme complexes, cRNA-injected oocytes were incubated in the presence of 5 µg/ml brefeldine A (Alexis Corp.) during a 24-h pulse period as described previously (21). Under these conditions, - complexes are retained in the endoplasmic reticulum and  subunits remain in their core-glycosylated form. This permitted removal of the sugar moiety from  subunits by treating the immunoprecipitated samples with endoglycosidase H as described previously (21) and allowed reliable quantification of the non-glycosylated species. The dissociated immune complexes were separated by SDS-polyacrylamide gel electrophoresis and labeled proteins were detected by fluorography and quantified by densitometry with a LKB 2202 Ultrascan.

Pump Current Measurements and Determination of Apparent K⁺ and Na⁺ Affinities and Voltage Dependence-- 3 days after cRNA injection, Na,K-pump currents were measured by using the two-electrode voltage-clamp method. The total currents measured in oocytes expressing exogenous Na,K-pumps are assumed to be the sum of the currents mediated by endogenous and exogenous Na,K-pumps. To identify the component which is due to the expressed Na,K-pumps, pump currents were measured in parallel under the same conditions in non-injected oocytes and the mean values were subtracted from those obtained in cRNA-injected oocytes of the same batch. To determine the apparent K⁺ affinity of Na,K-pumps, oocytes were loaded with Na⁺, as described previously (22), which increased the intracellular Na⁺ concentration to about 70 mM (not shown). The electrophysiological measurements were done in the presence of external Na⁺ (in mM, 80 sodium gluconate, 0.82 MgCl₂, 0.41 CaCl₂, 10 N-methyl-D-glutamine (NMDG)-Hepes, 5 BaCl₂, 10 tetraethylammonium chloride, pH 7.4). The current induced by increasing concentrations of K⁺ (0.5, 1, 3, 5, 10 mM) was measured at 50 mV. The Hill equation (I) I_K = I_max/[1 + (K_1/2 K⁺/[K])^nH] was fitted to the data of the current (I_K) induced by various K⁺ concentrations ([K]) and yielded least-square estimates of the maximal current (I_max) and of the half-activation constant for K⁺ (K_1/2 K⁺). A Hill coefficient (nH) of 1.6 was used, as described previously (22).

Measurements of the half-activation constant for internal Na⁺ were performed as described previously (3). In brief, oocytes were injected with Na,K-ATPase  and  cRNAs along with cRNAs coding for , , and  subunits (0.3 ng/subunit/oocyte) of the rat renal epithelial Na⁺ channel (23). Injected oocytes were incubated for 3 days in a modified Barth's solution containing 10 mM Na⁺. Before measurements, oocytes were incubated in a Na⁺-free solution (50 mM NMDG-Cl, 40 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM NMDG-Hepes, pH 7.4), in order to maximally reduce the internal Na⁺ concentration.

From five to eight pairs of measurements of [Na]_i and of the K⁺-activated Na,K-pump current were performed successively on each oocyte. Between each pair of measurements, oocytes were exposed to a 100 mM Na⁺ solution (100 mM sodium gluconate, 1 mM MgCl₂, 0.5 mM CaCl₂, 10 mM Na-Hepes, pH 7.4) in the absence of amiloride and at a holding potential of 50 to 100 mV to increase intracellular Na⁺ concentrations.

Intracellular Na⁺ concentrations were calculated from the reversal potential of the amiloride-sensitive current obtained from I-V curves recorded in the absence of amiloride in a solution containing 5 mM Na⁺ (5 mM sodium gluconate, 0.5 mM MgCl₂, 2.5 mM BaCl₂, 95 mM NMDG-Cl, 10 mM NMDG-Hepes, pH 7.4). The K⁺-activated Na,K-pump currents (I_K) were measured in the presence of 80 mM external Na⁺ (see above), 10 mM K⁺, and 20 µM amiloride.

To identify the component of the current mediated by exogenous Na,K-pumps in cRNA-injected oocytes, the endogenous pump currents (I_Kendo) were measured in parallel on non-injected oocytes and the I_max,endo and the K_1/2 Na⁺_endo were determined by fitting Equation 2, I_Kendo = (I_max,endo/[1 + (K_1/2 Na⁺_endo/[Na]_i)^nH]) to the K⁺-induced currents (I_Kendo) and intracellular Na⁺ concentrations [Na]_i measured in non-injected oocytes. The values obtained were introduced into Equation 3, I_K = I_max,endo/[1 + (K_1/2 Na⁺_endo/[Na]_i)^nH]) + I_max,hum/[1 + (K_1/2 Na⁺_hum/[Na]_i)^nH] and separate I_max (I_max,hum) and K_1/2 Na⁺ (K_1/2 Na⁺_hum) for the expressed, human pumps were obtained by fitting the entire Equation 2 to the I_K and [Na]_i values measured in cRNA-injected oocytes. Parameter fitting was performed with a Hill coefficient (nH) of 3 as described previously (3).

The voltage dependence of the ouabain-sensitive currents of the human Na,K-pumps was investigated in non-injected oocytes and in oocytes expressing human Na,K-pump isozymes by measuring the currents activated by 10 mM K⁺ during a series of ten 200-ms voltage steps ranging between 130 and +50 mV, before and after the addition of 100 µM ouabain in the presence of 90 mM external Na⁺. Averaged currents of endogenous Na,K-pumps were subtracted from currents measured in oocytes expressing exogenous Na,K-pumps.

[³H]Ouabain Binding on Intact Oocytes-- Three days after cRNA injection, the total number of Na,K-pumps expressed at the cell surface was determined. For this purpose, oocytes were loaded with Na⁺ for 2 h at 19 °C in a K⁺-free solution (90 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, pH 7.4) before incubation with 1 µM ouabain (0.3 µM [³H]ouabain (Amersham Pharmacia Biotech, 34.2-50 Ci/mmol) plus 0.7 µM unlabeled ouabain) for 30 min at room temperature as described previously (24). In preliminary experiments, we have determined that ouabain binding to all isozymes reaches a plateau after 20 min which persists up to 1 h indicating that ouabain does not gain access to an internal pool of binding sites during the assay period. After incubation with ouabain, oocytes were washed three times with 20 ml of the above solution and individually transferred to vials and solubilized with 100 µl of 5% (w/v) SDS before counting. Non-injected oocytes of the same batch were analyzed under the same conditions to determine the specific endogenous ouabain binding and the nonspecific component.

[³H]Ouabain Binding Kinetics on Oocyte Microsomes-- Three days after cRNA injection, microsomes were prepared from oocytes as described previously (21). Protein concentrations of microsomes were determined by the method of Lowry (25). Ouabain binding experiments were carried out as specified in the figure legends in the absence or presence of various concentrations of K⁺ at 37 °C with various [³H]ouabain concentrations (from 10⁹ to 5 × 10⁸ M) in a medium containing 4 mM ATP, 4 mM MgCl₂, 100 mM NaCl, 30 mM imidazole/HCl, pH 7.4 (Na-ATP conditions), or in a medium containing 4 mM H₃PO₄, 4 mM MgCl₂, and 30 mM Tris-HCl, pH 7.4 (Mg-P_i conditions). After temperature equilibration, binding reactions were initiated by addition of oocyte microsomes (final concentration, 11 µg/ml) previously permeabilized by incubation with 0.15 µg of SDS/µg of protein for 25 min at 19 °C. After various time periods, aliquots containing 5 µg of protein were removed, rapidly filtered under vacuum on glass fiber filters (Whatman GF/C), and rinsed three times with 4 ml of an ice-cold buffer containing 100 mM NaCl and 30 mM imidazole/HCl, pH 7.4. Radioactivity bound to filters was counted after addition of 4 ml of scintillation solution (Emulsifior scintillator plus, Packard). Equilibrium binding was reached within 90 min and 5 h in the absence and presence of K⁺, respectively, with the lowest [³H]ouabain concentrations used. Ouabain binding experiments were performed under the same conditions on microsomes from non-injected oocytes of the same batch to determine the endogenous, oocyte ouabain binding and the nonspecific binding and the mean values were subtracted from ouabain binding data obtained with microsomes from cRNA-injected oocytes. Nonspecific binding which was determined by addition of a 1000-fold excess of unlabeled ouabain was not significantly different in different batches of oocytes and did not exceed 15% of the total binding.

The association and dissociation kinetics of ouabain to each isozyme were determined as specified in the figure legends. The dissociation rate constant (k₁) was calculated from the slope of ln B/B_eq versus time plots; B_eq = specific ouabain bound at equilibrium and B = specific ouabain bound at several time points after addition of an excess unlabeled ouabain. The observed first-order association rate constant (k_obs) of ouabain binding to each Na,K-ATPase isozyme was determined as the slope of ln((B_eq  B)/B_eq) versus time plots; B_eq = specific [³H]ouabain bound at equilibrium and B = specific ouabain bound at several time points. Knowing k_obs, the ouabain concentration ([ouab]) used for association experiments and the dissociation rate constant (k₁), we determined the association rate constant (k₊₁) by using the equation k_obs = k₊₁[ouab]  k₁.

All curve fittings and unpaired Student's t test were done with Kaleidagraph software. When appropriate, two-way ANOVA variance analysis was performed by using Prisma III software.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cellular Expression and Processing of Human Na,K-ATPase Isozymes in Xenopus Oocytes

To test the cellular expression and processing of human Na,K-ATPase isozymes in Xenopus oocytes, we injected 1, 2, or 3 cRNAs alone or together with 1, 2, or 3 cRNAs, subjected the oocytes to a 24-h pulse with [³⁵S]methionine and a 48-h chase period and followed the stability of the  subunits. As previously shown for amphibian Na,K-ATPase (21), human Na,K-ATPase  isoforms expressed without  subunits were degraded during the chase period (Fig. 1A, lanes 3-8) and only the newly synthesized, endogenous, oocyte  subunits which are intrinsically stable (26), were immunoprecipitated (compare lanes 4, 6, and 8 to lanes 1 and 2). On the other hand, co-expression with 1 (lanes 9-14), 2, or 3 subunits (data not shown) permitted for the formation of stable 1, 2, and 3 isoforms indicating that all 3 human  isoforms can assemble with the 3 human  isoforms. Quantification of  and  subunits immunoprecipitated with an -antibody under nondenaturing conditions which preserve - interactions revealed a ratio of 1, 2, and 3 over 1 isoforms close to 1 when corrected for the variable number of methionines present in the 1 and the various  isoforms (Fig. 1B, lanes 1-3). Finally, 3 days after cRNA injection, at the time point where functional assays were performed, the expression of all 9 - complexes was significantly higher (Fig. 1C, lanes 1-9) than that of Na,K-ATPase in non-injected oocytes (lane 10). Within a 72-h period, all  subunits were at least partially processed from a core glycosylated, endoplasmic reticulum form which was endoglycosidase H-sensitive (data not shown) to a fully glycosylated form which was endoglycosidase H-resistant indicating that the various - complexes were routed through a distal Golgi compartment to the plasma membrane. Altogether, these results show that human Na,K-ATPase isozymes are correctly processed and targeted in Xenopus oocytes and that, due to the rapid degradation of unassembled  subunits, the functional characteristics described in the following are exclusively determined by the presence of - complexes.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Cellular expression and processing of 9 possible, human Na,K-ATPase isozymes in Xenopus oocytes. A, human Na,K-ATPase  isoforms need assembly with  subunits for stable cellular expression. Xenopus oocytes were injected with 1, 2, or 3 cRNAs alone (lanes 1-8) or together with 1 cRNAs (lanes 9-14) and labeled with [35S]methionine. Digitonin extracts were prepared after a 24-h pulse and a 48-h chase period and  subunits were immunoprecipitated with a polyclonal -antibody, resolved by SDS-polyacrylamide gel electrophoresis, and revealed by fluorography. The positions of  subunits and of a protein of known molecular mass are indicated. B, human Na,K-ATPase - isozyme complexes expressed in Xenopus oocytes exhibit a stoichiometry of close to 1. Xenopus oocytes were injected with 1 cRNAs together with 1, 2, or 3 cRNAs and subjected to a 24-h pulse. To enable reliable quantification of  subunits, oocytes were incubated during the pulse with brefeldine A which prevents endoplasmic reticulum exit of - complexes and full glycosylation of the  subunit (see "Experimental Procedures"). - Complexes were immunoprecipitated with an -antibody under nondenaturating conditions and the immunoprecipitated samples were treated with endoglycosidase H to remove core sugars from the  subunit. Quantification of the  and the non-glycosylated  subunits revealed a 1/1 ratio of 1.3 ± 0.14 (mean ± S.E., n = 7), a 2/1 ratio of 1.1 ± 0.23 (n = 4), and a 3/1 ratio of 1.4 + 0.17 (n = 3) by taking into account that human 1, 1, 2, and 3 isoforms contain 23, 4, 10, and 7 methionines, respectively. The positions of 1 isoforms and the non-glycosylated (ng)  isoforms are indicated. C, human - isozyme complexes expressed in Xenopus oocytes are targeted to the plasma membrane. Xenopus oocytes were injected with 1, 2, or 3 cRNAs together with 1, 2, or 3 cRNAs (lanes 1-9) and labeled with [35S]methionine for 72 h. - Complexes were immunoprecipitated with an -antibody under nondenaturating conditions. The positions of  isoforms and the core-glycosylated (*) and the fully glycosylated ()  isoforms are indicated. The differences in the molecular masses of the core and fully glycosylated  isoforms correspond to the presence of 3, 8, and 2 putative glycosylation sites in 1, 2, and 3 isoforms, respectively. 3 Isoforms show two core-glycosylated species which are glycosylated on 1 and 2 glycosylation sites, respectively. ni, non-injected oocytes (lane 10).

Cell Surface Expression and Transport Properties of Human Na,K-ATPase Isozymes

Cell Surface Expression of Functional Isozymes-- To confirm the presence and the functionality of the 9 human - isozymes at the cell surface, maximal ouabain binding and pump current measurements were performed on intact oocytes. In oocytes expressing human - complexes, ouabain binding was 3-5-fold higher (Fig. 2A, lanes 2-10) than in non-injected oocytes (lane 1). The expression levels were similar for isozymes associated with different  isoforms confirming that all  isoforms can form functional pumps at the cell surface with each  isoform. The cell surface expression of human isozymes appears low compared with the large excess of human  isozymes synthesized over endogenous, oocyte  subunits (Fig. 1C). However, it has to be considered that the turnover of endogenous, oocyte  subunits is low (26) compared with that of exogenous  subunits and therefore the signal of biosynthetically labeled, immunoprecipitated  subunits does not necessarily reflect the endogenous Na,K-ATPase pool present in oocytes. Oocytes expressing exogenous Na,K-pump isozymes showed pump currents which were 2-4-fold higher (Fig. 2B, lanes 2-10) than those measured in non-injected oocytes (lane 1).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Cell surface expression and functionality of human Na,K-ATPase isozymes. Oocytes were not injected (ni) or injected with different combinations of  and  isoform cRNAs as indicated and incubated for 3 days. A, maximal ouabain binding to intact oocytes. After electrophysiological measurements (shown in B), oocytes were transferred into a K+-free solution and ouabain binding was determined as described under "Experimental Procedures." The results are expressed as the number of ouabain-binding sites per oocyte. B, maximal Na,K-pump current (Imax). Increasing concentrations of external K+ were added to Na+-loaded oocytes and the pump currents were measured by the two-electrode voltage-clamp technique as described under "Experimental Procedures." Imax values were determined by extrapolation of the K+ activation curves. Data for Imax and ouabain binding are mean ± S.E. from 10-12 oocytes of one out of two to four similar experiments.

Na,K-Pump Turnover Rates-- Assuming an identical stoichiometry, the ratio between the mean K⁺-activated current and the mean number of ouabain-binding sites is a measure of the transport turnover rates of Na,K-pumps. Human Na,K-pump isozymes had different turnover rates which, according to variance analysis, depended on the  isoform (p < 0.001) and not the  isoform (p = 0.56) present in the - complexes (Fig. 3, Table I). 1- complexes (lanes 2-4) had a similar turnover number than endogenous, oocyte Na,K-pumps (lane 1) which was higher than that of human 2- (lanes 5-7) or of 3- (lanes 8-10) complexes which had the lowest turnover rates.

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Turnover rates of human Na,K-ATPase isozymes. The turnover number (charges transported/s/molecule) of the different Na,K-ATPase isozymes was calculated for individual oocytes as the ratio between the total pump current (as total charges transported/s) and the maximal ouabain binding sites. Data are mean ± S.E. of two to four experiments shown in Fig. 2 after subtraction of the endogenous Na,K-pump component. The turnover numbers of all 1- complexes were significantly different from those of 3- complexes (p < 0.01).

Apparent K⁺ Affinity-- The apparent affinities for K⁺ (K_1/2 K⁺) of the 9 human Na,K-ATPase isozymes expressed in Xenopus oocytes were determined from K⁺ activation curves of the Na,K-pump current measured (Fig. 4, inset). K_1/2 values for K⁺ ranged between 0.9 and 2.7 mM (Fig. 4 and Table I). Variance analysis revealed that the K⁺ affinity of human Na,K-pumps is determined by both the  isoform (p < 0.0001) and the  isoform (p < 0.0001) present in the isozyme complex as well as by the particular combination of  and  isoforms (p < 0.0001). The most pronounced effect of this cooperative mechanism was observed in 2-2 complexes which exhibited a more than 2-fold increase in the K_1/2 value for K⁺ compared with 2-1 complexes.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   K+ activation of human Na,K-ATPase isozymes. Oocytes were injected with different  and  isoform cRNAs as indicated. The K+ activation of the Na,K-pump currents was measured as described in the legend to Fig. 2 and under "Experimental Procedures" and the K+ activation constants (K1/2 K+) were determined. The K+ activation of the endogenous, oocyte Na,K-pumps was determined in parallel and the averaged, endogenous pump currents were subtracted from Na,K-pump currents measured in individual cRNA-injected oocytes at different external K+ concentrations. Data are mean ± S.E. from 15 to 30 oocytes obtained from two to four different Xenopus females. Inset, representative examples of K+ activation curves of exogenous Na,K-pump currents (I) determined in single oocytes injected with 1 plus 2 (), 2 plus 2 (), 3 plus 2 () cRNAs. The Imax values are represented in Fig. 2.

Apparent Na⁺ Affinity-- The activation by internal Na⁺ was investigated for human 1-1, 2-1, and 3-1 complexes by using an electrophysiological technique involving the expression of the rat epithelial Na⁺ channel along with the Na,K-pumps. The presence of rat epithelial Na⁺ channel permitted to achieve a controlled, gradual increase in the intracellular Na⁺ concentration from 2 to 70 mM, to measure intracellular Na⁺ concentrations and to determine the Na⁺ dependence of Na,K-pump currents as shown in Fig. 5A. The maximal pump currents of the various Na,K-ATPase isozyme complexes (Fig. 5A, inset), extrapolated from Na⁺ activation curves (Fig. 5A), were similar to those extrapolated from K⁺ activation curves (Fig. 2). This result indicates that the lower pump current measured for 3- complexes compared with that measured for 1- and 2- complexes (Fig. 2B) is indeed due to a lower turnover rate and not to an inefficient activation by internal Na⁺. As shown in Fig. 5B and Table I, 1-1 complexes exhibited a high (K_1/2 Na⁺ = 7.3 ± 0.9 mM), 2-1 complexes an intermediate (K_1/2 Na⁺ = 11.8 ± 2.9 mM), and 3-1 complexes a low (K_1/2 Na⁺ = 30 ± 5.2 mM) apparent affinity for internal Na⁺.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Na+ activation of human Na,K-ATPase isozymes. Oocytes were not injected (ni) or injected with 1 plus 1, 2 plus 1, or 3 plus 1 cRNAs of human Na,K-ATPase together with  and  and  cRNAs of the rat epithelial Na+ channel, and the Na+ activation of the Na,K-pumps was determined as described under "Experimental Procedures." A, representative examples of Na+ activation curves of Na,K-pump currents (I) determined in non-injected oocytes () or in oocytes injected with 1 plus 1 (), 2 plus 1 (), 3 plus 1 () cRNAs. Inset, Imax extrapolated from Na+ activation curves. B, Na+ activation constants (K1/2 Na+) of the endogenous, oocyte (lane 1) and of the exogenous, human Na,K-pump isozymes (lanes 2-4). To determine the Na+ activation of the exogenous, human Na,K-pumps, the currents which were mediated by the exogenous Na,K-pumps in cRNA-injected oocytes were calculated as described under "Experimental Procedures." To avoid artifactual results due to the low Na,K-pump current of the 3-1 complexes, only oocytes were analyzed which exhibited at least 2 times higher currents than those measured in non-injected oocytes. Data are mean ± S.E. from 7 to 11 oocytes from 3 different Xenopus females. Lane 1 versus lane 4, p > 0.05; lane 2 versus lane 3, p > 0.05; lanes 2 and 3 versus lane 4, p < 0.05.

Voltage Dependence-- The voltage dependence of the ouabain-sensitive currents was investigated in oocytes expressing human 1-1, 2-1, and 3-1 complexes (Fig. 6). The human 1-1 and 2-1 pump currents were voltage-sensitive over the whole potential range. The I-V curve profile of the human 1-1 pump currents was similar to that obtained for the endogenous Na,K-pumps (data not shown). The 2-1 pump complexes exhibited the most voltage-sensitive currents which at low membrane potentials of about 130 mV were nearly abolished. On the other hand, 3-1 complexes produced pumps that were not significantly affected by voltage changes over the whole potential range.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Voltage dependence of the human Na,K-ATPase isozymes. Oocytes were not injected (ni) or injected with 1 plus 1 (), 2 plus 1 (), or 3 plus 1 () cRNAs of the human Na,K-ATPase. K-activated and ouabain-sensitive Na,K-pump currents were determined at different membrane potentials as described under "Experimental Procedures." Averaged endogenous pump currents were subtracted from individual pump current measurements of cRNA-injected oocytes. Since 3-1 complexes have low pump currents, we only analyzed oocytes which had at least 2-fold higher Na,K-pump currents than non-injected oocytes. Current values at 50 mV were used as reference to normalize data. Data are mean ± S.E. of 6 to 13 oocytes from two to four different batches. Inset, Imax values at 50 mV after subtraction of endogenous current.

Pharmacological Properties of Human Na,K-ATPase Isozymes

Ouabain Sensitivity-- Because equilibrium binding studies on intact oocytes may be compromised by internalization and degradation of Na,K-pumps and recycling of ouabain, the equilibrium binding constants (K_d) for [³H]ouabain was determined for each one of the 9 different Na,K-ATPase isozymes on oocyte microsomes. Scatchard plots of binding data showed that maximal ouabain binding on microsomes from cRNA-injected oocytes was 3-10-fold higher than that obtained on microsomes from non-injected oocytes (Fig. 7A). The maximal binding was not significantly influenced by the presence of different  isoforms (data not shown). After subtraction of binding data obtained on microsomes of non-injected oocytes, Scatchard plots obtained with microsomes from cRNA-injected oocytes were linear (Fig. 7A), reflecting a single population of [³H]ouabain binding sites for each Na,K-ATPase isozyme. Ouabain affinity measured in Na-ATP conditions in the absence of K⁺ was high and of similar magnitude, in the nanomolar range, for all Na,K-ATPase isozymes (Fig. 7B and Table I). However, 2- isozymes had significantly higher K_d values (12-23 nM) than 1- or 3- isozymes (5-7 nM) (Fig. 7B, compare lanes 4-6 to lanes 1-3 and 7-9). According to variance analysis, the  isoform (p < 0.001) and to a lesser extent also the  isoform (p = 0.023) present in the - complexes as well as the particular - combination (p = 0.016) affected the ouabain affinity of the Na,K-pumps.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Ouabain affinity of human Na,K-ATPase isozymes. Oocytes were not injected (ni) or injected with different  and  cRNAs as indicated. Three days later, microsomes were prepared as described under "Experimental Procedures." Ouabain binding measurements were carried out in Na-ATP conditions (see "Experimental Procedures") in the presence of 109 to 5 × 108 M [3H]ouabain for 5 h either in the absence or presence of 5 mM K+. A, representative Scatchard plots of ouabain binding data obtained with microsomes from non-injected oocytes () or from oocytes injected with 1 plus 1 (), 2 plus 1 (), and 3 plus 1 () cRNAs in the absence of K+. Data from equilibrium ouabain binding measurements obtained with microsomes from noninjected oocytes were subtracted from those obtained with microsomes from injected oocytes. One out of four similar experiments is shown. B, equilibrium dissociation constants (Kd) of the human Na,K-ATPases isozymes in the absence (white bars) or presence (black bars) of 5 mM K+ as calculated from Scatchard plots. Data are mean ± S.E. of two to three experiments done in triplicate on two to three different microsomal preparations. Lanes 4 and 5 (K+) versus lanes 1-3 and 7-9 (K+) p < 0.01.

K⁺ is known to antagonize ouabain binding. For 1- complexes, 5 mM K⁺ induced a larger increase in K_d values for ouabain (3-4-fold) than for 2- and 3- complexes (2-3 fold). As an exception, for 2-2 complexes, the K_d value was not influenced by the presence of K⁺ (Fig. 7B, lane 5) which may reflect the lower apparent K⁺ affinity of these complexes (Fig. 4).

Association and Dissociation Rate Constants of Ouabain Binding-- To investigate in more detail the ouabain binding kinetics, we determined the association rate constant (k₊₁) and the dissociation rate constant (k₁) for all 9 human Na,K-ATPase isozymes.

Fig. 8A shows representative dissociation kinetics of ouabain for 1-1, 2-1, and 3-1 complexes which were similar to those obtained with isozymes containing 2 or 3 isoforms. The dissociation rate constants of ouabain for all 9 Na,K-ATPase isozymes were calculated from the slopes of the dissociation plots and are summarized in Fig. 8B and Table I. - complexes formed with 1 and 3 isoforms had slow dissociation rate constants corresponding to half-lives (t_1/2) between 30 and 80 min, whereas those formed with 2 isoforms had rapid dissociation kinetics with a t_1/2 of about 4-5 min.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Dissociation rate constants (k1) of ouabain binding to human Na,K-ATPase isozymes. Microsomes were prepared from non-injected oocytes or from oocytes injected with different  and  cRNAs. After incubation of microsomes for 1 h with 5 × 108 M [3H]ouabain under Na-ATP conditions (see "Experimental Procedures"), unlabeled ouabain (5 × 105 M, final concentration) was added to initiate ouabain dissociation (time 0) and ouabain binding was determined after various time periods. Ouabain binding due to endogenous, oocyte Na,K-ATPase was subtracted from data obtained on microsomes from cRNA-injected oocytes. A, representative experiment of dissociation kinetics obtained with microsomes from oocytes injected with 1 plus 1 (), 2 plus 1 (), and 3 plus 1 () cRNAs. The data are represented in a ln B/Beq versus time plot; Beq = specific ouabain bound at equilibrium and B = specific ouabain bound at several time points. B, dissociation rate constants (k1). k1 values were calculated as the slopes of plots shown in A. Data are mean ± S.E. of three experiments done in triplicate. Lanes 4, 5, 6 versus lanes 1-3 and 7 and 8, p < 0.01.

Representative examples of [³H]ouabain association kinetics for 1, 2, and 3 isoforms associated with 1 isoforms are shown in Fig. 9A. The time required to reach equilibrium binding was in the range of 10 min (2-) and 60 min (1-, 3-). The observed association rate constants (k_obs) were calculated from the slopes of plots shown in Fig. 9B and the association rate constants (k₊₁) (Fig. 9C and Table I) as described under "Experimental Procedures." Similar to dissociation kinetics, association kinetics of ouabain to Na,K-ATPase isozymes followed the order 2 3 > 1. According to variance analysis, neither association (p = 0.66) nor dissociation (p = 0.12) rate constants of ouabain binding were influenced by the associated  isoform. The K_d values calculated from the ratio k₁/k₊₁ were close to those measured by equilibrium binding for the different isozymes (1-, 1-3 nM; 2-, 4-5 nM; 3-, 2.5-3 nM) which supports that human Na,K-ATPase isozymes have similar, low K_d values for ouabain. On the other hand, our results clearly indicate that despite similar K_d values, the association and dissociation kinetics of ouabain differ significantly among the different  isoforms.

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Association rate constants (k+1) of ouabain binding to human Na,K-ATPase isozymes. Microsomes were prepared from non-injected (ni) oocytes or from oocytes injected with different  and  cRNAs. A, representative experiment of association kinetics obtained with microsomes from oocytes injected with 1 plus 1 (), 2 plus 1 (), and 3 plus 1 () cRNAs. Ouabain binding was carried out in Na-ATP conditions (see "Experimental Procedures") in the presence of 108 M [3H]ouabain and determined after various time periods. Ouabain binding due to endogenous, oocyte Na,K-ATPase was subtracted from data obtained on microsomes from cRNA-injected oocytes. Shown is one out of three experiments done in triplicate on three different microsomal preparations. B, ln (Beq  B)/Beq versus time plot of data shown in A. Beq = specific [3H]ouabain bound at equilibrium; B = specific ouabain bound at several time points. C, association rate constants k+1. k+1 values were calculated as described under "Experimental Procedures." Data are mean ± S.E. of three experiments done in triplicate.

K⁺ Antagonism of Ouabain Binding-- The K⁺ antagonism of digitalis binding to