Selective Assembly of Na,K-ATPase α₂β₂ Heterodimers in the Heart

Abstract

The Na,K-ATPase α₂ subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca²⁺, whereas α₁ has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α₂ in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α₂ is preferentially expressed with β₂ in T-tubules of cardiac myocytes, forming α₂β₂ heterodimers. We have expressed human α₁β₁, α₂β₁, α₂β₂, and α₂β₃ in Pichia pastoris, purified the complexes, and compared their functional properties. α₂β₂ and α₂β₃ differ significantly from both α₂β₁ and α₁β₁ in having a higher K_0.5K⁺ and lower K_0.5Na⁺ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K⁺ and shift of the E₁P-E₂P conformational equilibrium toward E₁P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α₂β₂ and α₂β₃ over α₁β₁ (range 22–33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K⁺ affinity of α₂β₂ could allow an acute response to raised ambient K⁺ concentrations in physiological conditions and explain the importance of α₂β₂ for cardiac muscle contractility. The high sensitivity of α₂β₂ to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α₂β₂-selective digoxin derivatives for reducing cardiotoxicity.

Introduction

The Na,K-ATPase plays a key role in cardiac muscle contractility by regulating the cytosolic Ca²⁺ concentration, via the Na⁺/Ca²⁺ exchanger, and hence the excitation-contraction coupling in cardiac myocytes (1). Three isoforms of the Na,K-ATPase α subunit and three isoforms of the β subunit are expressed in cardiac muscle, and their expression levels vary between species (2–4). In addition, the regulatory FXYD1 subunit and possibly other FXYD proteins are expressed in cardiac muscle (5, 6). Although α₁ is the most abundant α subunit isoform in cardiomyocytes in the majority of species, the α₂ isoform is functionally more important for cardiac muscle contractility (7–11). The mechanisms underlying distinct roles of α₂ and α₁ isoforms in the heart are unclear. In smooth and skeletal muscle, the α₂ subunit is concentrated at the junctions between the tubular membrane and sarcoplasmic reticulum in close proximity to the Na⁺/Ca²⁺ exchanger and other components of the excitation-contraction complex (12), resulting in more efficient regulation of contraction by the α₂ subunit in contrast to uniformly distributed α₁ subunit. However, the published data on localization of the two isoforms in cardiac myocytes is inconsistent. For example, preferential T-tubular localization of α₂ but uniform plasma membrane localization of the α₁ have been reported by several groups (11, 13), whereas quite the opposite distribution of the isoforms was reported (3), and uniform distribution of both α₁ and α₂ subunits has also been reported (14). Measurements of the Na,K-ATPase function of isoforms in cardiac myocytes suggest that the α₂ subunit is more concentrated in T-tubular membranes than in the external sarcolemma, whereas the α₁ subunit is equally distributed in the plasma membrane (11, 15, 16).

Assembly with the β subunit is required for maturation, trafficking, membrane insertion, and transport activity of the α subunit (17), and three β isoforms differentially regulate these processes. Three β subunit isoforms differ from each other in the number of N-linked glycans, and recombinant addition or removal of N-glycans has been shown to alter the polarized sorting of the Na,K-ATPase (18, 19). The three β subunit isoforms differentially modulate voltage dependence of the Na,K-ATPase activity and the apparent affinity of the enzyme for Na⁺, K⁺ and inhibitors (20, 21). β₁, but not β₂ or β₃, undergoes post-translational modifications, namely glutathionylation and palmitoylation, and β₁ glutathionylation induced by oxidants decreases the Na,K-ATPase activity (22, 23). Thus, the specific assembly with particular β isoforms could account for distinct physiological roles of α₁ and α₂ isoforms. However, it is not known whether the α₂ subunit has a preference for a particular β isoform in cardiac myocytes.

To examine possible functional differences between α₂β_(1–3) isoforms, we have expressed human α₂ with all three human β subunits in Pichia pastoris, purified the complexes, and compared their functional characteristics and inhibitor sensitivity. Previous work has demonstrated some features of α₂β₂ and α₂β₃ when expressed in Xenopus oocytes and SF-9 insect cells (20, 24). Experimentally, the purified complexes are advantageous in that they allow characterization of the functional properties and inhibitor selectivity of each isoform separately (25–27) and also detailed mechanistic properties of ion binding and conformational changes (28–30).

The inhibition of the Na,K-ATPase by digitalis CGs⁵ has been used for years to treat heart failure. CGs increase the force of cardiac muscle contraction by reducing the inward Na⁺ gradient that decreases Ca²⁺ extrusion via the Na⁺/Ca²⁺ exchanger (NCX1), leading to increased Ca²⁺-induced Ca²⁺ release from the sarcoplasmic reticulum during excitation-contraction coupling. As a toxic side effect, excessive inhibition of the Na,K-ATPase increases bulk intracellular Na⁺ concentration, excessive accumulation of Ca²⁺ ions (i.e. “calcium overload”), and “spontaneous” Ca²⁺ release from sarcoplasmic reticulum that can trigger cardiac arrhythmias (1, 31). The preferential role of α₂ in excitation-contraction coupling suggests that α₂-specific inhibitors might be able to induce an ionotropic effect without triggering Ca²⁺ overload and arrhythmias. Some years ago, we demonstrated that some natural CGs, such as digoxin and digitoxin, exhibit a moderate intrinsic selectivity for α₂ over α₁, whereas aglycones, such as digoxigenin and digitoxigenin, show no selectivity (25). Thus, the isoform selectivity was attributed to the sugar moiety, especially the third digitoxose. It was proposed that modification of the third sugar could raise selectivity for α₂. Indeed, chemical modification of the third digitoxose residue of digoxin by periodate oxidation and reductive amination by primary amines, R-NH₂, produced perhydro-1,4-oxazepine derivatives with enhanced selectivity of inhibition for α₂β₁ over α₁β₁ (27). Most recently, we have described perhydro-1,4-oxazepine digoxin derivatives with various straight chain, branched, and cyclic or heterocyclic aliphatic substitutions and shown that compounds with four carbon substitutions (cyclobutyl (DcB), methyl cyclopropyl (DMcP), and isobutyl (DiB)) showed an especially high selectivity for α₂β₃/α₁β₁. α₂β₃ is the principal Na,K-pump isoform in non-pigmented cells of ciliary epithelium. We have shown that the digoxin derivatives with enhanced selectivity for α₂β₁ and especially α₂β₃ efficiently reduce intraocular pressure when applied topically to rabbit eyes (26, 27).

We present evidence here that the α₂ and β₂ isoforms preferentially assemble with each other in the heart and reside predominantly in the T-tubules. By systematic analysis of properties of purified α₂β₂ in comparison with α₁β₁, α₂β₁, and α₂β₃, we demonstrate distinctive functional properties and isoform-selective inhibition of α₂β₂, which explain the important role of α₂ for myocardial contractility and the pharmacological potential of α₂β₂-selective CGs.

Results

Distribution of Na,K-ATPase α₁, α₂, β₁, and β₂ Subunits in Rat and Human Heart

Normal rat or human frozen heart sections were used to study the intracellular localization of the Na,K-ATPase subunit isoforms by immunofluorescence. In rat cardiomyocytes, the Na,K-ATPase subunits were differentially distributed (Fig. 1A). The α₁ isoform was less abundant in the T-tubular membranes than in the external sarcolemma. Conversely, the α₂ isoform was more abundant in the T-tubular membranes than in the sarcolemma, consistent with previously published results (11, 13, 21). Both isoforms are expressed at the intercalated discs, but α₁ subunit expression is more pronounced. The immunofluorescence pattern with the Na,K-ATPase β₂ antibody was similar to that of the α₂ antibody with major expression observed in the T-tubules. In frozen sections of human hearts, the β₁ isoform was present more abundantly in the sarcolemma than in the T-tubular membranes, whereas the β₂ isoform was present exclusively in the T-tubules (Fig. 1B). These results demonstrate a differential localization of the Na,K-ATPase subunits with the α₂ and β₂ Na,K-ATPase subunits following the same T-tubule-specific expression pattern and the α₁ and the β₁ subunits ubiquitously distributed but more abundant in the sarcolemma.

FIGURE 1.

Na,K-ATPase β₂ and α₂ subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α₁ and β₁ subunits are localized in both sarcolemma and T-tubules. A, frozen sections of rat heart were double-stained by using mouse antibodies against α₁ subunit (green) and rabbit antibodies against α₂ subunit (red) (top panels) or by using mouse antibodies against α₁ subunit (green) and rabbit antibodies against β₂ subunit (red) (bottom panels). Anti-mouse Alexa Fluor 488-conjugated secondary antibodies were used to detect anti-α₁ primary antibodies, and anti-rabbit Alexa Fluor 633-conjugated secondary antibodies were used to detect anti-β₂ and anti-α₂ primary antibodies. The arrows show localization of the α₁ subunits, but not of α₂ and β₂ subunits, in the sarcolemma. The arrowheads show co-localization of α₁ and β₂ subunits or α₁ and α₂ subunits in T-tubules. B, frozen sections of human heart were double-stained by using mouse antibodies against β₁ subunit (green) and rabbit antibodies against β₂ subunit (red). Anti-mouse Alexa Fluor 488-conjugated secondary antibodies were used to detect anti-β₁ primary antibodies, and anti-rabbit Alexa Fluor 633-conjugated secondary antibodies were used to detect anti-β₂ primary antibodies. The arrows show localization of the β₁ subunits, but not of β₂ subunits, in the sarcolemma. The arrowheads show co-localization of β₁ and β₂ subunits in T-tubules. The stealth-like arrowheads show the T-tubules in which the β₂ subunits, but not the β₁ subunits, are present.

Because the Na,K-ATPase is a crucial component in regulating postnatal cardiac function (32), we analyzed whether the Na,K-ATPase subunits are also selectively expressed during embryogenesis. Paraffin-embedded sections of mouse embryos (embryonic day 12.5) were analyzed by immunofluorescence (Fig. 2). The Na,K-ATPase α₁ subunit was expressed ubiquitously (top and bottom left panels), whereas the α₂ and β₂ were mostly restricted to the heart (top and bottom right panels, respectively).

FIGURE 2.

The Na,K-ATPase α₂ subunit and β₂ subunit are preferentially expressed in the mouse embryonic heart in contrast to the ubiquitously expressed α₁ subunit. A, paraffin-embedded sections of mouse embryos (embryonic day 12.5) were double-stained by using mouse antibodies against α₁ subunit (left panels) and rabbit antibodies against α₂ subunit or β₂ subunit (right panels). Anti-mouse Alexa Fluor 488-conjugated secondary antibodies were used to detect anti-α₁ primary antibodies, and anti-rabbit Alexa Fluor 633-conjugated secondary antibodies were used to detect anti-α₂ or anti-β₂ primary antibodies. The rectangles show the embryonic hearts. B, a scheme demonstrating the assembly of the images shown in A. Each rectangle outlined by a dotted line represents an individual confocal microscopy image taken at ×10 magnification.

The Na,K-ATPase α₂ and β₂ Subunits Are Selectively Co-immunoprecipitated from Mouse Heart

To analyze the composition of the Na,K-ATPase heterodimers present in heart microsomal membranes, proteins were co-immunoprecipitated with an α₂ subunit-specific antibody, and the presence of α₁, β₁, β₂, and β₃ subunits was analyzed by Western blotting. To prevent the overlap of the β subunits bands with the band corresponding to the heavy chain of the immunoprecipitating antibody, the immunoprecipitated proteins were treated with PNGase F before SDS-PAGE. Immunoprecipitation of the α₂ subunit resulted in the co-immunoprecipitation of the β₂ subunit and minor amounts of β₁ and β₃ subunits (Fig. 3A, left). No α₁ subunits were detected in the immunoprecipitation with the α₂-specific antibody. In contrast, when the α isoform-nonspecific antibody was used, both α₁ and α₂ subunits were immunoprecipitated, and approximately equal amounts of all of the three β subunits were co-immunoprecipitated (Fig. 3A, right). Conversely, immunoprecipitation with a β₂-specific antibody resulted in co-immunoprecipitation of the α₂ but not the α₁ subunit (Fig. 3B). Taken together, these results suggest that the α₂ and β₂ subunit isoforms associate predominantly with each other but not with other isoforms expressed in the heart.

FIGURE 3.

The Na,K-ATPase α₂ subunit and β₂ subunit preferentially interact with each other in mouse heart. A, Western blotting analysis of proteins immunoprecipitated and co-immunoprecipitated from the detergent extracts of mouse heart microsome membranes by using either the α₂-specific antibodies (left panels) or the α-nonspecific antibodies (right panels) shows preferential co-immunoprecipitation of the β₂ subunit with the α₂ subunit. B, Western blotting analysis of the immunoprecipitated β₂ subunit and co-immunoprecipitated α₂ subunit isoforms shows that the α₂ subunit is preferentially co-precipitated with the β₂ subunit. Input lanes contain 10% of the extract used for immunoprecipitation. To prevent an overlap of the β subunit bands with the heavy chain band of the antibodies used for immunoprecipitation, the immunoprecipitated proteins were treated with PNGase F before SDS-PAGE. IP, immunoprecipitation; DG, deglycosylated.

Expression and Purification of Na,K-ATPase α₂β₂

α₂β₂ and α₂β₃ were expressed in P. pastoris as described under “Experimental Procedures.” Under optimal expression conditions, specific ouabain binding for the α₂β₂ clone was 8 ± 1 pmol/mg protein and 10 ± 2 pmol/mg for the α₂β₃ clone, respectively. The addition of DMSO to the culture medium, which was reported to increase expression of GPCRs in P. pastoris, did not increase expression (33).

Both isoforms were purified via the N-terminal His tag of the β subunit by metal affinity chromatography on BD-Talon beads and reconstituted with purified human FXYD1 on the BD-Talon beads, as described in Experimental Procedures.⁶ The purity of α₂β₂ and α₂β₃ was comparable with that of purified human α₁β₁ and α₂β₁ complexes. As depicted in Fig. 4, at least five bands of β₂ were observed, in comparison with two bands for β₃ and β₁. The two bands of β₁ were shown previously to represent two glycosylated versions of the β₁ subunit of the Man_X-GlcNAc₂ type, typical for P. pastoris (34). An increase in heterogeneity of glycoforms of β₂ as compared with β₃ and β₁ is consistent with the presence of eight N-glycosylation sites in β₂ and only two and three in β₃ and β₁, respectively. When deglycosylated by PNGase treatment, all three β isoforms migrated at their expected molecular mass of approximately 35, 31, and 33 kDa for β₁, β₃, and β₂, respectively.

FIGURE 4.

Expression of purified Na,K-ATPase isoforms. Coomassie-stained SDS-PAGE of purified isoforms (5 μg/lane). For deglycosylation, samples were denatured and treated with PNGase F for 60 min at 37 °C.

Recent studies have demonstrated that α₂β₁ is less stable to thermal and detergent-mediated inactivation than α₁β₁, due to suboptimal interaction with phosphatidylserine (35). We investigated the relative effect of the β subunit on Na,K-ATPase isoform stability by thermal inactivation of [³H]ouabain binding to the membranes and Na,K-ATPase activity of the purified proteins, as described in previous publications (see for example Refs. 29 and 65). By both criteria, the α₂ complexes were significantly less thermally stable than α₁, and α₂β₂ was somewhat less stable than α₂β₁ and α₂β₃. Thus, the relative instability of the α₂β_(1–3) complexes is a feature attributable primarily to the α₂ subunit. The differences between β isoforms, β₂ < β₃ ≈ β₁, are rather small.

Functional Properties of Purified α₁β₁, α₂β₁, α₂β₂, and α₂β₃ Complexes

The specific Na,K-ATPase activity of the purified isoforms was highest for α₁β₁ (16.4 ± 0.7 μmol/mg/min), followed by similar values for α₂β₁ (10.9 ± 0.6), α₂β₃ (10.7 ± 1.9), and α₂β₂ (8.4 ± 1.4; Table 1, column 2). Note that FXYD1 itself inhibits the Na,K-ATPase activity of the purified human α₁β₁ by about 25% and α₂β₁ by about 15% compared with the αβ complexes alone (30). One kinetic property showing large differences between the isoform complexes was the K_0.5K⁺ for activation of Na,K-ATPase activity, with values of 1.5 ± 0.1 mm for α₁β₁ and 2.7 ± 0.1 mm for α₂β₁, whereas the K_0.5K⁺ for both α₂β₂ and α₂β₃ was much higher, with apparent K_0.5 values of 7.4 ± 0.2 and 6.4 ± 0.5 mm, respectively (Table 1, column 4). Sodium titrations for activation of Na,K-ATPase activity revealed that K_0.5Na⁺ was not different between α₁β₁ and α₂β₁, whereas K_0.5Na⁺ for α₂β₂ and α₂β₃ was significantly lower (Table 1, column 3). We have also looked at the affinity for inhibition of Na,K-ATPase activity by vanadate, which is a phosphate analogue that binds to the E₂(2K) conformation, mimicking the transition state E₂P2K during dephosphorylation (36). All three α₂ isoforms have a higher K_i vanadate compared with α₁β₁ (K_i = 0.48 μm), and the effects are greatest with β₂ and β₃ in the order α₂β₂ (K_i = 34 μm) > α₂β₃ (K_i = 19 μm) > α₂β₁ (K_i = 3.5 μm) (Table 1, column 5).

TABLE 1.

Functional properties of purified Na,K-ATPase complexes

Specific activity, K_0.5Na⁺, K_0.5K⁺, and K_i vanadate of purified Na,K-ATPase complexes are shown. Values represent averages of at least three different experiments ± S.E. The reaction medium contained sodium plus potassium as indicated, 1 mm ATP, 3 mm MgCl₂, 25 mm histidine, pH 7.4, 1 mm EGTA, 0.01 mg/ml SOPS, 0.001 mg/ml cholesterol, and 0.005 mg/ml C₁₂E₈. Maximal Na,K-ATPase activity was measured in the presence of 120 mm NaCl, 20 mm KCl. For ion titrations, ATPase activity was measured in medium containing 80 mm KCl and 0–120 mm NaCl or 120 mm NaCl and 0–40 mm KCl. Ionic strength was maintained constant with choline chloride. K_0.5 values were obtained from least square fits of the data points to the Hill equation. K_i vanadate was determined in medium containing 120 mm NaCl, 20 mm KCl, and 1 mm ATP, and data points were fitted to a one-side inhibition model.

Isoform complexes	Specific Na,K-ATPase activity	K0.5Na+ (nH)	K0.5K+ (nH)	Vanadate Ki
	μmol/mg/min	mm	mm	μm
α1β1	16.4 ± 0.7	16 ± 0.4 (1.7 ± 0.1)	1.5 ± 0.1 (1.8 ± 0.1)	0.5 ± 0.1
α2β1	10.9 ± 0.6	17.7 ± 0.5 (1.9 ± 0.2)	2.7 ± 0.1 (2.0 ± 0.2)	3.5 ± 0.3
α2β2	8.4 ± 1.4	9.8 ± 0.7 (1.8 ± 0.2)	7.4 ± 0.2 (1.7 ± 0.1)	34.0 ± 2.0
α2β3	10.7 ± 1.9	13.0 ± 0.2 (1.9 ± 0.2)	6.4 ± 0.5 (1.8 ± 0.2)	19.0 ± 1.5

A simple explanation of the raised K_0.5K⁺ in Na,K-ATPase activity assays could be that the β₂ and β₃ subunit reduce the binding affinity of 2K ions for their extracellular sites. K⁺ and Na⁺ binding was determined by using the electrochromic shift dye RH421 in a medium of fixed ionic strength (containing also 5 mm magnesium ions) (Fig. 5 and Table 2) (29, 30, 37). The inset of Fig. 5A shows the typical RH421 responses upon the addition of Na⁺ ions (E₁-E₁(3Na)) and then ATP (to E₂P) and K⁺ ions (E₂(2K)) for the α₂β₂ complex, as explained in recent papers (29). By varying Na⁺ or K⁺ concentrations, equilibrium titrations of either 3Na⁺ binding to cytoplasmic sites or 2K⁺ binding to extracellular sites are readily obtained, leading to curves such as those in Fig. 5, A and B. The binding parameters derived from best fits of the curves to the Hill equation are collected in Table 2, where K_0.5Na⁺ and K_0.5K⁺ represent the intrinsic binding affinity for 3Na⁺ and 2K⁺ ions, respectively. Evidently, the intrinsic binding affinity for 3Na⁺ ions is the same for all of the isoform complexes. By contrast, in these conditions, the binding affinity of 2K⁺ ions to α₂β₁ is significantly lower than to α₁β₁, and the binding affinity for both α₂β₂ and α₂β₃ is further strongly reduced compared with α₂β₁. Note that, compared with K_0.5K⁺ for α₁β₁, the K_0.5K⁺ for α₂β₂ or α₂β₃ is almost an order of magnitude higher. Because vanadate binds primarily to the E₂(2K) conformation, the differences in K_i for vanadate inhibition of Na,K-ATPase activity could be secondary to the differences in K_0.5K⁺ values, with the same order for the K_i as for K_0.5K⁺ values (α₁β₁ < α₂β₁ < α₂β₃ < α₂β₂; Table 1).

FIGURE 5.

Sodium and potassium binding and conformational changes of the isoforms measured with RH421. A, equilibrium titration of sodium binding to the E₁ conformation. The inset shows a standard experiment and fluorescence changes associated with ion binding and release to α₂β₂ (sodium binding to E₁, sodium release and conformational transition to E₂P, and potassium binding). B, equilibrium titration of potassium binding to E₂P. C, stopped-flow trace of the E₁Na3 → E₂P transition of α₂β₂ fitted to a double exponential function. The average of 15 traces is shown. D, stopped-flow trace of the α₂β₂ E₂(Rb2)ATP → E₁Na3ATP transition fitted to a single exponential function.

TABLE 2.

Binding affinities of sodium and potassium detected with RH421

Sodium or potassium titration curves in Fig. 5 were fitted to the Hill equation. The K_0.5 and n_H values represent averages from three experiments ±S.E.

Isoform	K0.5Na+ (nH)	K0.5K+ (nH)
	mm	mm
α1β1	7.7 ± 0.5 (1.7 ± 0.13)	0.6 ± 0.04 (1.8 ± 0.1)
α2β1	8.0 ± 0.3 (1.9 ± 0.2)	1.5 ± 0.1 (1.5 ± 0.1)
α2β2	8.1 ± 0.3 (1.7 ± 0.2)	5.1 ± 0.1 (1.6 ± 0.1)
α2β3	7.3 ± 0.6 (1.8 ± 0.1)	4.8 ± 0.6 (1.5 ± 0.2)

An alternative or additional explanation to that just given is that raised K_0.5K⁺ of the α₂β₂ and α₂β₃ complexes is caused by an α₂β₂- or α₂β₃-induced shift in poise of the E₁-E₂ conformational equilibrium toward E₁ or E₁-P. This explanation would also be consistent with the parallel reduction in apparent K_0.5Na⁺ and increase in K_i for vanadate (Table 1, column 3). A direct test of the relative effects of β₁, β₂, and β₃ on the conformational changes was made by measuring the rates of the E₁P(3Na) → E₂P and E₂(2Rb)ATP → E₁3Na·ATP transitions using RH421 in stopped-flow experiments, as described in our recent publications (29, 30). Traces for the α₂β₂ complex are shown in Fig. 5, C and D, and the rate constants for all of the isoform complexes are collected in Table 3. These data show directly that for α₂β₁, the rates of E₁P(3Na) → E₂P are indeed slower than for α₁β₁, and for both α₂β₂ and α₂β₃, the rate is still slower than for α₂β₁.

TABLE 3.

Rates of conformational changes for the different isoform complexes determined by stopped-flow measurements

All rates were measured at 23 °C as described under “Experimental Procedures.” The turnover rate was calculated from the function (a × b)/(a + b).

Isoform	a. E1P(3Na) →E2P	b. E2(2Rb)ATP → E13 NaATP	Turnover rate	Source
	s−1	s−1	s−1
α1β1	170.0 ± 8.0 (n = 3)	15.5 ± 1.2 (n = 4)	14.2	Ref. 52
α2β1	91.3 (n = 2)	10.2 (n = 2)	9.2	Ref. 52
α2β2	58.4 (n = 2)	10.3 (n = 2)	8.8	This work
α2β3	52.5 (n = 2)	10.5 (n = 2)	8.8	This work

The rates of E₂(2Rb)ATP → E₁3Na·ATP for α₂β₁, α₂β₂, and α₂β₃ are all significantly slower than for α₁β₁, but they are indistinguishable from each other. The turnover rate in s⁻¹ for each isoform was calculated from the expression (a × b)/(a + b), where a is the rate of E₁P(3Na) → E₂P and b is the rate of E₂(2Rb)ATP → E₁3Na·ATP (Table 3), assuming that the rates of phosphorylation E₁Na + ATP → E₁P(3Na) and dephosphorylation E₂P2Rb → E₂(2Rb) are fast and do not significantly limit the turnover rate. The calculated value for α₁β₁ (14 s⁻¹) is greater than for α₂β₁, α₂β₂, and α₂β₃, which are not significantly different from each other (9.17, 8.76, and 8.75 s⁻¹, respectively; average of 8.89). The ratios of the calculated turnover rates for α₁β₁/α₂β₁, α₁β₁/α₂β₂, and α₁β₁/α₂β₃ are essentially the same, with an average of 1.6. These ratios are close to those of Na,K-ATPase activities in Table 1, validating the assumptions that underlie the calculation of the turnover rates.

Inhibition of Na,K-ATPase Isoforms by Perhydro-1,4-oxazepine Derivatives of Digoxin

As reported recently, chemical modification of the third digitoxose residue of digoxin (by periodate oxidation and reductive amination with R-NH₂) to produce perhydro-1,4-oxazepine derivatives increases selectivity of inhibition for α₂β₁ and α₂β₃ over α₁β₁ (26, 27). Here we have compared inhibition of α₁β₁ and all three complexes α₂β₁, α₂β₂, and α₂β₃ by several derivatives described previously (26) and six new ones (DAz, DTh, DcB2,2dM, DMSM, DEMS, and DESA). The structures of the substituents, abbreviated names, and masses of all of the derivatives are given in Fig. 6. Fitted K_i values and selectivity ratios for all of these compounds are summarized in Table 4. Compounds that have the highest selectivity, compared with digoxin itself, are indicated by double asterisks, and compounds with significant but lower selectivity are shown with a single asterisk. There is a clear peak for the cyclobutyl derivative with four carbon atoms, DcB, with 16.9-, 22.2-, and 33.6-fold selectivity for α₂β₁, α₂β₂, and α₂β₃ over α₁β₁, respectively. Overall, Table 4 shows that aliphatic substituents with four carbon are most selective (DcB > DMcP > DiB), whereas the substituents with three carbons (DiP ≈ DcP > DP) are also quite selective, and the selectivity ratios decline for substituents with 5, 6, or 7 carbon atoms. The sulfonyl derivatives DMSM, DEMS, and DESA also showed strongly increased selectivity over α₁β₁ in the order α₂β₃ > α₂β₂ > α₂β₁.

FIGURE 6.

Substituent structures, abbreviated names, and predicted and found masses of perhydro-1,4-oxazepine derivatives of digoxin.

TABLE 4.

K_i values and selectivity ratios for the inhibition of purified Na,K-ATPase isoforms by perhydro-1,4-oxazepine derivatives of digoxin

The reaction medium contained 130 mm NaCl, 5 mm KCl, 3 mm MgCl₂, 25 mm histidine, pH 7.4, 1 mm EGTA, 0.01 mg/ml SOPS, 0.001 mg/ml cholesterol, and 0.005 mg/ml C₁₂E₈. Each experiment was carried out at least three times. The calculated K_i values represent averages ± S.E. *, compounds with significantly higher selectivity (K_i α₁β₁/α₂β_1–3) than digoxin (>4- and <10-fold). **, compounds with the highest selectivity (K_i α₁β₁/α₂β_1–3) compared with digoxin (>12-fold).

CG	Ki ± S.E.				Selectivity			n
	α1β1	α2β1	α2β2	α2β3	α1β1/α2β1	α1β1/α2β2	α1β1/α2β3
	nm
Digoxin	268.0 ± 13.8	58.7 ± 5.4	58.0 ± 1.9	42.8 ± 3.0	4.5	4.6	6.2	7
DMe	103.0 ± 5.6	15.3 ± 1.2	20.4 ± 1.8	10.8 ± 0.6	6.7*	5.1	9.5*	7
DEt	137.9 ± 12.6	23.2 ± 0.9	16.4 ± 1.6	14.4 ± 1.3	5.9	8.3*	9.5*	4
DP	87.7 ± 7.9	18.3 ± 1.7	10.5 ± 1.8	9.8 ± 1.1	4.8	8.3*	8.8*	5
DiP	149.0 ± 20.7	28.9 ± 1.7	16.7 ± 1.9	10.3 ± 1.8	5.1	8.9*	14.4**	4
DcP	109.0 ± 6.2	14.6 ± 11.6	13.0 ± 1.3	8.1 ± 1.36	7.5*	8.5*	13.4**	3
DiB	92.0 ± 8.9	20.6 ± 1.4	10.0 ± 0.8	5.8 ± 0.6	4.4	9.0*	16.0**	5
DMcP	95.8 ± 13.7	18.3 ± 1.6	8.0 ± 0.8	4.3 ± 0.6	5.2	12.0**	22.2**	4
DcB	135.0 ± 11.0	8.0 ± 1.3	6.0 ± 1.0	4.0 ± 0.15	16.9**	22.2**	33.6**	3
DAz	222.0 ± 11.6	52.0 ± 1.7	28.0 ± 3.8	26.5 ± 2.3	4.2	7.7*	8.4*	3
DTh	260.0 ± 41.0	108.0 ± 22.6	170.0 ± 10.4	196.0 ± 34.8	2.4	1.5	1.3	3
DcPe	138.0 ± 21.0	33.4 ± 7.5	33.5 ± 11.9	27.6 ± 9.5	4.1	4.1	5.0	3
DcHe	70.4 ± 4.1	15.2 ± 3.7	15.3 ± 2.9	11.7±.5	4.6	4.6	10.1*	3
DcB2,2dM	102.0 ± 4.0	49.0 ± 18.0	39.0 ± 6.0	45.0 ± 14.0	2.1	2.6	2.3	3
DMcB3,3dM	31.6 ± 0.5	8.6 ± 1.4	5.1 ± 0.5	3.9 ± 0.7	3.7	6.2	8.2*	5
DMSM	944.0 ± 123.0	137.0 ± 9.8	123.0 ± 7.3	89.0 ± 8.7	6.9*	7.7*	10.6*	3
DEMS	464.0 ± 14.0	49.2 ± 1.9	31.7 ± 3.2	24.7 ± 2.1	9.4*	14.6**	18.8**	3
DESA	301.0 ± 23.0	38.9 ± 2.2	31.5 ± 4.4	20.1 ± 0.9	7.7*	9.5*	15**	4

Potassium-Cardiac Glycoside Antagonism

CGs bind with high affinity to E₂P, and potassium binding causes rapid dephosphorylation to E₂(2K), which has a much lower affinity for CGs than E₂P (38). Because K_0.5K⁺ of α₂β₂, α₂β₃ and also α₂β₁ for activating Na,K-ATPase are higher than for α₁β₁ in the order α₂β₂ > α₂β₃ > α₂β₁ > α₁β₁ (Table 1), a higher selectivity for α₂β₂ and α₂β₃ and also α₂β₁ might be due, at least partially, to a weaker K⁺-CG antagonism. To assess the effect of K⁺-CG antagonism more systematically, we have measured K_i values for digoxin, the digoxin derivatives and ouabain at increasing K⁺ concentrations from 2.5, 5, 10, and 20 mm K⁺. Fig. 7 presents the data for digoxin, but very similar data were obtained for the isobutyl derivative of digoxin (DiB) and also ouabain (not shown). For α₁β₁, the K_i values increased by 3–4-fold in the range of 2.5–20 mm K⁺, respectively. By contrast, the K_i values for α₂β₁ were much less affected by the increased potassium concentration, and inhibition of α₂β₂ and α₂β₃ was essentially unaffected by potassium ions in the range 2.5–20 mm. Consequently, the selectivity for inhibition of α₂β₂ over α₁β₁ by digoxin (or other CGs) significantly increased over the range 2.5, 5, 10, and 20 mm potassium from 3.67, 4.77, 11.6, to 15.0, respectively. The differences in K⁺-digoxin antagonism were also investigated more directly by K⁺-[³H]digoxin displacement assays (Fig. 8). Yeast membranes harboring either α₁β₁, α₂β₁, α₂β₂, or α₂β₃ were equilibrated with [³H]digoxin and subsequently incubated with increasing amounts of KCl (up to 150 mm). In the conditions of digoxin binding (with vanadate/magnesium but sodium-free), the apparent K_0.5K⁺ for digoxin displacement was 0.62 mm for α₁β₁ (see also Ref. 25), 1.8 ± 0.1 mm for α₂β₁, 2.8 ± 0.1 mm for α₂β₂, and 3.2 ± 0.5 mm for α₂β₃. Furthermore, at high K⁺ concentrations, displacement of digoxin binding was incomplete for α₂β₁, α₂β₂, and α₂β₃, the remaining fraction at saturating potassium being 20.3 ± 1.5, 26 ± 1.8, and 30.3 ± 1.5% of control, respectively. Thus, K⁺-CG antagonism reflects both the affinity for potassium ions and the maximal degree of displacement by potassium ions.

FIGURE 7.

Potassium-digoxin antagonism depicted for α₁β₁, α₂β₁, α₂β₂, and α₂β₃. Inhibition of purified Na,K-ATPase isoforms by digoxin was measured, and K_i values were plotted against the potassium concentration in the assay medium. Error bars, S.E.

FIGURE 8.

Potassium-[³H]digoxin antagonism. Yeast membranes harboring α₂β₁, α₂β₂, or α₂β₃ Na,K-ATPase were incubated with varying concentrations of KCl, and residual [³H]digoxin binding was measured. The lines represent Hill fits. Data points for α₁β₁ were taken from Ref. 25. Error bars, S.E. from 3 experiments.

Overall, in conditions similar to the extracellular physiological medium (140 mm sodium, 5 mm potassium or with potassium elevated to 20 mm), K-CG antagonism is prominent for α₁β₁, weak for α₂β₁ and negligible for α₂β₂ and α₂β₃. Note, however, that, in similar conditions, the selectivity ratios for all six derivatives DiB, DMcP, DcB, DMSM, DEMS, and DESA for α₂β₃/α₁β₁ > α₂β₂/α₁β₁ > α₂β₁/α₁β₁ are significantly greater than for digoxin itself (Table 4). This shows that reduced K-CG antagonism can indeed account only partially for the increased selectivity for α₂β₃ and α₂β₂, whereas the structure of the derivatives is a crucial element determining the isoform selectivity.

Discussion

We discuss here the data showing selective assembly of α₂β₂ in cardiac myocytes and distinct functional properties and isoform-selective inhibition of human α₂β₂ and α₂β₃, together with possible molecular explanations and physiological or pharmacological implications. Comparisons of functional properties and inhibition of α₂β₁ with α₁β₁ reveal the differences between α₂ and α₁, whereas comparisons of α₂β₂ or α₂β₃ with α₂β₁ reveal the influence of β₂ or β₃ compared with β₁. As one general conclusion, it is the combined effects of α₂ and β₂ (or β₃) that give rise to the distinctive functional properties and isoform-selective inhibition of α₂β₂ (or α₂β₃).

Specific Assembly of α₂β₂ in Heart

Three of the four Na,K-ATPase α subunit isoforms and all three β subunit isoforms are expressed in the heart. Although α₁ is the most abundant α subunit isoform, the α₂ isoform rather than the α₁ isoform plays a key role in cardiac muscle contractility by regulating the cytosolic Ca²⁺ concentration in cardiac myocytes. The transient rise in Ca²⁺ concentration associated with electrical excitability then triggers cardiac muscle contraction (1). Despite extensive studies of isoform-specific properties of the Na,K-ATPase, the reasons for differential roles of α₂ and α₁ isoforms in cardiac muscle contractility are not fully understood.

The data presented here indicate that the α₂ and β₂ isoforms preferentially assemble with each other in the heart as detected by co-immunoprecipitation (Fig. 3). These data are consistent with previous reports on selective assembly (17) and co-purification of α₂ and β₂ isoforms in the brain (39). Immunofluorescence data in rat and human heart sections indicate that both α₂ and β₂ isoforms specifically localize to the T-tubular membranes, whereas α₁ and β₁ isoforms are distributed in both T-tubular and external sarcolemma membranes (Fig. 1). The data on preferential T-tubular localization of the α₂ and ubiquitous distribution of the α₁ are in agreement with previous reports on measurements of the isoform-specific Na,K-ATPase activity in cardiac myocytes (11, 15, 16, 40) and several immunofluorescence reports (11, 13). On the other hand, our data contrast with several other reports on immunodetection of the Na,K-ATPase α isoforms in the heart (3, 14). The uniform distribution of the β₁ isoform has been reported previously (3), whereas this is the first report describing the specific localization of the β₂ isoform in the heart.

It seems paradoxical that there are two isoform complexes, α₂β₂ and α₂β₃, with similar functional properties, as shown in this paper, but in the heart, α₂ assembles with β₂ rather than with β₃. Assembly with a particular β isoform is known to affect trafficking and polarized sorting of the α subunit (19, 41, 42), suggesting that association with the β₂ can be important for the specific location of the α₂ isoform in the T-tubules. Particularly, recombinant addition of N-glycosylation sites to the β₁ subunit has been shown to alter localization of the Na,K-ATPase from the basolateral to the apical domain of the plasma membrane in gastric epithelial cells (19). It is possible that eight N-glycans versus two or three in the β₃ or β₁ isoform play a role in this specific targeting of the α₂ to the microdomains of T-tubular membranes that contain Na⁺/Ca²⁺ exchanger and are proximal to other components of Ca²⁺-regulating complex, which can explain the importance of the α₂ isoform in cardiac muscle contraction.

Another difference between β₃ and β₂ is a role of β₂ in cell adhesion (39, 43). Whether this adhesive role is required for specific location of α₂β₂ in T-tubules is not clear, but α₂β₂ differs from α₂β₃ in this crucial aspect.

Functional Properties of the α₂β₁, α₂β₂, and α₂β₃ Isoform Complexes

The major functional differences between α₂β₂ and α₂β₃ when compared with α₂β₁, and especially α₁β₁, is the raised K_0.5K⁺ for activating Na,K-ATPase, reduced K_0.5Na⁺, and turnover rate (Table 1). These results confirm previous evidence that α₂ raises K_0.5K⁺ compared with α₁ when it is complexed with β₁ (20, 24, 44) and now show that both β₂ and β₃ lower K_0.5Na⁺ as well as raising K_0.5K⁺ when complexed with α₂, in comparison with complexes with β₁. The RH421 experiments show that the principal mechanism of the effects of α₂ versus α₁ and β₂ and β₃ versus β₁ to raise K_0.5K⁺ is reduction of the intrinsic binding affinity for potassium ions at the extracellular surface, with K_0.5K⁺ values in the order α₁β₁ < α₂β₁ < α₂β₃ ≤ α₂β₂, respectively (Fig. 5 and Table 2). In RH421 experiments, the potassium affinity is determined in the presence of 50 mm sodium, and, in principle, the reduced affinity for K_exc ions could reflect an increased affinity for Na_exc and competition with K_exc. Nevertheless, displacement of digoxin by potassium ions in the absence of sodium ions (K_0.5K⁺ 0.6 ± 0.07 mm for α₁β₁, 1.8 ± 0.1 mm for α₂β₁, 2.8 ± 0.1 mm for α₂β₂, and 3.2 ± 0.5 mm for α₂β₃) shows that there is a true and large reduction in intrinsic K_exc affinity (Fig. 8). The vanadate titrations with K_i values in the order α₁β₁ < α₂β₁ < α₂β₃ < α₂β₂ (Table 1) are consistent with the order of decreasing affinities for potassium ions.

Compared with α₁β₁, α₂β₁, α₂β₂, and α₂β₃ significantly reduced the rate of the conformational transition E₁P(3Na) → E₂P, with the order α₁β₁ > α₂β₁ > α₂β₂ ≈ α₂β₃ (Fig. 5 and Table 3). In steady-state Na,K-ATPase conditions, a shift of the E₁P(3Na) → E₂P conformational equilibrium toward E₁P would contribute to the higher K_0.5K⁺ as well as lower K_0.5Na⁺ (and higher K_i vanadate) (Table 1). Note that the reduced K_0.5Na⁺ of α₂β₂ and α₂β₃ compared with α₁β₁ and α₂β₁ is not explained by a change in intrinsic binding affinity for sodium ions (see Fig. 5A).

α₂β₁ also displayed a reduced rate of E₂(2Rb)ATP→E₁(3Na)ATP when compared with α₁β₁, but there was no further decrease in α₂β₂ and α₂β₃. This finding explains the reduced turnover rate for all α₂ complexes compared with α₁. The different β isoforms appear to have no isoform-selective influence on the turnover rate, which is governed by the slow rate-determining step E₂(2Rb)ATP → E₁(3Na)ATP, reduced by α₂, and not by the faster E₁P(3Na) → E₂P transition.

In the absence of high resolution structures of the α₂ complexes, one can only speculate on possible explanations of the kinetic effects of α₂ and β₂ or β₃, such as the reduced K_exc binding affinity. Using chimeras of Na,K-ATPase and H,K-ATPase, the extracellular domain was identified as the main modifier of the apparent potassium affinity (for displacing bound ouabain) (45), and the main interaction site was shown to be an SYGQ motif in the M7-8 loop of α (46). In an older study (47), the extracellular domain of β₁ was suggested to cover the extracellular domain of the α subunit and control access to the Rb(K) binding sites. In general, this concept appears compatible with the molecular structures of Na,K-ATPase (α₁β₁) (48–50). Thus, compared with α₁β₁, the K_exc entry and exit pathway may be more accessible in α₂β₁ itself and even more so in α₂β₂ and α₂β₃, leading to the large decrease in K_exc binding affinity.

Digoxin Derivatives with Strong Selectivity for α₂β₂ and α₂β₃ Complexes

The conclusion from all of the experiments in Table 4 is that digoxin perhydro-1,4-oxazepine derivatives with four carbon substitutions (cyclobutyl (DcB), methyl cyclopropyl (DMcP), and isobutyl (DiB)) have the highest selectivity for α₂β₃/α₁β₁ and α₂β₂/α₁β₁ compared with compounds with 1–3 or 5 or a greater number of carbon atom substitutions. This confirms the data in Ref. 26 for α₂β₃/α₁β₁ and extends them to α₂β₂/α₁β. The optimal size of the aliphatic substituents (four carbon atoms), cyclic and non-cyclic, may indicate a size restriction of the space between the α and β subunits. Replacement of one methylene group in the cyclobutyl DcB with a single NH (DAz) or sulfur (DTh) atom strongly reduces selectivity, whereas the three new sulfonyl derivatives, DMSM, DEMS, and DESA, showed enhanced selectivity for α₂β₂ and α₂β₃ and to some extent also α₂β₁, relative to digoxin itself (Table 4). Thus, the highest selectivity for the α₂β₂ and α₂β₃ isoform complexes depends crucially on the structure of the derivatives, providing the strongest evidence for selective interactions of the substituent groups with β₂ and β₃, respectively.

We have attempted to explain the selectivity of DcB for α₂β₃ and α₂β₂ with molecular docking models, starting with a structure of renal Na,K-ATPase (α₁β₁) with bound digoxin (51) (Protein Data Bank code 4RET) (Fig. 9). The models display the optimal positions found for DcB relative to digoxin and emphasize the major attractive interactions, particularly with the β subunit. The modeling supports the experimental result whereby DcB binds to α₂β₃ with the highest potency and selectivity, showing a hydrophobic interaction between the DcB-cyclobutyl moiety and β₃Val-88. There is also an electrostatic interaction of the protonated nitrogen of perhydro-1,4-oxazepine ring with Asp-889 of α₂. In the case of α₂β₂, to which DcB also binds with good potency and selectivity, the modeling shows a hydrogen bond interaction between the protonated perhydro-1,4-oxazepine ring nitrogen and β₂Glu-89 and also an additional hydrogen bond between the lactone ring and the structural water molecule located at the bottom of the binding site. It is noticeable that the steroid and lactone moieties are somewhat rotated compared with the digoxin itself. By contrast with the α₂β₃ and α₂β₂ models, in the α₁β₁ model, the steroid-lactone moiety of DcB almost exactly overlaps that of digoxin and displays only a weak interaction with β₁Gln-84. This result also appears to be consistent with the experimental result for α₁β₁ which displays a much lower potency for DcB and only a small difference from digoxin itself.

FIGURE 9.

Models for docking of DcB to α₁β₁, α₂β₂, and α₂β₃. DcB was docked into homology models of human Na,K-ATPase isoforms derived from the porcine E2P·Mg·digoxin structure (Protein Data Bank code 4RET) as described under “Experimental Procedures.” α and β subunits are shown in green and blue ribbon representations. Transmembrane helices 3 and 5 of the α-subunit are removed for clarity. Digoxin (yellow) and DcB (purple) are shown in a stick representation, and water and a magnesium ion are shown as spheres. The residues of the β subunit closest to the cyclobutyl moiety, β₁Gln-84, β₂Glu-89, and β₃Val-88, are shown as sticks, and distances to DcB are indicated.

Taken together, it is evident that both the α and the β subunits determine the selectivity of the digoxin derivatives, and the β subunit, in particular, has favorable interactions with the substituted third sugar residue.

Physiological Role of α₂β₂

In relation to the physiological function, major differences of α₂β₂ compared with α₁β₁ include the high K_0.5K⁺ values (low affinity) for extracellular potassium ions, a somewhat lower turnover rate, and a significantly lower K_0.5Na⁺. Although the current experiments have been done with detergent-soluble purified proteins, a very similar effect of α₂β₂ to strongly raise K_0.5K⁺ values for extracellular potassium ions compared with α₁β₁ and also α₂β₁ has been described with the proteins expressed in Xenopus oocytes (20, 21, 52). A property that we cannot assess in experiments with detergent-soluble proteins is the dependence of activity on membrane potential. As described previously (20), α₂β₁ shows a steeper dependence on voltage than α₁β₁ or α₃β₁, and recent work shows that α₂β₂ shows a particularly steep dependence (21). The voltage dependence of the pump current in physiological conditions is mainly a reflection of Na⁺_exc-mediated competition with K⁺_exc leading to inhibition at negative potentials. The conditions of our experiments are equivalent to those in cells at 0 mV membrane potential, and compared with physiological conditions, the raised K_0.5K⁺ of α₂β₂ represents, if anything, an underestimated value compared with α₁β₁. At physiological K⁺_exc of 4.5 mm and resting membrane potentials in the heart or skeletal muscle or brain glial cells (−70 to 90 mV), α₂β₂ is largely inactive. Thus, α₂β₂ acts as a “reserve pump,” which responds to acutely increased K⁺_exc or positive membrane potentials by increasing its rate and moderating the changes in K⁺_exc and then restoring the ion gradients after the change (see Refs. 52 and 53). For example, increased activity of skeletal muscles leads to loss of potassium ions and a large increase in K⁺_exc, which can reach as high as 8.3 mm in serum, 10–12 mm in muscle interstitial fluid, and locally as high as 25 mm in T-tubules (54). In heart muscle, the α₂β₂ is also almost inactive at resting potentials but becomes acutely activated during raised cardiac activity due to the fact that the membrane potential is positive for a significant fraction of the time during the extended action potentials (21). As in skeletal muscle, the potassium concentration in T-tubules can be presumed to rise significantly. The lower K_0.5Na⁺ of α₂β₂ should also contribute significantly to rapid restoration of the sodium gradient associated with increased activity and raised cytoplasmic sodium. Due to coupling of the sodium fluxes mediated by α₂β₂ with sodium and calcium fluxes mediated by NCX1, the kinetic features of α₂β₂ described are expected to play an important role in regulation of the calcium dynamics of active versus resting cardiac muscle. Indeed, an important role of α₂ overexpression in calcium dynamics in myocytes, associated with a decreased K_0.5Na⁺, has been proposed recently (55), although it was not known which β isoform is coupled with α₂. The α₁β₁ complex maintains ion gradients in resting conditions but is not suited for the regulatory role of α₂β₂ just discussed because the potassium sites (K_0.5K⁺ = 1.5 ± 0.1 mm) are almost saturated even in resting conditions, and the voltage dependence of α₁β₁ is shallow.

Pharmacological Implications

As discussed in the Introduction, an α₂-selective CG could be an efficient inotropic agent. The present findings have the additional interesting implication that an α₂β₂-selective CG, such as the digoxin derivatives described here, could also have reduced cardiotoxicity compared with digoxin.

For many years, digoxin was used routinely to treat heart failure, due to its inotropic and chronotropic effects, but it is now used much less on account of the narrow therapeutic range and cardiotoxicity resulting from the well known phenomena of calcium overload and cardiac arrhythmias (31). The incidence of digitalis toxicity has decreased in parallel with its decreased use (56). The main cause of digitalis toxicity is accumulation of digoxin, secondary to decreased renal function, and a major exacerbating factor is hypokalemia, which is prevalent in subjects treated with diuretics in addition to digitalis (56) (for a recent study, see Ref. 57). Indeed, due to the potentiation of digitalis toxicity by hypokalemia, it was even recommended, in the past, to treat severe cases by raising serum potassium (58).

The salient present finding is that inhibition of α₁β₁ by digoxin and all other CGs is strongly antagonized by raising potassium in the range of 2.5–20 mm, whereas α₂β₂ is unaffected (Fig. 7). Assuming that digoxin toxicity in vivo, exacerbated by hypokalemia, is associated with excessive Na,K-pump inhibition, this implies that toxicity is indeed mediated by α₁β₁. Conversely, the insensitivity of inhibition of α₂β₂ to potassium (2.5–20 mm) would imply that α₂β₂ does not play a major role in digitalis toxicity. This conclusion fits well with the rationale that α₂β₂-selective derivatives could be effective positive inotropic agents and also have reduced toxic effects.

Considerable evidence exists for the presence of endogenous CG-like compounds in mammalian tissues, such as ouabain or marinobufagenin, that may serve to regulate Na,K-ATPase activity (59–61). Our unpublished experiments show a higher sensitivity of α₂β₂ over α₁β₁ for ouabain (K_i = 63.7 ± 9.8 versus 153.0 ± 11.6 nm, respectively),⁷ suggesting that endogenous ouabain-like compounds may bind more efficiently to α₂β₂ isoform and thus specifically regulate the α₂β₂ ion pumping activity or α₂-dependent signaling pathways. In particular, α₂-mediated signaling may explain a specific role of the α₂ subunit in the modulation of blood pressure under stress conditions (60–63).

Conclusions

In summary, our data demonstrate the specific association of the Na,K-ATPase α₂ isoform with the β₂ and the specific intracellular location of the α₂β₂ heterodimer. The distinct functional properties of human α₂β₂ are consistent with an important regulatory role in cardiac muscle contraction. Furthermore, isoform-selective inhibition by digoxin derivatives, such as DcB, suggests that they could be safer cardiac inotropic agents compared with digoxin itself.

Experimental Procedures

Materials

n-Dodecyl-β-d-maltopyranoside (catalogue no. D310) and C₁₂E₈ (25% (w/w), catalogue no. 0330) were purchased from Anatrace, and BD-Talon metal affinity resin was from Clontech (catalogue no. 635503). 1-Stearoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (SOPS) was purchased from Avanti Polar Lipids. PiColorLock^TM was purchased from Innova Bioscience, and RH421 was from MoBiTec. All other reagents were purchased from Merck or Sigma-Aldrich at the highest quality level available.

Primary Antibodies

For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α₁ subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β₁ subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents). The polyclonal antibodies used were Na,K-ATPase α₂ subunit (rabbit, 1:200; Millipore) and Na,K-ATPase β₂ subunit (rabbit, 1:200; Millipore).

For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α₁ subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β₂ subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β₃ subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.). Na,K-ATPase β₁ subunit polyclonal antibody (rabbit; 1:5000) was a generous gift of Dr. W. James Ball, Jr. (University of Cincinnati).

Methods

Confocal Microscopy

Confocal microscopy images were acquired using the Zeiss LSM 510 laser scanning confocal microscope and ZEN 2009 software.

Isolation of Membrane Fractions from Mouse Heart

Mouse heart was homogenized with a tight Dounce homogenizer (Wheaton, Millwille, NY). Cell debris was removed by centrifugation (2000 × g, 10 min). The cleared homogenate was layered onto a 42% sucrose solution in 10 mm PIPES, 2 mm EGTA, 2 mm EDTA, pH 7.0, and spun in a Beckman SW28 swinging bucket rotor at 25,000 rpm for 1 h at 4 °C. The fraction at the interface of buffer/sucrose was collected and diluted to a total volume of 15 ml of 10 mm PIPES, 2 mm EGTA, 2 mm EDTA, pH 7.0. Membranes were spun down by centrifugation in a Beckman 75Ti rotor (35,000 rpm, 4 °C, 1 h). The pellet was resuspended in 10 mm PIPES/Tris buffer containing 2 mm EGTA and 2 mm EDTA, pH 7.0, by homogenization with a 2-ml Teflon homogenizer (Wheaton). The membranes were aliquoted, flash-frozen, and stored at −80 °C. Proteins were extracted by incubating membranes with 50 mm Tris, pH 7.5, containing 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and complete protease inhibitor mixture (1 tablet/50 ml at 4 °C for 30 min). Membrane extracts were clarified by centrifugation (100,000 × g, 1 h) at 4 °C. Where indicated, protein extracts were treated by PNGase F from Flavobacterium meningosepticum (New England Biolabs) according to the manufacturer's instructions before loading on SDS-PAGE.

Immunoprecipitation

Protein extracts from mouse heart membrane fractions (100–300 μg of protein) were incubated with 30 μl of the protein A-agarose suspension (Roche Diagnostics) in a total volume 1 ml of the extraction buffer at 4 °C with continuous rotation for at least 3 h (or overnight) to remove the components that non-specifically bind to protein A. The precleared cell extract was mixed with 10 μl of polyclonal antibodies against the Na,K-ATPase α₂ subunit (Millipore) or 10 μl of polyclonal antibodies against the Na,K-ATPase α subunit (64) or 10 μl of polyclonal antibodies against the Na,K-ATPase β₂ subunit (Millipore) and incubated with continuous rotation at 4 °C for 60 min. After the addition of 30 μl of the protein A-agarose suspension, the mixture was incubated at 4 °C with continuous rotation overnight. The bead-adherent complexes were washed three times on the beads and then eluted as described previously (65).

Where indicated, the bead-adherent proteins were treated with PNGase F. Deglycosylation by PNGase F was performed by incubation of the bead-adherent proteins with 1 μl of PNGase F in 30 μl of 50 mm sodium phosphate, pH 7.5, containing 1% Nonidet P-40 at 37 °C for 1 h. After incubation with glycosidases, the reaction mixture was separated from the beads. The adherent proteins were eluted from the beads by incubation in 30 μl of 2× SDS-PAGE sample buffer for 5 min at 80 °C. To account for possible dissociation of immunoprecipitated proteins from the beads during deglycosylation, the eluted proteins were combined with the reaction mixture. After separation by SDS-PAGE, the immunoprecipitated and co-immunoprecipitated proteins were analyzed by Western blotting by using appropriate antibodies.

Western Blotting Analysis

1–10 μg of proteins extracted from mouse heart membranes or 5–20 μl of proteins eluted from the protein A-conjugated agarose beads were loaded onto 4–12% gradient SDS-polyacrylamide gels (Invitrogen). Proteins were separated by SDS-PAGE, transferred onto a nitrocellulose membrane (Bio-Rad), and detected by Western blotting analysis as described previously (65).

Immunofluorescent Staining

Mouse embryo sections and frozen tissue sections on FDA standard frozen tissue rat or human arrays (BioChain) were incubated with Dako Protein Block serum-free solution (Dako Corp.) for 30 min. Immunofluorescent staining was performed by a 1-h incubation with the primary antibodies followed by a 1-h incubation with Alexa Fluor 633- or Alexa Fluor 488-conjugated anti-mouse or anti-rabbit antibodies (Invitrogen).

Plasmid Construction for the Expression of α₂β₂ α₂β₃ Na,K-ATPase

Generation of pHil-D2 expression vector containing cDNA of human α₁ and His₁₀-tagged porcine (p) or human (h) β₁ was described previously (35). cDNAs of human β₂ and β₃ in pSD5 were a gift from K. Geering (University Lausanne, Switzerland). Open reading frames and flanking regions of human β₂ and β₃ were amplified by PCR using primers containing BglII and SalI cleavage sites. The resulting fragments were subcloned into pHil-D2-hα₂/His₁₀-pβ₁ to create pHil-D2-hα₂/His₁₀-hβ₂ and pHil-D2-hα₂/His₁₀-hβ₃, respectively. Correct integration and sequence was confirmed by sequencing.

Yeast transformation and clone selection have been described in detail (35). P. pastoris SMD1165 was grown in BMG (100 mm potassium phosphate, pH 6, 1.34% yeast nitrogen base, 4 × 10⁻⁵% biotin, 0.3% glycerol) to OD 6–8, and expression was induced in BMM (100 mm potassium phosphate, pH 6, 1.34% yeast nitrogen base, 4 × 10⁻⁵% biotin, 0.5% methanol added daily). Yeasts were transformed with linearized pHil-D2-human α₂-human His₁₀-β₂ or human His₁₀-β₃, and His⁺/Mut^S clones were selected and grown at 20 °C for 3 days in baffled Erlenmeyer flasks, as described previously for α₁β₁ and α₂β₁ (35, 66). Under these conditions, only weak expression was observed for α₂β₂ and α₂β₃. It was then determined that expression of both α₂β₃ and α₂β₂ was transient and peaked between 15 and 19 h for α₂β₃ and between 16 and 28 h for α₂β₂. Further screening of expression temperature revealed 23 °C to be optimal. This transient expression was not observed for α₁β₁, α₂β₁, and α₃β₁ that were stably expressed for 3–5 days, which also resulted in a higher cell density and protein yield per liter of culture. To overcome this limitation for α₂β₃ and α₂β₂, the following three-phase growth/induction protocol was used. In phase I, glycerol batch cultivation, yeasts were grown in BMG in an aerated 10-liter vessel until OD reached 6–8. In phase II, the glycerol-fed batch phase, 0.05% glycerol/h was added to the culture. Glycerol feeding was continued until OD reached 13–15. Extending the fed batch to higher cell densities often led to excessive foaming and a loss of cells. In phase III, the induction phase, expression was induced by adding 0.5% methanol/day. No increase of OD was observed during this phase. All three phases were carried out at 23 °C. In addition to the increase in cell density, fed batch cultivation increased expression levels of Na,K-ATPase. Membranes prepared from cells grown in baffled spinner flasks showed >30% higher specific ouabain binding when compared with membranes from cells that did not undergo fed batch cultivation. The same increase was observed in Western blots of these membranes, indicating that most of the expressed protein was properly folded and functional.

Expression and Purification of α₁β₁FXYD1, α₂β₁FXYD1, α₂β₂FXYD1, and α₂β₃FXYD1 Complexes

The experiments have utilized the purified detergent-soluble αβFXYD1 complexes rather than the αβ complexes alone, because FXYD1 strongly stabilizes the proteins against thermal inactivation (67) and does not affect inhibition by ouabain or digoxin (25, 68). Membrane preparation and His tag purification on BD-Talon beads of recombinant human α₁β₁FXYD1 and α₂β₁FXYD1 Na,K-ATPase were done essentially as described previously (69–71). Yeast membrane expression and purification of recombinant α₂β₂ and α₂β₃ Na,K-ATPase were similar except where indicated (for details, see “Results”). Human FXYD1 was expressed in Escherichia coli and purified and reconstituted with αβ complexes on the beads to yield αβFXYD1 complexes (35, 67, 71). Elution buffers consisted of 200 mm imidazole, 100 mm NaCl, 20 mm MOPS/Tris, pH 7.4, 0.1 mg/ml C₁₂E₈, 0.01 mg/ml cholesterol, and 0.07 mg/ml SOPS. 25% glycerol was added, and the soluble protein complexes were stored at −80 °C.

Biochemical Assays

ATPase activity assays as well as titrations with NaCl, KCl, and vanadate were performed as described previously (29, 35) using PiColorLock^TM malachite green assay (Innova Bioscience). For ion titrations, activity was measured at varying concentrations of sodium plus choline chloride and KCl (80 mm), with constant total ionic strength (170 mm total). K_0.5Na⁺ and K_0.5K⁺ values were obtained by fitting the data to the Hill equation using KaleidaGraph (Synergy Software) (30).

Inhibition of Na,K-ATPase activity and [³H]ouabain binding and K-[³H]digoxin displacement assays were performed as reported (25). For derivation of K_i values, percentage inhibition of Na,K-ATPase activity, VCG/V0, was calculated, and K_i values were obtained by fitting the data to the function, VCG/V0 = K_i/([CG] + K_i) + c. Inhibition was estimated in 3–7 separate experiments, and average K_i values ± S.E. were calculated (27).

Equilibrium and Stopped-flow Fluorescence Measurements Using RH421

Equilibrium fluorescence experiments were carried out in a Varian fluorimeter at room temperature. 10 μg of purified Na,K-ATPase was added to 1 ml of 20 mm MOPS/Tris, pH 7.2, 5 mm MgCl₂, and 200 nm RH421. NaCl, ATP, and KCl where then added successively. NaCl and KCl titrations were performed as described previously (37) with emission and excitation wavelength set to 580 and 680 nm with a 5-nm slit width.

Stopped-flow measurements were made using an Applied Photophysics SX20 system (see Ref. 29). Using a combined xenon/mercury lamp, excitation wavelength was 577 nm, and fluorescence was measured at ≥665 nm using cut-off filters. Solutions were mixed 1:1 using ∼120 μl/syringe, and the temperature of the measurements was set to 23 °C. All solutions were buffered to pH 7.2 using MOPS/Tris, and ionic strength was kept constant at 120 mm for all measurements using choline chloride.

For measurement of E2(2Rb)ATP → E1·3NaATP, syringe 1 contained 20 μg/ml enzyme non-covalently labeled with 200 nm RH421 in 20 mm RbCl, 1 mm EDTA and was mixed with 80 mm NaCl, 2 mm ATP, 1 mm EDTA in syringe 2. For measurement of E1·3Na → E2P, 10 μg/ml Na,K-ATPase non-covalently labeled with 200 nm RH421 in 100 mm NaCl and 4 mm MgCl₂ in syringe 1 was mixed with 1 mm ATP in the same solution in syringe 2.

Data were fitted using KaleidaGraph (Synergy Software). Ion titrations were fitted using the Hill equation,

where n is the Hill coefficient, [ion] is the free concentration of the respective ion, and K_½ is the concentration required to obtain the half-maximal fluorescence signal.

Stopped-flow traces were fitted to a monoexponential function,

or double exponential function,

where A is the amplitude of the fluorescence signal, k is the rate of the reaction, and c is the equilibrium fluorescence level after the reaction is complete. All values are expressed as averages of 2–4 experiments.

Molecular Modeling

Homology modeling of human Na,K-ATPase α₂β₁, α₂β₂, and α₂β₃ isoform complexes was carried out using the template α₁β₁ with bound digoxin (Protein Data Bank code 4RET) as described previously (26). The final model exhibits the highest Profiles 3D score (72) and the lowest number of Ramachandran violations (73). Rather consistent profiles were observed for each model (α₂β₁, α₂β₂, and α₂β₃), demonstrating that the human Na,K-ATPase models were reasonable and could be employed for the further docking study. The magnesium ion and three structural waters were positioned in each final model. Models were eventually refined by energy minimization using the CHARMm force field (74).

Molecular Docking

Molecular docking of DcB to the different human Na,K-ATPase isoform models was carried out in Discovery Studio version 4.0 (Biovia, Dassault Systemes, San Diego, CA) with CDOCKER, which is an implementation of a CHARMm-based docking tool using a rigid receptor (75). The DcB ligand was prepared before docking using the Prepare Ligand module to evaluate ionization states for a given pH, isomers, and tautomers, correct bad valences, and generate three-dimensional conformations. The digoxin bioactive binding conformation was copied from the 4RET crystal structure and positioned into each model. The model binding site was defined as a sphere with radius that stays within 15 Å from the geometric centroid of the digoxin ligand using the Define and Edit Binding Site tool.

DcB was docked into the active site of each Na,K-ATPase human model. Different ligand orientations were generated, and for each orientation, the CHARMm energy (interaction energy plus ligand strain) and the interaction energy alone were calculated. The ligand orientations were sorted by CHARMM energy, and the top scoring (most negative, thus favorable to binding) orientations were retained. The final orientations selected were chosen based on their docking scores, which favor interactions with amino acids from the β subunit.

Synthesis of Digoxin Derivatives

Detailed protocols for synthesis, purification, and analysis of perhydro-1–4-oxazepine derivatives of digoxin have been described previously (26, 27, 76).

Author Contributions

M. H. designed and performed cloning, expression, and biochemical experiments; analyzed data; and wrote and edited the manuscript. E. T. designed and performed experiments, analyzed data, and edited the manuscript. Y. N. performed expression and biochemical experiments and analyzed data. S. P. F. designed and performed experiments, analyzed data, and edited the manuscript. R. J. K. provided mouse embryos. E. B. Z. performed the molecular modeling. E. B.-D. performed molecular cloning and expression experiments. L. A. D. aided in conceptual experimental design and wrote sections of the manuscript. Z. F. wrote the section on digitalis toxicity. J. H. K. provided the isoform-nonspecific antibody against α subunit and edited the manuscript. G. S. edited the manuscript. D. M. T. synthesized digoxin derivatives. A. K. performed experiments and analyzed data. O. V. designed the study, performed experiments, analyzed data, and wrote the manuscript. S. J. D. K. designed and coordinated the study, planned experiments, and wrote the manuscript.