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
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ABSTRACT |
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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
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INTRODUCTION |
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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
-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).
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METHODS |
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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
water1 · 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 is used to
represent the change in ion content over an interval during which the
change was proportional to time; hence,
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.
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RESULTS |
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The duck red blood cell activated by a -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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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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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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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 Lic
Rbc vs.
(
Nac
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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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
Lic
Rbc, saturated at 10 mM
extracellular Rb concentration
[Rb]o (Fig. 7).
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DISCUSSION |
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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 -adrenergic blocker (16). They can swell
or shrink in the presence of catecholamine, but only when physiological
gradients are significantly perturbed (17). Thus
-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 (Rbc
Lic) is equal to the excess
loss of K over Na (
Kc
Nac) under a variety of ionic
conditions (Fig. 2). 2) In high-Na
cells, the excess uptake of Li over Na
(
Lic
Rbc) is equal to the excess loss of Na over K (
Nac
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
(oE3E4
iE3).
The completely empty form can also undergo this transition (oE0
E0
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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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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1.
Altamirano, A. A.,
G. E. Breitwieser,
and
J. M. Russell.
Vanadate and fluoride effects on Na+-K+-Cl cotransport in squid giant axon.
Am. J. Physiol.
254 (Cell Physiol. 23):
C582-C586,
1988
2.
Benjamin, B. A.,
and
E. A. Johnson.
A quantitative description of the Na,K,2Cl cotransporter and its conformity with experimental data.
Am. J. Physiol.
273 (Renal Physiol. 42):
F473-F482,
1997
3.
Canessa, M.,
C. Brugnara,
D. Cusi,
and
D. C. Tosteson.
Modes of operation and variable stoichiometry of the furosemide-sensitive Na and K fluxes in human red cells.
J. Gen. Physiol.
87:
113-142,
1986[Abstract].
4.
Chapman, J. B.
A kinetic interpretation of "variable" stoichiometry for an electrogenic sodium pump obeying chemiosmotic principles.
J. Theor. Biol.
95:
665-678,
1982[Medline].
5.
Duhm, J.
Furosemide-sensitive K (Rb) transport in human erythrocytes: modes of operation, dependence on extracellular and intracellular Na, kinetics, pH dependency, and the effect of cell volume and N-ethylmaleimide.
J. Membr. Biol.
98:
15-32,
1987[Medline].
6.
Duhm, J.
Modes of furosemide-sensitive K (Rb) transport in human (and rat) erythrocytes: effects of [Na]o, [Na]c, [K]c, pH, cell volume, and NEM.
Federation Proc.
46:
2383-2385,
1987.
7.
Flatman, P. W.
Sodium and potassium transport in ferret red cells.
J. Physiol. (Lond.)
341:
545-557,
1983[Abstract].
8.
Flatman, P. W.
Stoichiometry of net sodium and potassium fluxes mediated by the Na-K-Cl cotransport system in ferret red cells.
Q. J. Exp. Physiol.
74:
939-941,
1989[Medline].
9.
Forbush, B.,
and
M. Haas.
Evidence for occlusion of anions and cations on the Na,K,Cl-cotransport system isolated from shark rectal gland (Abstract).
Biophys. J.
55:
422a,
1989.
10.
Funder, J.,
and
J. O. Wieth.
Determination of sodium, potassium, and water in human red blood cells: elimination of sources of error in the development of a flame photometric method.
Scand. J. Clin. Lab. Invest.
18:
151-166,
1966[Medline].
11.
Garay, R. P.,
N. Adragna,
M. Canessa,
and
D. Tosteson.
Outward sodium and potassium cotransport in human red cells.
J. Membr. Biol.
62:
169-174,
1981[Medline].
12.
Geck, P.,
C. Pietrzyk,
B.-C. Burckhardt,
B. Pfeiffer,
and
E. Heinz.
Electrically silent co-transport of Na, K, and Cl in Ehrlich cells.
Biochim. Biophys. Acta
600:
432-447,
1980[Medline].
13.
Haas, M.
The Na-K-Cl cotransporters.
Am. J. Physiol.
267 (Cell Physiol. 36):
C869-C885,
1994
14.
Haas, M.
Volume-Sensitive and Catecholamine-Stimulated Ion Transport Pathways in Duck Red Cells (PhD thesis). Durham, NC: Duke Univ., 1982.
15.
Haas, M.,
and
T. J. McManus.
Bumetanide inhibits [Na-K-2Cl] cotransport at a chloride site.
Am. J. Physiol.
245 (Cell Physiol. 14):
C235-C240,
1983[Abstract].
16.
Haas, M.,
and
T. J. McManus.
Effect of norepinephrine on swelling-induced potassium transport in duck red cells: evidence against a volume-regulatory decrease under physiological conditions.
J. Gen. Physiol.
85:
649-667,
1985[Abstract].
17.
Haas, M.,
W. F. Schmidt III,
and
T. J. McManus.
Catecholamine-stimulated ion transport in duck red cells. Gradient effects in electrically neutral [Na-K-2Cl] cotransport.
J. Gen. Physiol.
80:
125-147,
1982[Abstract].
18.
Hall, A. C.,
and
J. C. Ellory.
Measurement and stoichiometry of bumetanide-sensitive (2Na: 1K: 3Cl) cotransport in ferret red cells.
J. Membr. Biol.
85:
205-213,
1985[Medline].
19.
Johnson, E. A.,
and
J. M. Kootsey.
A minimal mechanism for Na/Ca exchange: net and unidirectional Ca fluxes as functions of ion composition and membrane potential.
J. Membr. Biol.
86:
167-187,
1985[Medline].
20.
Kracke, G. R.,
M. A. Anatra,
and
P. B. Dunham.
Asymmetry of Na-K-Cl cotransport in human erythrocytes.
Am. J. Physiol.
254 (Cell Physiol. 23):
C243-C250,
1988
21.
Kregenow, F. M.
An assessment of the cotransport hypothesis as it applies to the norepinephrine and hypertonic responses.
In: Osmotic and Volume Regulation, edited by C. B. Jorgensen,
and E. Skadhauge. Copenhagen: Munksgaard, 1978, p. 379-396.
22.
Kregenow, F. M.
Transport in avian red cells.
In: Membrane Transport in Red Cells, edited by J. C. Ellory,
and V. L. Lew. New York: Academic, 1977, p. 383-426.
23.
Lytle, C.
Activation of the avian erythrocyte Na-K-Cl cotransport protein by cell shrinkage, cAMP, fluoride, and calyculin-A involves phosphorylation at common sites.
J. Biol. Chem.
272:
15069-15077,
1997
24.
Lytle, C.
Na-K-2Cl and K-Cl Cotransport in Duck Red Cells (PhD thesis). Durham, NC: Duke Univ., 1988.
25.
Lytle, C.,
and
T. J. McManus.
A minimal model of Na-K-Cl cotransport with ordered binding and glide symmetry (Abstract).
J. Gen. Physiol.
88:
36a,
1986.
26.
McManus, T. J.
Na,K,2Cl cotransport: kinetics and mechanism.
Federation Proc.
46:
2378-2381,
1987.
27.
McManus, T. J.,
and
M. Haas.
Catecholamine stimulation of K/K (K/Rb) exchange in duck red cells (Abstract).
Federation Proc.
40:
484,
1981.
28.
McManus, T. J.,
M. Haas,
L. C. Starke,
and
C. Lytle.
The duck red cell model of volume-sensitive chloride-dependent cation transport.
Ann. NY Acad. Sci.
456:
183-186,
1985[Medline].
29.
Miyamoto, H.,
T. Ikehara,
H. Yamaguchi,
K. Hosokawa,
T. Yonezu,
and
T. Masuya.
Kinetic mechanism of Na+, K+, Cl-cotransport as studied by Rb+ influx into HeLa cells: effect of extracellular monovalent ions.
J. Membr. Biol.
92:
135-150,
1986[Medline].
30.
O'Grady, S. M.,
H. C. Palfrey,
and
M. Field.
Characteristics and functions of Na-K-Cl cotransport in epithelial tissues.
Am. J. Physiol.
253 (Cell Physiol. 22):
C177-C192,
1987
31.
Palfrey, H. C.,
P. W. Feit,
and
P. Greengard.
cAMP-stimulated cation cotransport in avian erythrocytes: inhibition by "loop" diuretics.
Am. J. Physiol.
238 (Cell Physiol. 7):
C139-C148,
1980[Abstract].
32.
Palfrey, H. C.,
and
P. Greengard.
Hormone-sensitive ion transport systems in erythrocytes as models for epithelial ion pathways.
Ann. NY Acad. Sci.
372:
291-308,
1981[Medline].
33.
Payne, J. A.,
C. Lytle,
and
T. J. McManus.
Osmotic and ionic equilibria in human red cells: effects of foreign anions.
Am. J. Physiol.
259 (Cell Physiol. 28):
C819-C827,
1990
34.
Russell, J. M.
Cation-coupled chloride influx in squid axon. Role of potassium and stoichiometry of the transport process.
J. Gen. Physiol.
81:
909-925,
1983[Abstract].
35.
Russell, J. M.
Na, K, Cl cotransport in squid axon.
Federation Proc.
46:
2390-2391,
1987.
36.
Schmidt, W. F., III,
and
T. J. McManus.
A furosemide-sensitive cotransport of Na plus K into duck red cells activated by hypertonicity or catecholamines (Abstract).
Federation Proc.
33:
1457,
1974.
37.
Schmidt, W. F., III,
and
T. J. McManus.
Ouabain-insensitive salt and water movement in duck red cells. I. Kinetics of cation transport under hypertonic conditions.
J. Gen. Physiol.
70:
59-79,
1977
38.
Schmidt, W. F., III,
and
T. J. McManus.
Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport.
J. Gen. Physiol.
70:
81-97,
1977
39.
Stein, W. D.
Transport and Diffusion Across Cell Membranes. Orlando, FL: Academic, 1986.
40.
Tanford, C.,
J. A. Reynolds,
and
E. A. Johnson.
Thermodynamic and kinetic cooperativity in ligand binding to multiple sites on a protein: Ca activation of an ATP-driven Ca pump.
Proc. Natl. Acad. Sci. USA
82:
4688-4692,
1985[Abstract].