I. INTRODUCTION

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The Mg²⁺-dependent, Na⁺- and K⁺-activated ATPase (EC 3.6.1.37; Ref. 165) is the molecular basis of the Na⁺-K⁺ pump in animal cell membranes. The elucidation of the amino acid sequence of the Na⁺-K⁺-ATPase -subunit (105, 163) and of the -subunit (106, 162) of various species (180) has prompted numerous studies on ATPase molecules modified by mutagenesis and heterologously expressed in various cells to investigate the relationship between structure and function of the Na⁺-K⁺-ATPase (98, 117, 180). Furthermore, the existence of Na⁺/K⁺ isozymes has been studied in a large variety of species and tissues (19, 174). The Na⁺-K⁺-ATPase consists of at least two subunit proteins in stoichiometric amounts, the - and -subunit. The -subunit exhibits a molecular mass of ~110 kDa and probably spans 10 times the cell membrane. Both NH₂ and COOH termini face the cytoplasma. The -subunit contains the binding sites for ATP, Na⁺, K⁺, cardiac glycosides, specific inhibitors of the enzyme, and the phosphorylation site. Thus the -subunit is largely responsible for the catalytic, transport, and pharmacological characteristics of the ATPase. The smaller -unit, with a molecular mass of ~50 kDa (depending on the degree of glycosylation), has only one transmembrane domain. The COOH terminus is located at the large ectodomain of the subunit, whereas the NH₂ terminus is exposed to the cytoplasma. The activity of the Na⁺-K⁺-ATPase requires the -subunit. The subunit modulates the transport characteristics of the ATPase and plays an important role in the maturation and proper membrane insertion of the Na⁺-K⁺-ATPase (19). It is still unclear whether the enzyme in vivo works as an()-monomer or an ()₂-diprotomer (154). A small, hydrophobic protein of ~12 kDa, termed the -subunit, copurifies with the - and -subunits of the Na⁺-K⁺-ATPase. It has been found in various tissues of different species (124). The physiological function of the -subunit is not yet known. Like other cellular proteins, - and -subunits are expressed in various isoforms. At present four -subunits (₁-₄) and three -subunits (₁-₃) have been identified. The ₁, ₂, and ₃ are expressed in a variety of tissues, whereas the ₄-protein has been detected so far only in the rat testis (186). Both the - and -isoforms of the Na⁺-K⁺-ATPase are expressed in a tissue-specific pattern (reviewed in Ref. 19). As to the -subunits, the ₁-isoform is expressed ubiquitously, whereas the ₂-expression is predominant in cardiac and skeletal muscle, brain, and adipocytes. The ₃-isoform is abundant in neural tissues and in the ovary (19, 186). The tissue-specific expression of ATPase isoforms can be altered during development and by hormones. Any combination between one of the -subunits ₁-₃ and one of the -subunits ₁-₃ may result in an active Na⁺-K⁺-ATPase isoform (27). The isoforms differ in their kinetic characteristics with regard to activation by Na⁺, K⁺, ATP, and inhibition by cardiac glycosides. For example, the rat ₁₁-isoform expressed in Sf9 insect cells shows a higher Na⁺ and K⁺ affinity but a lower affinity for ATP and a lower sensitivity toward cardiac glycosides than the ₃₁-isoform of the Na⁺-K⁺-ATPase (19). Because of their different kinetic characteristics and tissue-specific expression, the various Na⁺-K⁺-ATPase isoforms probably meet different physiological demands. On the one hand, the ₁₁-isoform may be a general, housekeeping enzyme, since it is ubiquitously expressed and exhibits suitable kinetic properties with relatively high Na⁺ and K⁺ affinities. On the other hand, ₃-isoforms are especially suited to restore the Na⁺ and K⁺ gradients across the cell membrane of electrically excitable cells due to their lower Na⁺ and K⁺ affinities and higher ATP affinity. With regard to the heart, the expression of the Na⁺-K⁺-ATPase isoforms is species specific. There is a marked variation of ₂- and ₃-expression among the species, whereas the ₁-isoform of the ATPase is present in cardiac tissue of all species studied. For instance, ventricular cells from adult human or macaque hearts express three -isoforms, ventricular myocytes from the adult rat heart contain mainly ₁- and ₂-isoforms of the Na⁺-K⁺-ATPase, whereas the sheep heart expresses only the ₁-isoform (175). Whether guinea pig ventricular cells exclusively contains the ₁-isoform (175) or additional ₂ (62) is still controversial. Because the -subunits are specific cardiac steroid receptors exhibiting different sensitivity for these drugs, this controversy echos in contradictory reports on the number of different glycoside receptors present in guinea pig cardiac ventricular myocytes (see below). For further information about the isoforms of the Na⁺-K⁺-ATPase, the reader is referred to pertinent reviews (19, 174).

The Na⁺-K⁺ pump maintains the Na⁺ and K⁺ gradients between the cytosol and extracellular medium. The maintenance of the gradients is a prerequisite for the ionic homeostasis of the cells, for cell volume regulation, and for secondary active transports of amino acids, sugars, bile acids, neurotransmitters, ions, and other solutes across the cell boundary. Furthermore, in electrically excitable cells, creation and maintenance of Na⁺ and K⁺ gradients across the membrane are required for the generation of the resting potential and the generation and propagation of action potentials. It is clear that Na⁺-K⁺ pumping is of prime functional significance in cells displaying relatively frequent electrical discharges over a long period of time, like cardiac cells. Our present understanding of Na⁺-K⁺ pumping is outlined in the Post-Albers cycle (3, 137-139). For detailled information about the experimental basis and the scientific elaboration of the concept, the reader is referred to excellent, introductory reviews (72, 111). The simplified scheme of the pump cycle, shown in Figure 1, may facilitate the appreciation of the findings and ideas discussed below. According to the Post-Albers cycle, the Na⁺-K⁺ pump essentially exists in two conformations, E₁ and E₂, which may be phosphorylated (E₁-P; P-E₂ in the Fig. 1) or dephosphorylated. In the E₁ ATP conformation, the cation-binding sites of the pump face the cytoplasm and preferentially bind Na⁺, whereas in the P-E₂ confirmation the binding sites face the extracellular space and preferably bind extracellular K⁺ (K) (or its congeners). Some binding sites are located at the bottom of an "access channel" and not just at the surface of the pump molecule. During the Na⁺ or K⁺ translocation across the cell membrane carried out by the pump, there are states [(Na₃) E₁-P and E₂ (K₂) in Fig. 1] in which the transported cations are "occluded" in the pump molecule and unable to interchange with ions in the surrounding media.

View larger version (16K): [in this window] [in a new window]   Fig. 1. An electrostatic model of the Na+-K+ pump. The conformation E1 of the pump faces the cytosol and exhibits at the surface two negatively charged binding sites for which intracellular Na+ and K+ compete with different affinities. A third Na+-specific neutral binding site is located inside the pump molecule. To reach the binding site the ion has to migrate through a narrow "access channel." Phosphorylation of the pump by ATP induces Na+ occlusion [(Na3)E1-P]. Transition to the E2 conformation opens the access to the extracellular medium via a narrow access channel. Binding of two K+ probably occurs at the bottom of the channel. It causes dephosphorylation and K+ occlusion [E2(K2)]. Release of two K+ into the cytol completes the pump cycle. [From Heyse et al. (89), by copyright permission of The Rockefeller University Press.]

Electrogenicity denotes the characteristic of a biological transport mechanism to produce electrical current. The concept of electrogenic Na⁺-K⁺ pumping slowly emerged during the 1950s (176). However, a most influential paper on active Na⁺-K⁺ transport in axons of Sepia and Loligo (94) lent little support to this idea. Connelly (26) was the first to conclude on the basis of experimental evidence that the Na⁺-K⁺ pump in nerve fibres is electrogenic. "... since 1960 more and more evidence has accumulated showing that the pump is probably always at least partly electrogenic, with more sodium being extruded than potassium taken up" (176). Today, it is generally accepted that the Na⁺-K⁺ pump of animal cells is electrogenic and generates the pump current I_p. Since, under physiological conditions, three Na⁺ are removed from the cell but only two K⁺ are taken up per pump cycle, I_p is an outward current. The existence of an electrogenic Na⁺-K⁺ pump in cardiac cells was first suggested to explain the high temperature sensitivity of the cardiac resting potential (29). The early experimental data demonstrating electrogenic Na⁺ pumping in nerve and muscle have been reviewed in detail several years ago (119, 176). The experimental evidence for the electrogenicity of the cardiac Na⁺-K⁺ pump from multicellular preparations has been presented in various reviews (45, 47, 63, 65, 181).

The introduction of new methods and techniques into the electrophysiology of the Na⁺-K⁺-ATPase during the last two decades has markedly improved our knowledge of electrogenic Na⁺ pumping. The isolation of single cardiac cells (140) rendered possible I_p measurements (48) by means of patch-clamp techniques (77). Reliable I_p measurements require patch pipettes with a large tip diameter of ~4-5 µm and a low resistance (<2 M). In addition, membrane currents other than I_p have to be suppressed by adequate experimental conditions. The measurements demonstrated the activation of I_p by various intra- and extracellular cations and established the voltage dependence of I_p under a variety of conditions. Furthermore, they revealed the existence of transient pump currents in the Na⁺ and K⁺ limb of the Na⁺-K⁺ pump cycle (130, 135). Clearly, electrophysiological investigations of the Na⁺-K⁺ pump in noncardiac preparations have likewise produced exciting new insight into active cation transport. For example, studies of I_p in Xenopus oocytes (110, 147) and combined electrophysiological and tracer flux measurements in squid axons (53, 144) provided important data for our current understanding of structural and functional properties of the Na⁺-K⁺ pump in animal cells (111, 145, 150, 180).

The electrogenicity of ion pumps cannot be considered an epiphenomenon which is inevitably linked to ion pumping. Although the electrical potential difference set up by the pump across the cell membrane is thermodynamically equivalent to the simultaneously generated osmotic gradient, the former and the latter display quite different kinetic characteristics. As a consequence, the pump-generated membrane potential is, under many conditions, more efficient as a driving force for secondary active transports than the osmotic (ionic) gradient produced by the pump (see Ref. 111, p. 13-14). In addition, the pump current I_p directly affects the automaticity, the resting and action potential, and thereby the conduction of electrical impulses in excitable membranes. I_p is a direct indicator of Na⁺-K⁺ pumping, since the coupling ratio 3Na⁺:2K⁺:1ATP per pump cycle remains constant under a variety of conditions including changes of the intracellular Na⁺ or extracellular K⁺ concentration and of the membrane potential. This applies for cardiac (46, 65) and noncardiac tissues (reviewed in Ref. 32). Measurements of I_p by means of electrophysiological methods offer the possibility to study the Na⁺-K⁺ pump in its physiological environment, i.e., in the cell membrane separating two compartments of different ionic compositions (intra- and extracellular space) and to measure pump-mediated Na⁺ and K⁺ fluxes. In cardiac myocytes as in most animal cells, a membrane potential exists across the cell membrane. Because a translocation of electrical charge across the membrane constitutes the pump current, the I_p amplitude must depend on the membrane potential. The interaction between the electrogenic Na⁺-K⁺ pump and the membrane potential is best studied by electrophysiological techniques. The measured voltage dependence of external and internal cation binding to the cardiac pump has inspired our imagination of the molecular shape of the Na⁺-K⁺ pump as a channel-like structure. Furthermore, electrophysiological studies have rendered possible the identification of additional partial reactions in the pump cycle displaying voltage sensitivity. They also revealed effects of Na⁺-K⁺ pumping on currents produced by cardiac ionic channels or transporters in the vicinity of pump molecules. A major advantage of electrophysiological methods for studies on Na⁺-K⁺ pump-generated Na⁺ and K⁺ fluxes across the sarcolemma over, for instance, tracer measurements is the much better time resolution (up to the microsecond range). Corresponding measurements of I_p provided new insights into the kinetics of the interaction between the Na⁺-K⁺ pump and drugs with hitherto unrivalled precision.

This paper reviews some characteristics of the Na⁺-K⁺ pump as a current-generating molecule in single cardiac cells. The pump current has been investigated in cells isolated from various regions of the heart of different mammalian species. These regions differ in morphology and function. They include the primary pacemaker (sinoatrial node), the cardiac conducting system (Purkinje fibres), and the working myocardium (atrial and ventricles). Especially the ventricular myocytes are easy to isolate and exhibit a high density of pump molecules in the cell membrane. Their cellular geometry is adequate for studies on the Na⁺-K⁺ pump by means of patch-clamp techniques. For these reasons, several investigations that are pivotal for our understanding of the electrogenicity of the Na⁺-K⁺ pump have been carried out on these cells.

	II. ACTIVATION OF THE CARDIAC PUMP CURRENT BY MONOVALENT CATIONS

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A.  Activation of I_p by Intracellular Na⁺

1.  Mean affinity constant values for I_p activation by intracellular Na⁺ solution vary according to the experimental conditions

Earlier studies in multicellular cardiac preparations showed that intracellular Na⁺ (Na) activates I_p (45, 65). However, due to experimental problems, a quantitative relationship between [Na⁺]_i and I_p was difficult to establish. Whole cell recording from single cardiac cells certainly facilitated investigations on this issue, and, during the last decade, various mean affinity constant (K_0.5) values for half-maximal I_p activation by intracellular Na⁺ (Na) have been reported. In view of the variable numbers published, the following points should be kept in mind. Apart from species differences (including the expression of different Na⁺-K⁺-ATPase isoforms), the experimental procedure chosen for estimation of a K_0.5 value is likely to affect the result. A low tip restistance (~1 M) of the patch pipette is a prerequisite for correct measurements of I_p as a function of [Na⁺]_i (121). There is evidence suggesting the existence of a "fuzzy space" (112), in which the ionic concentration deviates from that in the bulk cytosolic solution (23, 159). As to the cardiac I_p-[Na⁺]_pip relationship, it was demonstrated that the subsarcolemmal [Na⁺] is not always controlled by the [Na⁺] of the pipette solution. This is true not only during strong I_p activation but also in the steady state, at least in certain cells (16). Furthermore, a relatively high patch-pipette resistance might cause an additional Na⁺ gradient across the cytosol if active Na⁺/K⁺ exchange is strongly activated. Thus a thoughtful procedure is required to obtain reliable results. In addition, intracellular K⁺ are known to be competitive inhibitors of Na⁺ at intracellular Na⁺-binding sites of the Na⁺-K⁺ pump (73). Consequently, one would expect a lower [Na⁺]_pip for half-maximal I_p activation (K_0.5 value) from measurements where the main cation of the pipette solution is a weaker competitor than K⁺ [Cs⁺ or even tetraethylammonium ion (TEA⁺)]. It might be helpful to remember these points when reading the data presented in Table 1. They were mainly obtained at 30-37°C. Since, under physiological conditions, K⁺ is the main cation in cardiac cells, the "physiological" K_0.5 value for I_p activation by Na may be in the range of ~20 mM Na.

View this table: [in this window] [in a new window]   Table 1. Activation of Ip by Na

Figure 2 compares the I_p activation as a function of [Na⁺]_pip using pipette solutions containing different main cations in guinea pig ventricular myocytes at 0 mV holding potential. Obviously, half-maximal I_p activation occurs at higher [Na⁺]_pip if Cs⁺ instead of TEA⁺ or N-methyl-D-glucamine ion (NMDG⁺) is used as main pipette cation.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Activation of pump current (Ip) as a function of pipette Na+ concentration ([Na]pip) in guinea pig ventricular myocytes. Ip normalized to the respective amplitudes at 50 mM [Na]pip. The curves obey a Hill equation. The principal cation in the pipette solution affects Ip activation. [Data from Nakao and Gadsby (131) () and Barmashenko et al. (9) (, ).]

In summary, cardiac I_p is activated by intracellular Na⁺. The [Na⁺]_pip reported for half-maximal I_p activation varies widely according to the experimental procedure and the ionic conditions chosen.

Whether or not binding of intracellular Na⁺ to the cardiac Na⁺-K⁺ pump is voltage sensitive is still a point of controversy. The sensitivity of the pump in guinea pig ventricular cells to [Na⁺]_pip in the range between 3 and 50 mM increased with depolarization (131). An e-fold drop of the K_0.5 value (i.e., [Na⁺]_pip for half-maximal I_p activation) was estimated for a depolarization by 250 mV. However, the effect was present only in Na⁺-rich superfusion media but absent in Na⁺-free solution where the apparent affinity of the pump to Na seemed to be voltage independent. Consequently, it was concluded that cytoplasmic Na⁺ binding to the cardiac Na⁺-K⁺ pump is voltage insensitive (131). The weak membrane potential dependence of the K_0.5 value seen in myocytes superfused with Na⁺-rich media was ascribed to a voltage-dependent redistribution of enzyme intermediates in the pump cycle. In line with the findings described above, an increasing sensitivity of the pump to low [Na⁺]_pip (5 mM) was observed with depolarization in sheep cardiac Purkinje cells superfused with Na⁺-rich medium (66). Similarly, an increased apparent Na⁺ affinity of the pump to low [Na⁺]_pip with depolarization was reported for guinea pig atrial myocytes superfused with Na⁺-containing or Na⁺-free solution (109). However, little evidence for voltage-dependent internal Na⁺ binding between 20 and 85 mM Na was found during whole cell recording from rat ventricular cells in Na⁺-containing medium (167). The results described so far were obtained with pipette solutions containing K⁺ or Cs⁺, which may compete with Na⁺ for internal cation binding sites of the Na⁺-K⁺ pump and may thereby obscure the mechanism of Na⁺ binding. In a more recent study, TEA⁺ or NMDG⁺ were used as noncompetitive main cations in the pipette solution, and at 5 mM Na, voltage-sensitive binding of internal Na⁺ to the pump of guinea pig ventricular myocytes was clearly shown in Na⁺-free solution (9). Figure 3 is from this work and demonstrates that the K_0.5 value for I_p activation by [Na⁺]_pip varies with membrane potential in myocytes containing either NMDG⁺ (A) or TEA⁺ (B) as the main cation species. Thus experimental evidence is accumulating that the apparent affinity of the cardiac Na⁺-K⁺ pump to intracellular Na⁺ is voltage dependent and increases with depolarization. This conclusion is supported by observations from experiments on cell-free Na⁺-K⁺-ATPase systems (74, 89, 134, 171).

View larger version (8K): [in this window] [in a new window]   Fig. 3. Mean affinity constant (K0.5) values for Ip activation by Na are voltage dependent. K0.5 values for activation of Ip by Na in cells containing either N-methyl-D-glucamine ion (NMDG+) (A) or tetraethylammonium ion (TEA+) (B) are plotted versus membrane potential (V). In both cases K0.5 values decrease monotonically with depolarization. The fitted curves obey a Boltzmann equation [analogous to Equation 1 with -values of 0.16 for NMDG+ (A) and 0.15 for TEA+ (B); r2 = 0.948 (A) and 0.895 (B)]. [From Barmashenko et al. (9). Copyright 1999 The Physiological Society.]

B.  Activation of I_p by Extracellular K⁺ and Its Congeners

1.  Measurements in Na⁺-containing solution

Under physiological conditions, the Na⁺ pump of animal cells is activated by extracellular K⁺. The activation of the pump by external K⁺ follows sigmoid saturation kinetics that can be described by a Hill equation. Earlier studies on the pump activation by extracellular K⁺ in multicellular cardiac preparations have been reviewed in useful articles (45, 65). The majority of the data suggest half-maximal I_p activation by [K⁺]_o in the low millimolar range. Similar K_0.5 values were obtained from experiments on single cardiac myocytes. The data obtained at 30-37°C are presented in Table 2. The I_p activation by two K⁺ congeners, Tl⁺ and NH, was tested in rabbit Purkinje cells (15). The authors derived K_0.5 values of 0.4 mM Tl⁺ and 5.7 mM NH and noted no alteration in the shape of the I_p-Voltage (V) curve if equipotent concentrations of K⁺, Tl⁺, or NH were applied. The following relative potency of extracellular monovalent cations to activate I_p was observed in rabbit sinoatrial node cells: K⁺ Rb⁺ > Cs⁺ Li⁺ (151). The order of potency is the same as that observed for the activation of the isolated Mg²⁺-dependent, Na⁺- and K⁺-activated ATPase, which is the molecular basis of the Na⁺-K⁺ pump (155). In addition, the order of potency confirmed the sequence deduced from voltage-clamp measurement on guinea pig papillary muscles and sheep Purkinje fibers (39).

View this table: [in this window] [in a new window]   Table 2. Activation of Ip by K in Na+-containing solution

Since the early observation on erythrocytes it is known that external Na⁺ and K⁺ compete for common binding sites (138). As a consequence, the K_0.5 value for pump activation by extracellular K⁺ is appreciably lower in Na⁺-free than in Na⁺-containing media. Thus one would expect for cardiac cells a lower K_0.5 value for the I_p activation by K⁺ and its congeners in Na⁺-free than in Na⁺-containing solution. In fact, a K_0.5 value of 0.22 mM was derived for the activation of I_p by K in guinea pig ventricular myocytes superfused with Na⁺-free medium (holding potential 0 mV) (131). This is shown in Figure 4, where normalized pump currents are plotted versus [K⁺]_o. The holding potential is 0 mV. The K_0.5 value for I_p activation by K in these cells decreases from 1.5 mM K in Na⁺-containing solution to 0.22 mM K in Na⁺-free medium (131). Similarly, a K_0.5 value of 0.2 mM was reported for the I_p activation by K in rat and guinea pig ventricular cells (84). Furthermore, the maximum I_p amplitude that can be activated in Na⁺-free media by K and congeners declines with increasing K_0.5 values of the various monovalent cations. K_0.5 values for the I_p activation by external K⁺ or Tl⁺ of ~0.22 mM K and ~0.08 mM Tl were estimated in rat ventricular myocytes superfused with Na⁺-free solution (holding potential 0 mV) (13). In Na⁺-free media, the K_0.5 values for the activation of the pump current by K or K⁺ congeners in rabbit Purkinje cells amount to 0.05 mM Tl, 0.08 mM K, 0.4 mM extracellular NH, and 1.5 mM Cs (holding potential 0 mV) (18). The authors concluded that in general the K_0.5 values for the Na⁺-K⁺ pump activation in cardiac cells superfused with Na⁺-free solution are lower by a factor of 10-20 than those obtained in Na⁺-containing media. The same is true for the K_0.5 values reported from I_p measurements in squid axons (144) and Xenopus oocytes (133), although the absolute numbers are somewhat larger than those estimated for cardiac cells.

View larger version (8K): [in this window] [in a new window]   Fig. 4. Activation of Na+-K+ pump current in guinea pig ventricular myocytes at 0 mV by [K+]o at high [Na+]o (A) or at zero [Na+]o (B). In both cases, pump current amplitude at each [K+]o was normalized to control runs at 5.4 mM K. The graphs show mean values ± SE of the resulting relative pump currents plotted against [K+]o, and the curves show least-squares fits of the Hill equation to the unweighted data. [Na+]pip was 50 mM in all cells. A: data from 17 cells at high [Na+]o; best-fit parameters: maximal relative current = 1.30 ± 0.10; K0.5 = 1.54 ± 0.31 mM K, nH = 0.96 ± 0.13. B: data from 4 cells at zero [Na+]o; best-fit Hill equation parameters: maximal relative current = 1.03 ± 0.05, K0.5 = 0.22 ± 0.03 mM K, nH = 1.12 ± 0.14. [Adapted from Nakao and Gadsby (131).]

The negative slope of the I_p-V relationship in Xenopus oocytes is probably due to voltage-sensitive binding of extracellular K⁺ to the Na⁺-K⁺ pump (147). It is considered that K binding to the pump occurs at the bottom of a "high field, narrow access channel" (see Ref. 111, p. 74-83). To reach their binding sites, the K⁺ have to migrate through the channel within the electrical field across the cell membrane. The membrane potential thereby affects the local K⁺ concentration at the binding sites which, in general, differ from [K⁺] of the extracellular medium. Depolarization of the cell membrane decreases the local [K⁺] and increases the estimated (apparent) K_0.5 value for I_p activation by K. Vice versa, hyperpolarization increases the local [K⁺] at the binding sites and decreases the K_0.5 value. Under certain experimental conditions, the cardiac I_p-V relationship also exhibits a region of negative slope. This is true for myocytes superfused with Na⁺-containing solution (13, 15, 84, 167) or Na⁺-free (poor) media (13, 15, 18, 52), especially at low (<K_0.5 value) concentrations of K or its congeners. A detailled study on the activation of I_p by K, Tl, extracellular NH, and Cs at various membrane potentials (V_c) was carried out with rabbit cardiac Purkinje cells in Na⁺-free solution (18) (Fig. 5; see also Table 3). Figure 5 shows that the apparent affinity of the cardiac Na⁺-K⁺ pump to K, Tl, Cs, or extracellular NH depends on voltage. The affinity decreases with depolarization, whereas the corresponding K_0.5 values increase according to the Boltzmann equation

(1)

where K_{0.5(Vc=0 mV)} denotes the K_0.5 value at zero potential.  is a steepness factor, and F, V_c, R, and T have their usual meanings. K_{0.5(Vc=0 mV)} values for I_p activation by various external cations and the respective -values are collected in Table 3 (from Ref. 18). Because the steps subsequent to K binding in the K⁺ limb of the pump cycle are supposed to be voltage independent (135), it is assumed that the binding of K or its congeners to the Na⁺-K⁺ pump probably is the voltage-sensitive process involved. From the -values and the Hill coefficients derived, it can be estimated that the external activator cations sense ~0.2 of the membrane potential at their binding sites (see Ref. 150). In summary, the binding of K and ist congerners to the cardiac Na⁺-K⁺ pump is most probably voltage dependent.

View larger version (12K): [in this window] [in a new window]   Fig. 5. K0.5 values for Ip activation of rabbit cardiac Purkinje cells by external cations at various clamp potentials. The curves fitted to the data obey Equation 1 with parameters listed in Table 3. A: , K+; r2 = 0.93; , Tl+; r2 = 0.79. B: , NH; r2 = 0.98; , Cs+; r2 = 0.77. Note the different scales of the ordinates in A and B. [From Bielen et al. (18). Copyright 1993 The Physiological Society.]

View this table: [in this window] [in a new window]   Table 3. K0.5(Vc=0 mV) and -values for Ip activation by external cations in Na+-free solution

It is known from earlier studies on multicellular cardiac preparations that Li⁺ is a weak external pump activator cation (see Ref. 64 for references). As to isolated cardiac cells, I_p is activated by Li in single rabbit Purkinje cells (15). In isolated sinoatrial cells, Li is an ~14-fold weaker external activator than K (151). Interestingly, intracellular Li⁺ activates Li/K and Li/Li exchange via the Na⁺-K⁺ pump in rabbit ventricular myocytes (83). However, internal Li⁺ is a much less effective activator than intracellular Na⁺. The pump current density elicited by 160 mM Li was clearly lower than the current density measured at high [Na⁺]_pip. A K_0.5 value for I_p activation by [Li⁺]_pip of 36 mM was found for guinea pig ventricular myocytes superfused with Na⁺-free (choline⁺) medium containing 1 mM K⁺ (holding potential 20 mV) (87). Half-maximal I_p activation was observed at 23 or 73 mM Li in myocytes containing either 50 to 100 mM [Na⁺]_pip or 100 mM [Li⁺]_pip, respectively. Binding of Li to the pump is voltage sensitive. At the locus of their binding, Li⁺ sense ~0.2 of the membrane potential across the sarcolemma. Thus Li, like the other K⁺ congeners and K itself, probably binds to the Na⁺-K⁺ pump at the bottom of an access channel. Regions of positive and/or negative slope persist in the cardiac I_p-V curve under conditions where the intra- and extracellular cation binding sites of the Na⁺-K⁺ pump should be saturated by Li⁺. Therefore, mechanisms apart from binding/rebinding of the transported cations are voltage dependent and affect the shape of the cardiac I_p-V relationship (87).

Usually, in experiments designed to measure I_p, the Na⁺-K⁺ pump of cells is strongly activated by nearly saturating concentrations of Na and [K⁺]_o. The measured I_p densities depend not only on these ionic concentrations, but also on the membrane potential and the temperature at which the measurements were carried out. In addition, cells in culture often exhibit lower I_p densities than freshly isolated cells. Furthermore, the species of the main intracellular cation used (K⁺, Cs⁺, or NMDG⁺) affects the I_p density. Ionic species that do not compete with Na⁺ for intracellular binding sites of the pump (for instance NMDG⁺) tend to evoke higher I_p densities (166). It might be helpful for the reader of the data presented below to keep these points in mind. The first measurement of the I_p density in isolated guinea pig ventricular myocytes yielded 1-1.5 µA/cm² (48). The maximum I_p density of these cells were estimated to be ~1.6 µA/cm² (131). In sheep Purkinje cardioballs (1-3 days in culture), an I_p density of 1.1 µA/cm² has been observed (66). A maximum I_p of ~1.9 µA/cm² has been derived from I_p measurements in rabbit sinoatrial node cells (151). Guinea pig atrial cardioballs (1 day in culture) exhibit an I_p density of 0.66 µA/cm² (109), whereas rabbit ventricular myocytes display I_p densities between ~1.7 and ~2.2 µA/cm² (14, 75, 76). I_p densities reported for rat ventricular cells vary between 1.6 µA/cm² (166) and ~4.3 µA/cm² (101). All these numbers were obtained from measurements at membrane potentials greater than 45 mV and near body temperature. They agree reasonably well with earlier data for the active Na⁺ efflux via the cardiac Na⁺ pump obtained by various nonelectrophysiological methods (63). (An I_p density of 1 µA/cm² translates to a pump-mediated flux of ~30 pmol·cm²·s¹). They are also in line with previous estimates of I_p density in multicellular cardiac preparations (28). For comparison, simultaneous measurements of I_p density and active ²²Na efflux in squid axons yielded 0.89 µA/cm² and ~ 25 pmol·cm²·s¹ (144), again in accordance with earlier flux data (94).

From the maximum quantity of movable charge derived from measurements of transient pump currents in single cardiac cells (see sect. IVB), the pump site density of the myocytes has been estimated assuming a single charge (1.6 × 10¹⁹ C) to be transfered per pump molecule and the specific membrane capacitance to be 1 µF/cm². The first number obtained by this procedure for the pump site density of single cardiac cells was published by Nakao and Gadsby (130). According to the authors, the pump site density of guinea pig ventricular myocytes is ~1,200/µm². Later estimates include slightly higher numbers of 2,200 to 2,800 pumps/µm² for guinea pig ventricular cells (108) and ~ 2,600 sites/µm² for rat ventricular myocytes (35). These values are higher by a factor 2 to 4 than an earlier number derived from I_p measurements in a guinea pig multicellular ventricular preparation (28).

The maximum turnover rate of the charge transfer by the cardiac Na⁺-K⁺ pump can be calculated if the maximum I_p density and the pump site density are known. Accordingly, a maximum turnover rate of ~80/s has been obtained from the data mentioned above for guinea pig ventricular myocytes (51). A higher maximum turnover rate of ~200/s has been derived from measurements of transient pump currents in excised patches of rat ventricle cells (42). Both numbers apply to turnover rates at 0 mV and 36°C.

The pump site densities reported above are within the range of earlier estimates in a variety of cell species by means of different methods, mainly cardiac glycoside binding (see Table 3 in Ref. 32). These estimates vary considerably among the cell types, whereas the calculated turnover rates are much more similar and amount to ~100/s at body temperature (32).

	III. THE REVERSAL POTENTIAL OF THE CARDIAC PUMP CURRENT

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The free energy of intracellular ATP hydrolysis (G_ATP) fuels the active Na⁺/K⁺ transport by the Na⁺-K⁺ pump. It amounts to about 60 kJ/mol in many animal cells (31). The energy is used to transport 3 mol Na⁺ and 2 mol K⁺ against their respective concentration gradient (osmotic work) and the electric charge of 1 mol Na⁺ against the electrical field across the cell membrane (electrical work). Thus the physiological active Na⁺/K⁺ transport proceeds as long as

(2)

where a^o and aⁱ represent the activities of the transported ions at both sides of the cell membrane, E_m denotes the membrane potential, and R, T, and F have their usual meanings. According to Equation 2, there should be a membrane potential where G_ATP equals the energy required for the active cation transport. This potential is called the reversal potential (E_rev) of the Na⁺-K⁺ pump. No active Na⁺/K⁺ fluxes occur at E_rev, and I_p vanishes. If the membrane potential becomes more negative than E_rev, the Na⁺-K⁺ pump runs backward producing an active Na⁺ influx into the cell and generating an inwardly directed I_p. E_rev can be derived from Equation 2 by

(3)

where E_Na and E_K stand for the Nernst potential of the respective cation. From Equation 3, E_rev can be calculated to be

(3a)

Thus under physiological conditions E_rev is beyond the membrane potentials that are experimentally accessible. However, according to Equation 3, it is possible to shift E_rev to more positive membrane potentials by lowering G_ATP and steepening the ionic gradients. G_ATP is given by

(4)

where G denotes the standard free energy of ATP hydrolysis and [ATP], [ADP], and [P_i] represent the intracellular concentration of adenosine triphosphate, adenosine diphosphate, and inorganic phosphate, respectively. Thus a lower (less negative) G_ATP can be obtained by increasing the [ADP] · [P_i]/[ATP] ratio in the cell studied.

The procedure outlined above was first applied to squid giant axons, and an inwardly directed I_p was demonstrated (34). Under similar experimental conditions, an inwardly directed I_p was measured in guinea pig ventricular myocytes over a range of membrane potentials between +40 and 120 mV (6). Figure 6 presents some of the results . Figure 6A (bottom trace) demonstrates that the strophanthidin-inhibited pump current I_p of a guinea pig ventricular myocyte is inwardly directed under the conditions chosen. As shown in Figure 6B, I_p remains an inward current even at positive potentials. Figure 6C displays I-V curves before, during, and after application of the cardiac steroid, a specific inhibitor of the Na⁺-K⁺ pump. The I-V relationships were derived from the experiment illustrated in Figure 6A. Figure 6D exhibits the I_p-V curve of the backward running Na⁺-K⁺ pump obtained from the strophanthidin-sensitive current I_p at various potentials, as illustrated in Figure 6B. I_p was small at +40 mV, increased with hyperpolarization, and reached an apparent plateau level of about 0.32 µA/cm² near 100 mV. By means of a similar approach, an inward I_p between +30 mV and 110 mV that did not reach a plateau at the most negative membrane potential tested was observed in cardiac Purkinje cells. The I_p density amounted to 0.13 µA/cm² at 95 mV (71). Furthermore, E_rev shifted to more positive potentials at less negative G_ATP values. Shifting G_ATP to less negative values also diminished I_p over the entire voltage range studied. However, the concentration of extracellular Cs⁺ required for half-maximal I_p activation remained unchanged. Equation 3 predicts changes of E_rev by variation of the transmembrane gradients of the pumped cations at constant G_ATP. This prediction was verified (69). Flattening the ionic gradients increased I_p over the entire voltage range studied and shifted E_rev toward more negative potentials. Conversely, steepening the gradients diminished I_p and shifted E_rev to more positive potentials.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Voltage dependence of inward current generated by the backward-running Na+-K+ pump. A: chart recording of membrane potential (top) and membrane current (bottom); holding potential, 40 mV. The horizontal bar marks exposure to 0.5 mM strophanthidin (str). The K+-free external solution contained 5 mM Ba2+; the pipette solution was Na+ and Cs+ free and contained 145 mM K+, 5 mM MgATP, 5 mM Tris2ADP, and 5 mM phosphate. B: superimposed records of strophanthidin-sensitive currents for 80-ms pulses to +40, 0, 60, and 100 mV, obtained by subtracting each trace recorded in the presence of strophanthidin from the average of control traces recorded during pulses to the same potential just before and just after the exposure to strophanthidin. C: whole cell current-voltage relationships from the experiment in A, determined before (), during (), and after () exposure to strophanthidin. Ordinate, steady current levels; abscissa, membrane potential. D: current-voltage relationship of the backward-running Na+-K+ pump. Ordinate, steady levels of the strophanthidin-sensitive currents represented in B; abscissa, membrane potential. [Adapted from Bahinski et al. (6).]

The I_p-V curve of the backward running Na⁺-K⁺ pump was also studied in internally dialyzed squid axons under voltage clamp (143). There was a steady decline of I_p density from an apparent plateau at 80 to 100 mV (0.24 µA/cm²) to practically zero at +30 mV. In contrast to earlier observations (34), a negative slope of the I_p-V relationship was not found. In K⁺-free solution, an inwardly directed I_p was measured in Xenopus oocytes with a reduced intracellular [Na] and an augmented [ADP] · [P_i]/[ATP] ratio. The I_p density amounted to about 0.1 µA/cm² at 100 mV without an apparent plateau and declined with depolarization. Under these conditions the I_p reversal potential was obviously at positive membrane potentials (38).

In summary, the studies demonstrate under suitable conditions in various cell species a backward running Na⁺-K⁺ pump generating an inward I_p, in line with thermodynamic considerations.

	IV. VOLTAGE DEPENDENCE OF CARDIAC PUMP CURRENTS

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A.  Cardiac Steady-State I_p-V Relationships

1.  Basic characteristics of the I_p-V curve

Whole cell recording is a mode of the patch-clamp technique (77) which permits a much better control of the membrane potential and the ionic composition of the intracellular compartment than earlier voltage-clamp techniques applied to multicellular preparations. Gadsby et al. (48) were the first to study the voltage dependence of I_p by whole cell recording from isolated, single guinea pig ventricular cells in Na⁺-containing solution after minimizing passive Na⁺, Ca²⁺, and K⁺ currents. According to the authors, the I_p-V relationship of the myocytes is sigmoid in shape with a steep positive slope between 100 and 0 mV, a less steep slope at more negative potentials, and nearly no voltage dependence of I_p at positive membrane potentials (see also Fig. 7A, circles). A further careful study by the authors confirmed these characteristics of the cardiac I_p-V curve (51). However, a region of negative slope in the I_p-V curve was not observed. A decrease of I_p with hyperpolarization in guinea pig ventricular cells was also reported in 1987 by others (123) in abstract form. The pump current of adult isolated rat cardiac myocytes displays a similar voltage dependence as in guinea pig myocytes (167). This is also true for the voltage dependence of I_p in rabbit ventricular cells dialyzed with a pipette solution containing 80 mM Na⁺ (79). For unknown reasons, some authors were unable to detect any voltage dependence of I_p in guinea pig ventricular myocytes (127). In the cardiac conducting system, the I_p-V curve shows little voltage dependence at membrane potenials positive to 20 mV. The pump current declines with hyperpolarization (66).

View larger version (21K): [in this window] [in a new window]   Fig. 7. The effect of Na and Na on the Ip-V relationship of guinea pig ventricular myocytes. A: Na+-K+ pump current-voltage relationships determined in a single cell at 50 mM Na, 5.4 mM K, and [Na+]o of 150 mM (), then 1.5 mM (), and then 150 mM again (). Steady current levels were measured near the end of 160-ms voltage pulses from the holding potential 40 mV. Pump currents were obtained by subtracting currents recorded in the presence of 2 mM strophanthidin from the average of currents recorded just before, and just after, the brief exposure to strophanthindin. [Adapted from Gadsby and Nakao (50).] B and C: Ip-V relationships at various [Na+]pip in Na+-free solution. Na+-K+ pump currents normalized to cell capacitance are plotted versus membrane potential. B: Ip-V relationships at 0 mM (), 0.5 mM (), 2 mM (), 5 mM (), and 50 mM () Na in cells dialyzed with NMDG+-containing pipette solution (n = 4-8). C: corresponding Ip-V curves for myocytes containing TEA+ as the principal cation (n = 4-7). Symbols have the same meaning as in B. Note the absence of Ip in Na+-free pipette solutions and the voltage dependence of Ip at low (5 mM) Na. In contrast, Ip activated by 50 mM Na is voltage insensitive. [From Barmashenko et al. (9). Copyright 1999 The Physiological Society.]

Already in 1987 it was reported that lowering internal [Na⁺] diminishes the pump current of guinea pig ventricular cells in Na⁺-rich solution and shifts the I_p-V curve to the right (to more positive potentials), whereas the voltage dependence of I_p persists (50). However, in the absence of external Na⁺, lowering [Na⁺]_pip from 50 to 17 mM scaled down the I_p-V relationship of these cells without a marked shift toward more positive potentials (131). A similar shift of the normalized I_p-V curve to the right and a reduction of I_p if [Na]_pip was lowered from 50 to 5 mM occurred in sheep cardiac Purkinje cells superfused with Na⁺-rich medium (66). Reducing [Na⁺]_pip from 85 to 20 mM scales down the I_p-V relationship of rat myocytes at 145 mM Na without any shift of the I_p-V curve along the voltage axis (167). It seems likely that [Na⁺]_pip in these experiments was not low enough to produce this shift. By way of contrast, both a scaling down and a rightward shift of the I_p-V relationship were observed in guinea pig atrial myocytes superfused with Na⁺-free or Na⁺-containing media (109). Recently, a scaling down and a shift of the normalized I_p-V curve toward more positive potentials were reported from measurements on guinea pig ventricular cells in Na⁺-free solution after lowering [Na⁺]_pip from 50 to 5 mM (main cation in pipette: TEA⁺ or NMDG⁺) (9). Some of the findings are displayed in Figure 7, B and C. Lowering [Na⁺]_pip from 50 to 5 mM or below reveals the effect of [Na⁺]_pip on the cardiac I_p-V curve in myocytes containing NMDG⁺ (Fig.