A model of Na-K-2Cl cotransport based on ordered ion binding and glide symmetry

Christian Lytle1, Thomas J. McManus2, and Mark Haas2

1 Division of Biomedical Sciences, University of California, Riverside, California 92521; and 2 Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

In the duck red blood cell, Na-K-2Cl cotransport exhibits two modes of ion movement: net cotransport and obligate cation exchange. In high-K cells, the predominant exchange is K/K (or K/Rb). In high-Na cells, it becomes Na/Na (or Na/Li). Both represent partial reactions in which a fully loaded carrier releases part of its cargo, rebinds fresh ions, and returns back across the membrane fully loaded. Net cotransport occurs when the carrier unloads completely and returns empty. This mode has a fixed stoichiometry of 1Na:1K:2Cl under all conditions tested. The ion requirements of the two exchanges differ: K/K exchange requires only K and Cl outside but all three ions inside. Na/Na exchange requires all three ions outside but only Na inside. We propose a simple model in which the carrier can only move when either fully loaded or completely empty and in which the ions bind in a strictly ordered sequence. For example, externally, a Na binds first and then a Cl, followed by a K and a second Cl. Internally, the first on is the first off (glide symmetry), so the Na is released first and then the first Cl, followed by the K and finally by the second Cl. Only then can the empty form return to the outside to start a new cycle.

avian red blood cells; transport kinetics; bumetanide

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE AVIAN RED BLOOD CELL is the premier single cell model of Na-K-2Cl cotransport. Its ready availability, robust responses, and ease of experimental manipulation make it ideal for exploring the kinetic characteristics and intracellular signaling pathways controlling this complex transporter. When exposed to beta -adrenergic catecholamines, hypoxia, or a decrease in cell volume, a vigorous cotransport pathway is activated that moves Na, K, and Cl across the membrane in a tightly coupled, electrically neutral fashion, driven by the combined chemical gradients of the participating ions (17, 36-38). Both the catecholamine and cell shrinkage methods of stimulation result in phosphorylation of the 146-kDa cotransport protein at a common constellation of serine and threonine sites (23). Once phosphorylated, the cotransporter can bind inhibitors, such as bumetanide or furosemide, with high affinity (15, 31).

This study examined norepinephrine-activated, bumetanide-sensitive ion movements in ouabain-treated duck red blood cells. A comparison of net and unidirectional cation movements revealed two partial reactions of the cotransport cycle: K/K exchange in normal high-K cells and Na/Na exchange in high-Na cells. The internal and external ion requirements of each mode can be explained by a reaction cycle based on ordered binding and glide symmetry. A model incorporating these features is presented. Preliminary reports of this work have appeared elsewhere (25, 26).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Preparation of red blood cells. Blood was obtained from the brachial vein of healthy White Pekin ducks (Anas platyrhynchos). Occasionally, blood from two or more ducks was pooled. Red blood cells were washed three times, with removal of the buffy coat, in an ice-cold isotonic (323 mosmol/kg) solution containing 171 mM NaCl and 2.5 mM KH2PO4/K2HPO4 (pH 7.4 at 41°C). To achieve a steady state with respect to ion and water content, washed cells were routinely preincubated (1 h, 3% hematocrit, 41°C) in DFS medium containing (in mM) 6 KCl, 1.25 PO4, 20 N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES; Sigma Chemical, St. Louis, MO) titrated to pH 7.40 at 41°C with NaOH, 10 glucose, and sufficient NaCl to yield 323 ± 4 mosmol/kg.

Alteration of intracellular ion composition. The ratio of intracellular K concentration ([K]c) to [Na]c was altered by the nystatin technique (5) as modified for this system by Haas et al. (17). Washed cells were incubated (2.5% hematocrit, 1 h, 20°C) twice in a dialysis medium containing (in mM) 116 (Na+K)Cl, 50 sucrose, 5 glucose, 2.5 tetramethylammonium (TMA)-TES (pH 7.4 at 20°C), and 0.05 ouabain. Nystatin (18 µg/ml; B-grade mycostatin, Calbiochem) was added to the first incubation from a 20 mg/ml stock solution in dimethyl sulfoxide. Elution of the ionophore to restore native membrane permeability was accomplished by washing the cells six times at room temperature in 35 volumes of solution containing 0.1 mM ouabain and fortified with 0.25% bovine serum albumin (fraction V, Sigma). Na contamination of the 10% albumin stock solution was removed by passage over a cation exchange resin (AG 5OW-X, Bio-Rad). The desalted albumin was then titrated to pH 7.4 at 23°C with tris(hydroxymethyl)aminomethane base.

When only a modest increase in cell Na (10-60 mM) was desired, the cells were preincubated (2.5% hematocrit, 41°C) without nystatin in DFS supplemented with K (24 mM) and ouabain (0.1 mM). Under these conditions with the Na pump disabled, cell Na increased at a constant rate of 8.5 mM/h over 3.5 h, with an equivalent reduction in K so that cell volume remained normal. Once calibrated in this fashion, incubation periods could be chosen to yield any desired level of cell Na in the 10-60 mM range.

Cl substitution. Replacement of internal Cl was accomplished by two consecutive equilibrations (2.5% hematocrit, 0.5 h, 41°C) in DFS containing methanesulfonate (MSA) in place of Cl. Measurements of 36Cl/MSA exchange indicated that both anions attained equilibrium distribution across the cell membrane within 5 min. MSA, unlike other Cl substitutes (e.g., NO3, F, SCN), does not affect red blood cell Na, K, pH, or water content (33).

Inhibition of anion exchange. When Cl was to be replaced by MSA on only one side of the membrane, anion gradients and cell pH were stabilized during the final incubation by treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; Sigma) which irreversibly inhibits anion exchange and conductance in avian red blood cells (17). Optimal inhibition of anion exchange was achieved when 1) the cells were pretreated for 45 min in the dark with 50 µM DIDS, 2) MSA was used as the Cl substitute, and 3) 25 µM DIDS was also included in the final incubation medium. After this procedure, cells in a MSA medium containing bumetanide lost Cl only very slowly (0.83 ± 0.35 mmol · l cell water-1 · min-1, n = 7). Ascending thin-layer silica gel chromatography of commercially obtained DIDS (Sigma) using pyridine-acetic acid-water (10:1:40) revealed three spots (a major spot flanked by two minor ones). Neither DIDS nor its contaminants had a significant effect on Na-K-2Cl cotransport.

Transport assay. Sampling to measure ion movements was limited to a brief initial period of incubation during which the cell ion and water contents changed at linear rates. Cells were incubated (2.5% hematocrit, 41°C) in a solution containing (in mM) 20 TMA-TES (pH 7.4 at 41°C), 10 glucose, and 0.1 ouabain, plus other constituents as indicated (see Figs. 1-8). Aliquots of cell suspension containing ~200 mg cells were periodically removed and centrifuged in specially fabricated nylon tubes (10, 37). The packed cells were separated from the supernatant liquid by cutting the tube with a razor blade. Analysis for wet cell mass, dry cell mass, radioactivity, and ion content was carried out as previously described (17, 37). Inward cation movements were determined using 86Rb, 85Rb, 22Na, or Li. Outward cation movements were estimated from the net loss of Na and K from ouabain-treated cells. Concurrent measurement of bidirectional cation movements was therefore possible simply by estimation of the increase of Rb or Li and the decrease of K or Na in the cell compartment. One minute before the sampling period, norepinephrine was added to a final concentration of 10 µM, a dose 30 times that producing maximal cotransport activity. All the cotransport activity in this report was elicited by activation with norepinephrine and quantitated as the difference between ion levels measured in the presence and absence of 100 µM bumetanide (kindly provided by Dr. Peter Sorter of Hoffmann-LaRoche, Nutley, NJ). Bumetanide was added from a 100 mM stock solution freshly prepared in 105 mM TMA-OH. Transport was terminated by diluting the cell suspension into >40 volumes of an ice-cold stop solution (isotonic KCl containing 100 µM bumetanide).

Replacement ions. TMA, a nontransportable substitute for alkali metal cations, was obtained as the Cl salt from Eastman Organic Chemicals, recrystallized from absolute ethanol, and stored desiccated at -20°C. Gluconate salts were prepared by titration of 335 mM gluconolactone (Alfa Products) with alkali metal hydroxides (Alfa Products) to a stable neutral pH. MSA salts were prepared by titration of alkali metal hydroxides with methanesulfonic acid (15 M, 99.5%, Alfa Products). Tan discoloration of the gluconate and MSA stock solutions as well as a slight gray precipitate that formed in refrigerated Rb-MSA over time were removed (with no resulting change in osmolality) by passage through clean activated charcoal.

Ion analysis. Diluted perchloric acid extracts of the cells were assayed for Na, K, Li, and Rb by air-acetylene flame spectrophotometry using, a Perkin-Elmer model 460 spectrophotometer set in emission-chopping mode. Cl was analyzed coulometrically using a Radiometer CMT-10 Cl titrator. 22Na was counted in a Beckman Biogamma counter, and 86Rb and 36Cl were counted in a Packard 2000CA liquid scintillation counter.

Presentation of data. By convention, ion contents, water contents, and transport rates were calculated per kilogram of dry cell solid (10), which represents a fixed number of cells (14.7 × 1012; Ref. 24), thus providing a reference point independent of cell volume. The symbol Delta  is used to represent the change in ion content over an interval during which the change was proportional to time; hence, Delta  closely approximates the actual transport rate. Because the level of cotransport produced in response to in vitro catecholamine stimulation can vary in cells from different animals, data are presented from representative experiments, each of which was repeatedly confirmed.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

The duck red blood cell activated by a beta -adrenergic catecholamine has the highest rate of Na-K-2Cl cotransport of any red blood cell thus far examined (26). With such a robust response, it is possible to measure net ion movements quite accurately over a relatively brief interval. In these cells, Na-K-2Cl cotransport can be defined as the component of cation flux activated by norepinephrine, dependent on Cl, or inhibited by bumetanide, with equivalent results (15). Such measurements on cells incubated in a solution free of Na and K (16, 17) show that they lose co-ions in a fixed stoichiometric ratio (1Na:1K:2Cl). With all three co-ions present in the cell, the ratio of net loss is independent of the internal concentrations of Na and K.

Studies of a similar pathway in human red blood cells, however, have led to suggestions that the stoichiometry varies with internal ion composition (3) and that the system is "asymmetrical" in the sense that outward cotransport does not respond to internal ion changes in the same manner as inward transport does to external variations (20). To evaluate these possibilities in the duck cell, we designed experiments to test whether stoichiometry was independent of both internal and external ion concentrations, as well as the direction of net movement. The strategy was to measure bumetanide-sensitive net transport in cells of different composition in solutions containing mostly Na or mostly K (Table 1). The direction of net transport was determined by manipulation of the ion gradients. To produce net outward cotransport, internal Na was raised and external Cl was lowered (Table 1, experiments 1 and 2); to produce net inward cotransport, both Cl and Na were lowered in the cells (Table 1, experiments 3 and 4). Dissipation of anion gradients and recycling of cotransported Cl were prevented by pretreating the cells with DIDS. It is apparent from the net ion movements presented in Table 1 that a stoichiometric ratio of 1Na:1K:2Cl is maintained regardless of the direction and magnitude of net cotransport or of the composition of the internal and external compartments. Moreover, the stoichiometry was not affected by which ion gradient was responsible for driving net cotransport, offering further evidence of tight stoichiometric coupling. For example, outwardly directed K and Cl gradients drove Na from the cell against its gradient in experiment 1, whereas inwardly directed Na and Cl gradients in experiments 3 and 4 produced a net uptake of K against its gradient. In each case, the stoichiometric ratio was unvarying.

                              
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Table 1.   Stoichiometry of net cotransport

Transport stoichiometry in high-K cells: defining K/K exchange. Figure 1 demonstrates how the stoichiometry of cotransport can appear to be markedly different when unidirectional ion fluxes are measured. Figure 1, left, charts the time course of unidirectional Rb and 22Na uptake measured concurrently in the same cells. Addition of norepinephrine elicits a dramatic increase in cation movement, but only after a significant delay. This delay period, representing the time interval between addition of catecholamine and onset of the stimulated cation uptake, averaged 52 ± 14 s (n = 14) at 41°C. Both this delay period and the transport process itself were highly temperature dependent (data not shown).


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Fig. 1.   Time course of bumetanide-sensitive cation uptake in high-K cells. Norepinephrine (NE; 1 µM) was added at times indicated by arrows. Steady-state uptake rates (mM · kg cell solid-1 · min-1) were 5.25 for Rb and 2.75 for 22Na (left) and 2.43 for Rb and 1.50 for Li (right). Cation uptake in presence of bumetanide (left: Rb 0.05, 22Na 0.27; right: Rb 0.04, Li 0.31) was unaffected by NE. c, Intracellular; o, extracellular.

The same pattern of cation uptake was seen when external Na was replaced by Li (Fig. 1, right). In the duck red blood cell, Li interacts with the Na site of Na-K-2Cl cotransport, but at a much lower apparent affinity (apparent K1/2 for Na = 15 mM; apparent K1/2 for Li = 65 mM; Refs. 15, 25). This characteristic of Li transport is evident in Fig. 1, where Rb uptake driven by Li was less than one-half that driven by Na (note the difference in the ordinate between left and right). Another important characteristic of the system is also illustrated in Fig. 1. Unidirectional influx of Rb was nearly twice that of Na (or Li), a feature previously observed in duck red blood cells (38) as well as those from other birds (21, 32). Because this system is symmetrical (Table 1), one would expect unidirectional efflux to share this characteristic, which was confirmed, as is shown below (see Fig. 3).

Because the stoichiometric ratio of net uptake or loss is always 1Na:1K:2Cl, it follows that the excess uptake of Rb (or K) over Li (or Na) (Delta Rbc - Delta Lic) should exactly equal the excess loss of K over Na -(Delta Kc - Delta Nac). Figure 2 shows a plot of these parameters under several experimental conditions. The data fit a straight line with a zero intercept and a slope near unity, demonstrating that the two are equivalent under all conditions tested. Also K/Rb exchange does not depend on the presence of external Na or Li (experiments 3324 and 2748).


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Fig. 2.   Stoichiometry of K/Rb exchange. High-K cells were incubated in media containing various concentrations of Rb and Li. Rb uptake in excess of Li (Delta Rbc - Delta Lic) is plotted against K lost in excess of Na [-(Delta Kc - Delta Nac)], where each Delta  term represents 1 mM furosemide-sensitive or 0.1 mM bumetanide-sensitive component of uptake or loss; Delta t, change in time. Ouabain (0.1 mM) was present in all incubations.

Ion requirements of Na-K-2Cl cotransport and K/K exchange. With the use of this same approach, the internal and external ion requirements of Na-K-2Cl cotransport and K/Rb exchange were compared. Figure 3 shows the effect of removing external Rb, Li, and/or Cl on cation uptake (top) and loss (bottom) from moderately Na-loaded cells. The presence of a full set of co-ions on both sides of the membrane (Fig. 3, column 1) resulted in an uptake of Li equal to the loss of Na and an uptake of Rb equal to the loss of K, reflecting the fact that the ion gradients under these circumstances were balanced. Moreover, the excess uptake of Rb over Li is approximately equal to the excess loss of K over Na, again revealing the presence of K/Rb exchange. When external Li was removed (column 2), leaving Rb as the only transportable external cation, K/Rb exchange and outward cotransport (represented by net Na loss) persisted. When external Rb was removed (column 3), leaving Li as the only transportable cation, Li uptake and K/Rb exchange were extinguished, and net outward cotransport was doubled. After the elimination of K/Rb exchange, Na efflux became equal to K efflux. In the absence of external Rb, there is no significant uptake of Li, indicating that it cannot substitute for K. In the complete absence of external transportable cations (column 4), the 1:1 stoichiometric ratio for net efflux of Na and K was preserved, confirming a previous report (17). These findings also clearly show the presence of an obligate K/Rb exchange and also suggest that those transport units engaged in this exchange become available for net cotransport after the removal of external Rb or K. 


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Fig. 3.   Ion requirements of K/Rb exchange and cotransport. Bumetanide-sensitive components of Rb and Li uptake (top) and K and Na loss (bottom) were measured concurrently. To produce a moderately elevated level of internal Na, cells were preincubated in either Cl or methanesulfonate (MSA) media containing ouabain and then treated with DIDS, and finally test incubations (7 min, 41°C, 2% hematocrit) were carried out in media containing the ions indicated between top and bottom. When present, Rb, Li, and Cl concentrations were 50, 100, and 150 mM, respectively.

Another batch of cells was incubated in a medium in which all the Cl was replaced by MSA (Fig. 3, columns 5-8). Cl loss and cell pH change over the 7-min test period were minimized by pretreatment with DIDS. The same series of external cation omissions was performed. Outward cotransport of Na and K was unaffected by removal of external cations (columns 5-8). However, external Cl was clearly required for K/Rb exchange (compare columns 5 and 6 with columns 1 and 2). In one batch of cells, internal Cl was replaced by MSA ([Cl]c = 0). When these cells were incubated in the presence of both external Cl and Rb, both modes of cotransport, net efflux and K/Rb exchange, were abolished (compare column 9 with column 2). Hence, operation of the cotransporter in the net outward mode requires none of the co-ions externally, but K/Rb exchange is only possible when Cl is present on both sides of the membrane.

Although K/Rb exchange is independent of the presence of external Na (or Li), it is absolutely dependent on internal Na (Fig. 4). The effect of cell Na on Rb uptake is shown under conditions (Na-free media) in which Rb uptake reflects only K/Rb exchange and Na loss represents net outward cotransport. Both modes were abolished when cell Na was <1 mM and were half maximally activated at ~10 mM. Above 30 mM, the activation curves for both influx and efflux decline, an effect previously reported in human red blood cells (5).


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Fig. 4.   Effect of cell Na on bumetanide-sensitive Rb uptake and Na loss. Cell Na was increased at expense of K by timed preincubation with ouabain (0.1 mM). Initially, cell water content was 1.413 ± 0.012 kg water/kg cell solid. Horizontal bars extending from each point indicate change in cell Na levels over 5-min interval of test incubation.

Transport stoichiometry in high-Na cells: defining Na/Na (or Na/Li) exchange. When cells were prepared by the nystatin technique to be high in Na, a different pattern of ion movements was observed. Although the activation lag time following addition of norepinephrine (Fig. 5) was similar to that seen in high-K cells (Fig. 1), the influx of 22Na was much greater than influx of Rb. When external Na was replaced by Li, the rate of uptake dropped 50% (Fig. 5, right; note the scale difference in the ordinate) with no change in the pattern. When data were pooled from four separate experiments and the same approach employed in Fig. 2 was used, a plot of Delta Lic - Delta Rbc vs. -(Delta Nac - Delta Kc) yielded a straight line with a slope of one and an intercept of zero (Fig. 6). Thus, in high-Na cells, the excess uptake of 22Na (or Li) over Rb is equal to the excess loss of Na over K, indicating the presence of 1:1 Na/22Na (or Na/Li exchange). The dependence of Na/Li exchange on external Li fits the Michaelis-Menten equation, showing an apparent affinity of 86 mM, whereas the dependence of Na/22Na exchange on external Na reveals an apparent affinity of 18 mM (data not shown). These results are consistent with the difference in rates of Na and Li uptake in Fig. 5.


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Fig. 5.   Time course of bumetanide-sensitive cation uptake in high-Na duck red blood cells. Experimental design exactly mirrors that shown in Fig. 1, except that ratio of internal Na to K was reversed by nystatin preincubation technique.


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Fig. 6.   Stoichiometry of Na/Li exchange. These experiments were of a design similar to those shown in Fig. 2, except that ratio of internal Na to K was reversed. Li uptake in excess of Rb (Delta Lic - Delta Rbc) is plotted against Na loss in excess of K [-(Delta Nac - Delta Kc)]. Each Delta  term represents 0.1 mM bumetanide-sensitive component of uptake or loss. Ouabain (0.1 mM) was present in all incubations.

Co-ion requirements of Na/Na exchange. Although removal of external Na had no effect on K/Rb exchange (Fig. 3), internal Na (Fig. 4) was absolutely required. In contrast, the ion requirements of Na/Na exchange in high-Na cells were very different. Complete removal of external Rb abolished all bumetanide-sensitive cation uptake (Fig. 7). Addition of even a low concentration of Rb strongly accelerated Li uptake. External Rb activation of Na/Li exchange, represented by Delta Lic - Delta Rbc, saturated at 10 mM extracellular Rb concentration [Rb]o (Fig. 7).


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Fig. 7.   Activation of Na/Li exchange by external Rb. High-Na cells were incubated for 7 min in media containing (in mM) 0-15 Rb, 155 Li, and 150 Cl. Initially, cell water content was 1.36 ± 0.043 kg water/kg cell solid.

A conclusive test for the internal ion requirements of Na/Na exchange is experimentally problematic. Because both external Rb and Cl must be present externally for this mode to operate, their unavoidable entry into the cell during the period of observation complicates determination of internal ion requirements. To minimize this difficulty, we started at the beginning of the test period to monitor uptake continuously at the briefest possible intervals. High-Na cells were divided into three portions: one batch contained all three co-ions, another contained only Na and Cl, and in a third Cl was replaced by MSA. All cells were pretreated with DIDS to minimize Cl movement during the experiment. After norepinephrine activation, Na/22Na exchange was initiated by adding Rb, which is required for both cotransport and exchange in high-Na cells. The time course of uptake is shown in Fig. 8. Under the conditions of this experiment, bumetanide-sensitive Rb uptake represents inward Na-K-2Cl cotransport, whereas 22Na influx takes place by both cotransport and exchange. In Fig. 8, left, the external Rb dependence of Na/22Na exchange is again apparent. A similar pattern was observed in cells initially free of K (Fig. 8, middle), showing that Na/22Na exchange does not require that ion internally. For comparison, recall that K/Rb exchange is completely dependent on internal Na (Fig. 4). The result in Fig. 8, right, suggests that Na/22Na exchange can also take place in the absence of internal Cl. Although uptake of Rb and Cl rapidly increased their level in the cell, there was no indication that appearance of these ions accelerated uptake of either 22Na or Rb. In other experiments, we found that Na/22Na exchange absolutely requires external Cl and that the activation by this anion displays sigmoidal kinetics (25). Thus, although Na/22Na exchange clearly requires K and Cl externally, it has no need of them internally.


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Fig. 8.   Demonstration that 22Na/Na exchange does not require presence of K or Cl in cell. High-Na cells were prepared by nystatin preincubation technique to contain either 10 mM K (left) or <0.1 mM K (middle). Right: cell Cl was replaced with MSA. After treatment with DIDS, cells were incubated in a medium containing 75 mM 22Na and 146 mM Cl. After 1.2 min (arrows), 22Na/Na exchange was initiated by addition of Rb to a final concentration of 10 mM. After 3 min, net uptake via cotransport caused low-K cells to accumulate 2.1 mM Rb and low-Cl cells to accumulate 16.8 mM Cl.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

These experiments, and others previously published from this laboratory, demonstrate important characteristics of Na-K-2Cl cotransport. Incubated in their own plasma, duck red blood cells maintain steady states with respect to ions and water but rapidly lose volume in the presence of a beta -adrenergic blocker (16). They can swell or shrink in the presence of catecholamine, but only when physiological gradients are significantly perturbed (17). Thus beta -adrenergic hormones play a significant role in red blood cell volume maintenance in vivo by regulating the activity of Na-K-2Cl cotransport.

When the Na-K pump is inhibited by ouabain and the anion exchanger by DIDS in vitro, the observed stoichiometry of net movement through the pathway is 1Na:1K:2Cl, independent of the prevailing ratio of K to Na in the cytosol (18). In contrast, measurement of isotopic fluxes in normal high-K cells shows that K influx greatly exceeds Na influx (15, 17, 21, 27, 32, 38). However, measurement of unidirectional fluxes without regard to net movements can yield a misleading estimate of the true stoichiometry of cotransport.

Net cotransport shows an invariant stoichiometric ratio of 1Na:1K:2Cl, whether the three transportable ions are present on both sides of the membrane (Table 1) or only on one side (Fig. 3; Refs. 16, 17). Naturally, all three ions must be present ipsilaterally before net cotransport can occur (Fig. 3; Refs. 17, 37). Na-K-2Cl cotransport is electrically neutral in that it neither affects (22, 28) nor is affected by (17) the membrane potential. Consequently, the driving force that determines the magnitude and direction of net movements must be calculated from the chemical, rather than the electrochemical, potentials of all the transported ions (17). The observed stoichiometry is also confirmed by the hyperbolic dependence of external Na and K uptake and the sigmoidal dependence of Cl uptake (24, 31). Moreover, plots of activation by external Na, K, and Cl yield Hill coefficients close to 1.0, 1.0, and 2.0, respectively (24).

Although the stoichiometry of net cotransport is fixed, the ratio of unidirectional fluxes varies from 2K:1Na in high-K cells (Fig. 2) to almost 1K:5Na in high-Na cells (Fig. 5). Both influx and efflux show this pattern. The excess unidirectional flux of one cotransported cation over the other reveals the presence of a 1:1 obligate K/K exchange in high-K cells and Na/Na exchange in high-Na cells. The evidence for this conclusion is as follows. 1) If high-K cells are incubated in media containing Li and Rb in place of Na and K, the excess uptake of Rb over Li (Delta Rbc - Delta Lic) is equal to the excess loss of K over Na (Delta Kc - Delta Nac) under a variety of ionic conditions (Fig. 2). 2) In high-Na cells, the excess uptake of Li over Na (Delta Lic - Delta Rbc) is equal to the excess loss of Na over K (Delta Nac - Delta Kc), also under various ionic conditions (Fig. 6). 3) In the absence of any external transportable cations obligate exchange is eliminated, and all bumetanide-sensitive cation loss occurs via net Na-K-2Cl cotransport, regardless of which cation is predominant in the cell (Fig. 3; Refs. 17, 18).

The notion that catecholamine-activated, loop diuretic-sensitive, K/K (or K/Rb) exchange represents an intrinsic part of Na-K-2Cl cotransport in this cell was first proposed by Schmidt and McManus (38). They identified two components of Rb influx, only one of which required external Na. Based on this difference, two cation uptake mechanisms were proposed: Na-Rb cotransport (the [Na]o-dependent component) and K/Rb exchange (the [Na]o-independent component). Subsequent studies revealed the Cl dependence of K/Rb exchange (27) and details of its inhibition by bumetanide (15).

The concept that K/K exchange and Na/Na exchange are partial reactions of the Na-K-2Cl cycle rather than parallel modes of transport is based on several lines of evidence. 1) Both require ions not involved in the exchange, i.e., K/K exchange requires internal Na, and Na/Na exchange requires external Rb. 2) Both require Cl on at least one side of the membrane. 3) Although K/K exchange occurs in the absence of external Na, its kinetic characteristics are affected by that ion. For example, when normal high-K cells are incubated in the absence of external Na or Li, the apparent half maximum for activation of K/Rb exchange by external Rb is ~23 mM. Raising external Li to 100 mM reduces this number to 7 mM, close to the value for Rb activation of Na-K-2Cl cotransport (14). Also, raising internal Na markedly increases K/Rb exchange (Fig. 4), as well as its apparent affinity for external Rb. 4) K/K exchange, Na/Na exchange, and Na-K-2Cl cotransport are all equally sensitive to bumetanide (50% inhibitory concentration = 0.15 µM) (15, 31), but its inhibitory potency is enhanced by low concentrations of external Na, K, or Cl (14, 31). 5) Each mode (K/K exchange, Na/Na exchange, Na-K-2Cl cotransport) is equally stimulated by the same activating factors, such as catecholamine or cell shrinkage (27), and the time course of activation is similar (Figs. 1 and 5). 6) Na-K-2Cl cotransport and K/K exchange are equally temperature sensitive (24). Taken together, these results show that the exchanges are not only an intrinsic aspect of cotransport, but represent partial reactions of a cycle of ordered binding and debinding of the transported ions at the external and internal face of the membrane, as is discussed in A model of Na-K-2Cl cotransport.

Na-K-2Cl cotransport in other cells. Since the original demonstration of co-ion transport in the duck red blood cell (17, 36-38) and the Ehrlich ascites cell (12), it has been found to be widespread in animal cells (13). In most cases in which ion movements can be accurately estimated, such as the duck red blood cell and the Ehrlich cell, the stoichiometric ratio of net cotransport is 1Na:1K:2Cl. In human red blood cells, a 1Na:1K ratio has been reported for net mutually interdependent cation uptakes (20), as well as for net furosemide-sensitive cation losses (11). Estimates of cotransport stoichiometry in other cell types have had to rely on paired measurements of unidirectional ion fluxes, which show that K flux exceeds Na flux (human, rat, and bird red blood cell), that Na flux exceeds K flux (ferret red blood cell and squid axon), or that Na and K fluxes are similar (HeLa, Madin-Darby canine kidney, 3T3, and chicken embryo heart cells).

Discrepancies between net and unidirectional ion movements is a common feature of complex transporters, e.g., the Na-K pump (4) or the Na/Ca exchanger (19). Coupled transmembrane movement of multiple substrates necessarily involves a cycle of many steps. Uptake or loss by exchange rather than net transport can occur via partial reactions of the cycle. By definition, this phenomenon can only be detected by unidirectional fluxes. Our results show that two segments of the Na-K-2Cl cotransport cycle can perform obligate cation exchanges. The rates of both partial reactions are affected by internal and external substrate ions. Thus K/K exchange and Na/Na exchange contribute to unidirectional cation fluxes, depending on the concentrations of Na, K, and Cl on either side of the membrane. In duck red blood cells, and perhaps in other cells in which this phenomenon has been observed, the K/K and Na/Na exchanges are clearly responsible for the observed variation in unidirectional flux stoichiometries. In human red blood cells, the discrepancy between unidirectional and net furosemide-sensitive cation movements has been attributed to a K/K (or K/Rb) exchange operating in parallel with Na-K-2Cl cotransport (3, 5). This process appears to have all of the characteristics of the exchange we have observed in duck red blood cells. For example, it requires Cl and internal (but not external) Na and, in a Na-free medium, Rb increases K/Rb exchange and decreases outward Na-K-2Cl cotransport (5).

Most furosemide-sensitive K influx in human red blood cells can be attributed to either Na-K-2Cl cotransport (the [Na]o-dependent component) or K/K exchange (the [Na]o-independent component). Duhm (6) compared the relative rates of these 2 modes in cells from 18 donors. Although their sum varied threefold between individuals, K/K exchange represented a constant percentage (24.4 ± 2.5%) of the total flux. This shows that Na-K-2Cl cotransport and K/K exchange are closely linked, and the total level of expression in cells from different individuals may be controlled by other genetic or physiological factors. There also appear to be minor components of furosemide-sensitive cation transport in human red blood cells that cannot be attributed to Na-K-2Cl cotransport. For example, furosemide (but not bumetanide) inhibits a Na/Na exchange that occurs in media lacking K, as well as a portion of K efflux that occurs in cells lacking Na; unlike cotransport, these fluxes are stimulated when Cl is replaced with nitrate or when DIDS is added (3). Such findings show that furosemide is not a very specific inhibitor of Na-K-2Cl cotransport at doses (~1 mM) commonly employed in in vitro experiments (31).

In most cell types, the bumetanide-sensitive influx of K exceeds that of Na, probably owing to the presence of K/K exchange. However, this pattern is reversed in the ferret red blood cell, which exhibits an influx stoichiometry of 2Na:1K:3Cl (7, 18). The unusual influx stoichiometry in ferret red blood cells has been shown to result from the fact that these cells are normally high in Na (8, 24). Ferret red blood cells display Na/Na exchange with characteristics similar to what we have found in the high-Na duck red blood cell. When obligate exchange is rendered impossible by incubation in a Na-free medium and only net ion movements can occur, the stoichiometry of cotransport in the ferret is always 1Na:1K:2Cl (24).

Like the ferret red blood cell, the squid axon shows a stoichiometric ratio of 2Na:1K:3Cl (34, 35). However, this result does not appear to be attributable to cation exchange, because the influxes were measured into perfused axons lacking Na. Although their ratios appear to differ, cotransport in the squid axon and duck red blood cell nevertheless exhibit striking similarities. For example, both are equally sensitive to loop diuretics, greatly accelerated by removal of internal Cl, electrically neutral, dependent on internal ATP, volume sensitive, and operate bidirectionally (1, 34, 35).

A model of Na-K-2Cl cotransport. The results presented in this paper can be fit with a simple kinetic scheme (Fig. 9). Models with similar underlying assumptions have been developed to describe other types of transport (39). In this case, the cotransporter is depicted as a membrane-spanning protein with four ion binding sites arrayed in a pocket in such a way that they can load only in a strictly ordered sequence. Once the sites are fully occupied (E4), the transporter can oscillate between two major conformations, exposing it alternately to the intra- or extracellular medium (oE3left-right-arrow E4left-right-arrow iE3). The completely empty form can also undergo this transition (oE0left-right-arrow E0left-right-arrow iE0), a property dictated by the requirement for return of an empty carrier if net cotransport is to take place in either direction. However, to account for the phenomenon of cation exchange in this system, another requirement is that the empty carrier makes its transition at a much slower rate, as is discussed below. Although Fig. 9 is not intended to represent actual structure, the major conformational change of either form, fully loaded or empty, is depicted schematically as a jaw-closing or hinge-bending mechanism similar to that proposed for the sarcoplasmic reticulum Ca pump and other enzymes (40). Transition of partially loaded or electrically unbalanced intermediate forms appears to be forbidden. This feature is based on the fact that the pathway is electrically neutral, has an invariant stoichiometry, and shows no evidence of the independent movement of any other combination of the three ions.


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Fig. 9.   A model of Na-K-2Cl cotransport. Ten intermediate states of a membrane-spanning transporter or carrier are depicted. A specialized ion binding pocket is alternately exposed by a hinge type of mechanism to external (oE) and internal (iE) medium. Association of an ion with its site triggers a minor conformational change that occludes the bound ion and induces a new site to form with a selective affinity for the next ion in sequence. All binding is assumed to be at equilibrium. When all sites are occupied (E4), a major conformational change occurs, allowing bound ions to unload on opposite side of membrane in the order they were bound (first on, first off). When carrier is completely unloaded (E0), empty form can also undergo a major conformational change, allowing it to begin reloading ions at opposite side of membrane, thus facilitating net cotransport. This latter change, however, occurs at a much slower rate than when carrier is fully loaded. Partial unloading and reloading with return of fully loaded carrier can be detected by use of tracers or surrogate ions. Exchanges are a consequence of a kinetic barrier presented by rate-limiting movement of empty carrier. See text for further details.

A major feature of the model is that net cotransport in either direction can only occur after the ions bind in a fixed sequence. For the inward journey, they bind on the outside in the order Na, Cl, K, Cl. On the inside, they are released in the same order. This first-on, first-off behavior has been called glide symmetry (39). For the return journey, they bind in the reverse order (Cl, K, Cl, Na) and then are released in that order on the outside.

It is usually assumed in transport models that substrate binding achieves equilibrium and transitions between the intermediate forms are slower (39). Also, occupancy of a site in an ordered binding sequence induces formation of the subsequent site in the series. For example, in the initial loading of the empty carrier at the outer face of the membrane, binding of a Na in the pocket (oE0left-right-arrow oE1) promptly induces formation of a Cl site, and binding of that Cl (oE1left-right-arrow oE2) creates a site that can bind K, and so forth. Evidence that bumetanide inhibition of Na-K-2Cl cotransport results in occlusion of Na, K, and Cl (9) supports the idea that all four ions are occluded momentarily in a transitional state (E4) without access to either side of the membrane.

The requirement that the transition rate of the empty form (oE0left-right-arrow E0left-right-arrow iE0) must be slower than the fully loaded one (oE3left-right-arrow E4left-right-arrow iE3) means that the conformational change of the empty carrier is a kinetic barrier or rate-limiting step in the cycle. One consequence of this assumption is that obligate cation exchanges become possible. Partial reactions can occur in which a labeled or surrogate ion binds in the normal sequence on one side of the membrane, is transported via the fully loaded form to the other side, and then exchanges for an unlabeled congener, only to return rapidly the way it came. This is possible because the much slower transition rate of the empty carrier at the other end of the sequence limits the rate of net movement. Thus a full turn of the cycle does not occur, and all that is observed is an unproductive exchange of one kind of cation for another, e.g., a Rb for a K or a Li for a Na. If there were not a rate-limiting step in the cycle, cation exchanges would not be apparent and the mechanism would have only one mode of operation, net salt transport. If the empty and loaded forms undergo transition at equal rates and exchanges are minimized, more carriers become available for net cotransport. Indeed, in salt-transporting epithelia, such as the winter flounder intestine, Na-K-2Cl cotransport does not appear to have significant K/K exchange activity (30).

Study of these partial reactions has proved to be the key to the development of this model. Indeed, the entire sequence of binding and release can be deduced from the asymmetrical ion requirements of the exchanges. For example, K/K exchange is independent of external Na but requires external Cl. However, both ions must be present internally. Therefore, the partial reaction would have a fully loaded form generated on the inside cross the membrane, partially unload, exchange its K for an external K without unloading the Na, rebind the final Cl, and cross back to the inside. This implies that K binds externally distal to the Na site and just before the last Cl. Glide symmetry is shown by the fact that Na is required internally, indicating that it comes off before the K. On the other hand, Na/Na exchange does not require K or Cl internally, although both must be present in the medium, which again is consistent with the notion that Na is first off on the inside but last off on the outside.

Because both external and internal Cl sites are involved in the sequence, K/K exchange is actually an exchange of KCl for KCl, suggesting that an experimental test for Cl/Cl exchange should be informative. However, we find that, even after DIDS-sensitive anion exchange is >99% inhibited, the background flux of Cl is still too great to make such a test feasible. On the other hand, a readily testable prediction of the model is that, when internal Cl is held constant by DIDS and external Cl is varied, K/K exchange should show a hyperbolic dependence because only a single external Cl site is involved, whereas Na/Na exchange should show a sigmoidal dependence, reflecting the presence of two external Cl sites in the sequence. We have been able experimentally to confirm this prediction (Lytle and McManus, unpublished results).

In Fig. 10, the partial reactions of K/K and Na/Na exchange are highlighted graphically and the co-ion requirements are summarized. The predominance of one or the other of the two modes can be explained by the tendency of high internal K to drive the reaction by the principle of mass action from the point where it binds (iE1left-right-arrow iE2) to the point where it is released (oE3left-right-arrow oE2). On the other hand, high internal Na would tend to drive the reaction from where it binds (iE3left-right-arrow iE4) to its point of release (oE1left-right-arrow oE0). An important underlying feature of this phenomenon is the fact that physiologically all red blood cells have a high concentration of Cl that would facilitate the outward return of carriers in the exchange mode. In other types of cells in which Cl levels are much lower, however, the tendency would be for the carriers to complete the internal unloading process rather than participate in exchange, which may be another explanation of why it is difficult to demonstrate these exchanges in some epithelial systems (30).


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Fig. 10.   Using same scheme presented in Fig. 9, partial reactions responsible for K/K and Na/Na exchange are highlighted. A: K/K exchange (high K cells). B: Na/Na exchange (high Na cells).

Our demonstration that the cotransporter binds ions at the external face of the membrane in the order Na, Cl, K, Cl has been corroborated by kinetic studies on HeLa cells (29). Further support has also come recently from computer fits using experimental data from a number of studies of cotransporting cells (2). A series of simultaneous differential equations based on the scheme presented here were used in simulations of the reaction cycle that completely support the validity of our interpretation. This conceptually and kinetically simple model not only offers a plausible explanation for the hitherto unexplained discrepancy between unidirectional and net cotransport measurements but also provides the basis for more detailed studies of the structure, mechanism, and regulation of this complex and fascinating transport pathway.

    ACKNOWLEDGEMENTS

We particularly express our gratitude to Dr. W. D. Stein for an encouraging and supportive discussion of the model, as well as several substantive suggestions.

    FOOTNOTES

This study was supported by National Institutes of Health Grants GM-07171 and HL-28391, as well as North Carolina Heart Association Grants 1981-82-A-44, 1984-85-A-49, and 1986-87-A-26, the Commonwealth Fund, Sigma Xi, the Southern Medical Association, and the Walker P. Inman Fund.

M. Haas is the recipient of an Established Investigatorship Award from the American Heart Association.

Current address of M. Haas: Department of Pathology, The University of Chicago, Chicago, IL 60637.

Address for reprint requests: C. Lytle, Div. of Biomedical Sciences, University of California, Riverside, CA 92521.

Received 8 July 1997; accepted in final form 7 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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