Department of Biology, Syracuse University, Syracuse, New York
Submitted 12 November 2004 ; accepted in final form 14 March 2005
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ABSTRACT |
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Li+/H+ exchange; amiloride; Na+ substrate sites
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+.
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MATERIALS AND METHODS |
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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 5060% 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 15 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.
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RESULTS |
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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 K 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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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. K
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 K
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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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 (K = 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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DISCUSSION |
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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 K 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 K 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 K 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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GRANTS |
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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