Na+-inhibitory sites of the Na+/H+ exchanger are Li+ substrate sites

Philip B. Dunham, Scott J. Kelley, Paul J. Logue, Michael J. Mutolo, and Mark A. Milanick

Department of Biology, Syracuse University, Syracuse, New York

Submitted 12 November 2004 ; accepted in final form 14 March 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Amiloride-inhibitable Li+ influx in dog red blood cells is mediated by the Na+/H+ exchanger, NHE. However, there are substantial differences between the properties of Li+ transport and Na+ transport through the NHE. Li+ influx is activated by cell shrinkage, and Na+ influx is not, as we reported previously (Dunham PB, Kelley SJ, and Logue PJ. Am J Physiol Cell Physiol 287: C336–C344, 2004). Li+ influx is a sigmoidal function of its concentration, and Na+ activation is linear at low Na+ concentrations. Li+ does not inhibit its own influx; in contrast, Na+ inhibits Na+ influx. Li+ prevents this inhibition by Na+. Na+ is a mixed or noncompetitive inhibitor of Li+ influx, implying that both a Na+ and a Li+ can be bound at the same time. In contrast, Li+ is a competitive inhibitor of Na+ influx, suggesting Li+ binding at one class of sites on the transporter. Because the properties of Li+ transport and Na+ transport are different, a simple explanation is that Na+ and Li+ are transported by separate sites. The similarities of the properties of Li+ transport and the inhibition of Na+ transport by Na+ suggest that Li+ is transported by the Na+-inhibitory sites.

Li+/H+ exchange; amiloride; Na+ substrate sites


THE NA+/H+ EXCHANGER (NHE) is a widespread class of transporters responsible for regulating intracellular pH and volume. Na+ is exchanged for protons in an obligatory, electroneutral fashion (see Ref. 16 for a recent review). Nine isoforms have been described, NHE1–NHE5 localized in surface membranes (10, 17, 19, 22, 24) and NHE6–NHE9 in Golgi and post-Golgi compartments (3, 4, 6, 14, 15). NHE1 is the most widely distributed isoform (19) and is responsible for the regulation of pH in most cells (23). In addition, Na+ influx through NHE coupled to Cl influx through the HCO3/Cl exchanger promotes osmotically obliged water influx and restoration of cell volume in response to cell shrinkage. NHE1 is the isoform of dog erythrocytes (1), which are the object of this study.

NHE1 is activated by intracellular acidification by H+ binding to allosteric sites, shifting the set point for activation of NHE to a higher pH (Ref. 2; for an alternative view, see Ref. 11). Cell shrinkage also promotes H+ binding to these sites (7). We recently proposed external allosteric sites for Na+ involved in the regulation of NHE (5). In isotonic medium, external Na+ (Na) inhibits NHE at these allosteric sites, the Na+-inhibitory sites, at external Na+ concentrations ([Na+]o) >40 mM. Osmotic cell shrinkage reduces the apparent affinity of these inhibitory sites for Nao+, thereby activating NHE. Thus the allosteric H+ and Na+ sites function in complementary modes. When the allosteric H+ sites are unoccupied, the NHE flux is low and occupation of these sites leads to activation of NHE (2), whereas when the allosteric Na+ sites are occupied, the NHE flux is low and release of Na+ from these sites (by shrinkage) activates NHE (5).

Among monovalent cations, Li+ is transported by the Na+/H+ exchanger, but K+, Rb+, and Cs+ are not (9). Studies of rabbit renal cortex vesicles showed competition between Li+ and Na+ for the exchanger (8, 12). In addition, there is noncompetitive interaction of Li+ with separate sites on the exchanger (8). In another study, the noncompetitive interaction of Li+ was not observed (12). The difference in results was attributed to different techniques used for measuring exchanger activity: an indirect method (8) and 22Na+ fluxes (12). There is additional recent evidence for separate external binding sites for Na+ and Li+ on NHE. In fibroblasts with NHE mutated in transmembrane region TM4 with a 10-fold reduced affinity for Na+, the affinity of NHE for Li+ was unchanged (21).

We measured Li+ influxes mediated by the exchanger, the effects of Li+ on Na+-promoted exchange, and the effects of Na+ on Li+ fluxes. We suggested previously that the Na+ substrate sites are volume insensitive and that there is one site per transporter (5). The Na+-inhibitory sites appeared to be volume sensitive in that cell shrinkage reduced their apparent affinity for Na+ and there was more than one inhibitory site per transporter. The activation of NHE by shrinkage appeared to be due at least in part to the relief of inhibition by Na+ (5). Herein we provide evidence for Li+ binding to the Na+-inhibitory sites; surprisingly, the Na+-inhibitory sites appear to serve as transport sites for Li+.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Red blood cells. Blood was drawn from the jugular vein into heparinized containers from beagles maintained at Marshall Farms USA (North Rose, NY). The protocol was approved by the Institutional Animal Care and Use Committees (IACUCs) of both Marshall Farms and Syracuse University. The cells were used on the first or second day after the blood had been drawn. The red cells were washed free of plasma and white cells by performing three brief successive centrifugations and resuspensions in an isotonic medium (290 mosmol/kgH2O measured using a vapor pressure osmometer) containing (in mM) 150 NaCl, 5 glucose, and 10 Tris·HCl, pH 7.4. Hypertonic media were made by adding sucrose to the isotonic media.

Na+ influxes. Unidirectional influxes of Na+ were measured using 22Na+ as a tracer (counted using a Tracor Analytic Gamma Counting System). Cells in 150 mM NaCl medium were packed using centrifugation to 50–60% hematocrit and then added to media appropriate for the experiments at ~1% hematocrit and incubated at 37°C for 5 min. Na+ concentrations in the media were varied by replacement with N-methyl-D-glucamine (NMDG). The cells were then spun briefly and resuspended in the same flux media at ~5% hematocrit; these suspensions were used to measure the fluxes over 10 min, and all measurements were performed in triplicate. Unidirectional Na+ influx is linear for at least 8 min (5). Na+/H+ exchange was taken as the amiloride-inhibitable Na+ influx (1 mM amiloride). Two amiloride derivatives, 5-(N,N-hexamethylene)-amiloride and 5-(N-ethyl-N-isopropyl)amiloride, specific inhibitors of Na+/H+ exchange (13), were used to confirm that amiloride-inhibitable Na+ influx in dog erythrocytes is Na+/H+ exchange (5). The methods used to calculate the influxes were slight modifications of earlier methods (18). Fluxes are expressed as mmol/l cells/h when measured in isotonic media calculated using the hemoglobin concentration of the lysates. Fluxes measured in shrunken cells were corrected to the original physiological cell volume using the hemoglobin concentrations of the lysates, and these fluxes are expressed as mmol/original l cells/h.

Li+ influxes. The Na+/H+ exchanger mediates Li+/H+ exchange that is inhibited by amiloride with the same sensitivity as Na+/H+ exchange (8). Li+ influxes were measured, calculated, and expressed in the same way as Na+ fluxes, except that the measurement of the flux was based on chemical analysis of Li+ by atomic absorption spectrometry. Cell samples of ~0.04 ml of cells were washed three times in isotonic NMDG-Cl and lysed in 5 ml of deionized water. Analysis of the samples was performed using a PerkinElmer Analyst 100 atomic absorption spectrometer in the emission mode. Standards of 1–5 µM Li+ were used, and cell lysates were diluted to bring their Li+ concentrations into this range. Li+ influx at its maximum in shrunken cells (390 mosmol/kgH2O) at 150 mM external Li+ concentration ([Li+]o) was linear for at least 20 min (results not shown). Most fluxes were measured for 10 min.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Li+ influxes through the exchanger: effect of cell shrinkage. Figure 1 shows amiloride-inhibitable Li+ influxes at external Li+ concentrations from 0.2 to 100 mM in dog red blood cells in isotonic and hypertonic media (290 and 390 mosmol/kgH2O, respectively). Shown are the means of two experiments at each osmolality. For clarity, the curve for isotonic media is offset slightly to the right. The mean values were fitted to the Hill equation using a nonlinear least-squares procedure (Sigma Plot 2001; SPSS, Chicago, IL). The mean kinetic constants ± asymptotic SE for isotonic media were K1/2 = 9.7 ± 2.0 mM, Jmax = 14.1 ± 0.3 mmol/l cells/h, and nH (Hill coefficient) = 1.25 ± 0.08, and the mean kinetic constants ± asymptotic SE for hypertonic media were K1/2 = 11.6 ± 1.0 mM, Jmax = 33.5 ± 1.2 mmol/original l/cells/h, and nH = 0.98 ± 0.06. Shrinkage stimulated Jmax more than twofold. The curve in isotonic media was sigmoid, and the curve in hypertonic media was hyperbolic. The K1/2 values were similar, or perhaps the same, for the two conditions. The two individual estimates of K1/2 were 10.1 ± 2.9 and 9.3 ± 1.6 mM for isotonic media and 9.2 ± 1.1 and 15.0 ± 1.2 mM for hypertonic media.



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Fig. 1. Amiloride-inhibitable Li+ influxes in dog red blood cells as function of external Li+ concentration ([Li+]o) in isotonic and hypertonic media (290 and 390 mosmol/kgH2O, respectively). Shown are means of two experiments for each osmolality. The data were fitted to sigmoid functions. For clarity, the curve for isotonic media is offset slightly to the right. For isotonic media, the kinetic constants were K1/2 = 9.7 ± 2.0 mM, Jmax = 14.1 ± 0.3 mmol/l cells/h, and Hill coefficient (nH) = 1.25 ± 0.08. For hypertonic media, the kinetic constants were K1/2 = 11.6 ± 1.0 mM, Jmax = 33.5 ± 1.2 mmol/orig. l cells/h, and nH = 0.98 ± 0.06.

 
These results illustrate three differences between amiloride-inhibitable Na+ and Li+ influxes. 1) In isotonic media, Li+ did not inhibit Li+ influx; in contrast, Na+ >40 mM inhibited Na+ influx (5). 2) Li+ influx was stimulated by cell shrinkage, whereas Na+ influx was volume insensitive (5). 3) In isotonic media, Li+ influx is a sigmoid function of [Li+]o and Na+ influx is not a sigmoid function of [Na+]o.

Na+ and Li+ influxes at low concentrations. Experiments were performed to confirm the last point. Fig. 2 shows amiloride-inhibitable Na+ influxes at external Na+ concentrations up to 1 mM in isotonic and hypertonic media. The Na+ influxes up to 1 mM [Na+]o fit straight lines, which is not surprising, because the K1/2 for Na+ influx is 63 mM as shown in Fig. 4. There is no suggestion of sigmoidicity, confirming an earlier conclusion of one Na+ substrate site per transporter (5). There was no effect of shrinkage on Na+ influx, confirming its insensitivity to cell volume change (5).



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Fig. 2. Amiloride-inhibitable Na+ influxes in dog red blood cells at external Na+ concentrations ([Na+]o) from 0.05 to 1.0 mM. Measurements were performed in isotonic (open symbols) and hypertonic (closed symbols) media (290 and 390 mosmol/kgH2O, respectively). For clarity, the symbols for hypertonic media are offset slightly to the right. For isotonic and hypertonic media, the slopes were 2.29 (R2 = 0.999) and 2.13 (R2 = 0.999), respectively, and the intercepts at 0 Na+ influx were –0.028 and +0.021, respectively. Means ± SD from one experiment performed in triplicate; similar results were obtained in three other experiments of the same design. In this figure and elsewhere, SD is not shown when smaller than the points.

 


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Fig. 4. Amiloride-inhibitable Li+ influxes in dog red blood cells in isotonic media with varying [Li+]o ± 50 mM [Na+]o. Kinetic constants from fits to hyperbolic functions were control (0 [Na+]o), K1/2 = 9.0 ± 0.9 mM, Jmax = 18.1 ± 0.5 mmol/l cells/h; 50 mM [Na+]o, K1/2 = 14.2 ± 1.5 mM, Jmax = 14.7 ± 0.5 mmol/l cells/h. Means ± SD from one experiment performed in triplicate; similar results were obtained in one other experiment of the same design.

 
The Li+ influxes (Fig. 3) were sigmoid functions of [Li+]o and were increased by cell shrinkage more than sixfold at 1 mM Li+ (Fig. 3A). In the experiments shown in Fig. 1, cell shrinkage activated Li+ influx 2.5-fold at 1 mM Li+, the variability among experiments. The curves were sigmoid at both osmolalities. Although this may not be obvious for isotonic media in Fig. 3A, it becomes so when the curves are normalized (Fig. 3B) by setting both fluxes at 1 mM Li+ at 1.0. The sigmoid curves for Li+ influx show that there is more than one Li+ site per transporter, presumably a transport site and an activating site. The initial slope was lower in hypertonic media than in isotonic media, i.e., the hypertonic response can be more sigmoid (Fig. 3) and yet have a Hill coefficient of 1 (Fig. 1), whereas the isotonic response has a larger Hill coefficient and is less sigmoid. This is partly because the Hill equation is an approximation; if one uses the more general equation (e.g., equation VII-6 in Ref. 20), one can easily find parameters that generate curves as shown in Fig. 1 and in which the hypertonic response is more sigmoid. The increased sigmoidicity shows that shrinkage reduces the affinity for Li+ at least at one site.



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Fig. 3. Amiloride-inhibitable Li+ influxes in dog red blood cells at [Na+]o from 0.05 to 1.0 mM in isotonic (open symbols) and hypertonic (closed symbols) media. A: fluxes in mmol/l cells/h (isotonic) or mmol/orig. l cells/h (hypertonic). B: same data normalized by setting fluxes at 1.0 in both isotonic and hypertonic media at 1.0 mM Li+. The lines connect the points. Data are means ± SD from one experiment conducted in triplicate. Similar results were obtained in three other experiments of the same design.

 
Li+ influx vs. [Li+]o with or without Na+. Li+ influxes were measured at [Li+]o from 5 to 100 ± 50 mM [Na+]o in isotonic media (Fig. 4). Both curves showed saturation kinetics and were fitted by hyperbolic functions. Na+ was a mixed inhibitor of Li+ transport: Na+ caused nearly a 60% increase in the K1/2 for Li+, from 9.0 ± 0.9 to 14.2 ± 1.5 mM, and a ~20% reduction in Jmax, from 18.1 ± 0.5 to 14.7 ± 0.5 mmol/l cells/h. The effect on K1/2 was threefold that on Jmax. Because inhibition was mixed, Na+ binds to a site other than the Li+ substrate site. Because Jmax changes less than K1/2, the inhibitor, Na+, binds better to its site when there is no Li+ bound to the Li+ substrate site.

Na+ influx vs. [Na+]o with or without Li+. Na+ influxes were measured in hypertonic media (isotonic media + 120 mM sucrose, 415 mosmol/kgH2O) from 5 to 100 [Na+]o ± 5 mM [Li+]o (Fig. 5). The high osmolality was used to obtain a hyperbolic control curve for Na+ influx. Na at lower osmolalities inhibits Na+/H+ exchange, producing nonhyperbolic curves (5). The curves, means from three experiments (0 [Li+]o) or four experiments (5 mM [Li+]o), were fitted to hyperbolic functions. Li+ was a competitive inhibitor of Na+ influx through the NHE. K1/2 was 63 ± 5 mM [Na+]o at 0 [Li+]o and 155 ± 6 [Na+]o mM at 5 mM [Li+]o, a 2.5-fold increase in K1/2 caused by Li+. There was no effect of Li+ on Jmax (153 ± 6 and 159 ± 9 mmol/original l cells/h at 0 and 5 mM [Li+]o, respectively). Therefore, Na+ is a mixed inhibitor of Li+/H+ exchange (Fig. 4), and Li+ is a competitive inhibitor of Na+/H+ exchange (Fig. 5).



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Fig. 5. Amiloride-inhibitable Na+ influxes in dog red blood cells in hypertonic media (isotonic + 120 mM sucrose, 415 mosmol/kgH2O) with varying [Na+]o ± 5 mM [Li+]o. Kinetic constants from fits to hyperbolic functions were, for 0 [Li+]o, K1/2 = 63 ± 5 mM and Jmax = 153 ± 6 mmol/original l cells/h. For 5 mM [Li+]o, K1/2 = 155 ± 6 and Jmax = 159 ± 9 mmol/original l cells/h. Means ± SD from one experiment performed in triplicate.

 
The experiment shown in Fig. 4, Li+ influxes ± 50 mM Na, was performed in isotonic media, and the experiments shown in Fig. 5, Na+ influxes ± 5 mM Li, were conducted in hypertonic media. The experiment shown in Fig. 4 was repeated in hypertonic media (415 mosmol/kgH2O) (results not shown) and produced the same results as shown in Fig. 4. Therefore, conclusions can be drawn by comparing the results shown in Figs. 4 and 5, despite the different osmolalities.

Na+ influx vs. [Na+]o with Li+. The effect of Li+ at 5 mM on amiloride-inhibitable Na+ transport in isotonic media was determined at Na+ concentrations from 5 to 145 mM (Fig. 6). The curve for the influx was fitted by a hyperbolic function (K1/2 = 49.2 ± 3.8 mM; Jmax = 30.9 ± 1.0 mmol/l cells/h). There was no indication that [Na+]o >40 mM inhibited Na+ influx as it did in the absence of Li+ (5). A possible explanation is that Li+ binds to the Na+-inhibitory sites (5) and prevents inhibition of Na+ influx by Na+. The effect of Li+ is more complex, however, because the Jmax of Na+ influx shown in Fig. 6, 30.9 mmol/l cells/h, is far less than the Jmax of Na+ influx in shrunken cells with no Li+, 161 mmol/original l cells/h (5). The latter was measured in hypertonic media, but it was concluded that cell shrinkage affected not Na+ influx but the inhibition of Na+ influx by Na+ (5).



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Fig. 6. Amiloride-inhibitable Na+ influxes in dog red blood cells in isotonic media with varying [Na+]o + 5 mM [Li+]o. Kinetic constants from a fit to a hyperbolic function were K1/2 = 49.2 ± 3.8 mM and Jmax = 30.9 ± 1.0 mmol/l cells/h. Means ± SD from one experiment done in triplicate.

 
Effect of Li+ on Na+ flux through the exchanger varying both [Na+]o and [Li+]o. The effects of varying [Li+]o on Na+ influxes at three Na+ concentrations, 5, 25, and 50 mM, were determined in isotonic media. Figure 7 shows Dixon plots (20) of the results. The three sets of points fitted straight lines and the lines were extrapolated to about the same point on the x-axis (–15.5 ± 2.6 mM ± SD), indicating noncompetitive inhibition. This x-axis intercept is –Ki, so the Ki for Li+ is 15.5 mM, not far from the K1/2 value estimated for Li+ in Fig. 1.



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Fig. 7. Dixon plot of amiloride-inhibitable Na+ influxes in dog red blood cells with [Na+]o fixed at 5, 25, or 50 mM in isotonic media with varying [Li+]o up to 100 mM. Means from one experiment conducted in triplicate.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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We report the transport of Li+ by the Na+/H+ exchanger of dog red blood cells. NHE mediates Li+ influx in several systems (8, 9, 12, 21). The new finding described herein is that Li+ does not appear to be transported by Na+ substrate sites; instead, Li+ appears to be transported by the Na+-inhibitory sites reported earlier at which Na+ inhibits Na+ influx through the NHE (5).

Different characteristics of Li+ and Na+ transport indicate that the two ions are not transported by the same sites. Li+ transport is stimulated by cell shrinkage (Fig. 1), Li+ does not inhibit its own transport (Figs. 1, 3), and Li+ transport is a sigmoid function of external Li+ concentration (Figs. 1, 3). In contrast, Na+ >40 mM inhibits Na+ influx (5), Na+ influx is not stimulated by cell shrinkage (Fig. 2; see Fig. 7 in Ref. 5), and Na+ influx is a hyperbolic function of [Na+]o (5) and is fitted by a straight line at low Na+ concentrations (Fig. 2). The common characteristics of Li+ transport and Na+ inhibition of Na+ transport which indicate that Li+ is transported by the Na+-inhibitory sites are 1) cell shrinkage reduced the affinity of at least one Na+ inhibitory site for Na+ (5) and of one Li+ site for Li+ (Figs. 1, 3); 2) cell shrinkage modified both Li+ transport (Figs. 1, 3) and Na+ inhibition of Na+ transport, but not Na+ transport (5); and 3) addition of Li+ to isotonic media abolished the inhibition of Na+ influx by Na+ (Fig. 5).

The evidence indicates that in isotonic media, there is more than one Li+ site per transporter (Figs. 1, 3), but only one can be a transport site. Li+/H+ exchange, like Na+/H+ exchange, exchanges one Li+ for one H+. The additional Li+ site (or sites) must be regulatory sites, and the regulatory sites are volume sensitive; their affinity for Li+ is reduced by cell shrinkage (Figs. 1, 3), just as the affinity of the Na+ inhibitory site for Na+ is reduced by shrinkage (5).

Li+ is a competitive inhibitor of Na+ exchange, indicating that Li+ binds to Na+ substrate sites (Fig. 5). Na+ is a mixed inhibitor of Li+/H+ exchange (Fig. 4). Therefore, Na+ inhibits Li+ transport at a site that Li+ does not bind. The effect of Na+ on the K1/2 for Li+ was greater than its effect on Jmax, so Na+ binds preferentially to the Li+ substrate site.

In two earlier studies of Li+ and NHE in the same system, microvillus membrane vesicles isolated from rabbit renal cortex, conflicting results were reported (8, 12). In one study, NHE activity was measured from quenching of acridine orange fluorescence as an indirect measure of the rate of H+ transport (8). In that study, as we found in the present study, Li+ was transported more slowly than Na+ and the K1/2 for Li+ was lower than that for Na+. In addition, the previous inhibition studies suggested to those authors (8) that there was a cation binding modifier/regulatory site on the exchanger in addition to the transport site, consistent with our present results. Li+ was a noncompetitive inhibitor of Na+/H+ exchange. The results suggested the possibility that the binding of Li+ to this noncompetitive site could account for the slower Li+ flux rate, and that is also a possibility in our studies.

In another study of the same system, NHE was measured from 22Na+ influxes (12). The results showed a single external site that bound both Na+ and Li+. The K1/2 for Na+ was ~11 mM and the Ki for Li+ was ~2 mM, consistent with the acridine orange-based study and with our study that Li+ binds with higher affinity than Na+. An important difference between the previous investigators' work (8) and ours is that Ives et al. (8) found that Li+ was not competitive, whereas our study and that of Mahnensmith and Aronson (12) found that Li+ competitively inhibited Na+ transport. Mahnensmith and Aronson found no evidence for interaction of Li+ with an additional site, and our conclusions differ from theirs in this respect. Mahnensmith and Aronson (12) attributed the earlier evidence for an additional Li+-binding site (8) to the indirect measure of NHE. There was no evidence for interaction of Li+ with an additional site. It was noted that amiloride can decrease the response of acridine orange after changes in a pH gradient in renal microvillus vesicles. How the evidence for a noncompetitive effect of Li+ could result from such an effect was not made clear. It is difficult to discount the results suggesting the additional Li+ sites.

A more recent study presented evidence for separate external binding sites for Na+ and Li+ on NHE. In Chinese hamster fibroblasts, a mutation in TM4 (Phe162Ser) of NHE caused a 10-fold reduction in the affinity of NHE for Na+ (21). The apparent affinity of NHE for Li+ did not seem to have been changed, which is good evidence for separate external binding sites for Na+ and Li+ on NHE.

In the studies of microvillus membrane vesicles and Chinese fibroblasts, there was no indication of inhibition of Na+ influx by high [Na+]o. The preparation of the vesicles from homogenized cells (8, 9, 12) may have eliminated the necessary regulatory system. The fibroblasts were a mutant cell line lacking NHE that subsequently were transfected with a vector containing NHE1 (21). Mutagenizing the cells may have eliminated regulatory systems as well as the transporter, and transfection may have occurred with the transporter alone.

The major observations of the present study of Li+ and NHE are that Li+ sigmoidally activated Li+/H+ exchange, that Li+ did not inhibit Li+ influx at high concentrations in isotonic medium (as Na+ inhibits Na+ influx; see Ref. 5), and that the interaction between Li+ and Na+ was volume dependent and complex. Several models can explain these results. We can unify these observations in a working model with these properties. 1) The Li+ transport site and the Na+ transport site are different sites. 2) Li+ binds to a transport site and a regulatory site. 3) Li+ transport is stimulated by cell shrinkage. 4) Li+ prevents Na+ binding to the Na+-inhibitory sites, which may be the Li+ transport sites.


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 ABSTRACT
 MATERIALS AND METHODS
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R37 DK-33640 (to P. B. Dunham) and R01 DK-37512 (to M. A. Milanick).


    ACKNOWLEDGMENTS
 
We are grateful to Dr. John M. Russell for a critical reading of the manuscript.

Present address of M. J. Mutolo: Forensic Biology, School of Criminal Justice, Michigan State University, East Lansing, MI 48824.

Present address of M. A. Milanick: Department of Medical Pharmacology and Physiology, University of Missouri-Columbia School of Medicine, Columbia, MO 65211.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. B. Dunham, Biological Research Laboratories, Syracuse University, 130 College Place, Syracuse, NY 13244-1220 (e-mail: pbdunham{at}syr.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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