Emory University School of Medicine, Atlanta, Georgia 30322-3110
Submitted 31 December 2002 ; accepted in final form 27 March 2003
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ABSTRACT |
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sodium/lithium exchange; sodium,lithium-phosphate cotransport; human erythrocytes; kinetic model
The remaining known pathway is the ouabain-insensitive, phloretin-sensitive Na/Na and Na/Li exchange pathway. Na on the trans side is known to stimulate Li transport toward the trans side, and Na on the cis side inhibits Li transport from the cis side of the membrane. Thus it is believed that the ouabain-insensitive Na/Li countertransporter mediates the uphill Li efflux by secondary active transport, using a molecular mechanism shared with the Na/Na exchange pathway and using the electrochemical gradient for Na into the cell (15). The kinetics of Na/Li countertransport have been quantitatively described by a single transport site with Michaelis coefficients for Na and Li on the two sides of the erythrocyte membrane (Table 1). However, no explanation has been offered to explain the cooperative increase of Liout-activated 22Na efflux seen at Li concentrations >90 mM (see Fig. 12 in Ref. 26).
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Na-PO4 cotransport in the human erythrocyte has been quite
separately studied (28). This
mechanism transports at least 1.7 Na ions per PO4 (valence unknown)
into the cell. The reported kinetic characteristics of this pathway are also
shown in Table 1. Recently, we
have shown that the Na-PO4 cotransport in erythrocytes and K562
cells, an erythroleukemic cell line, is the most likely product of the BNP1
gene (GenBank accession no. I59302
[GenBank]
), first cloned from brain
(30). We also showed that Na
activation of 32PO4 influx is cooperative
(K outNa = 46 mM when
PO4 out = 0.3 mM) with a Hill coefficient of 1.9,
consistent with the 2:1 stoichiometry of the cotransport
(30).
The molecular basis for the ouabain-insensitive Na/Li countertransport is not known. In this study, we measured Li effluxes and influxes, 32PO4 influxes, and 22Na effluxes in human red blood cells. The results provide kinetic evidence that the Na-PO4 cotransporter of human red blood cells is also the most likely mechanism for Na/Li countertransport. We have shown that Liout can substitute for Naout to activate both Li and 32PO4 influx, that PO4 out inhibits Liin/Naout exchange, and that at low concentrations where 1:1 exchange of cations occurs, Liout and Naout do not activate 32PO4 influx. We propose a model in which Na/Li exchange is mediated by the high-affinity cation site of the Na/Li-PO4 cotransporter without PO4 and Na/Li-PO4 cotransport is mediated by the same protein but with both high- and low-affinity cation sites loaded with Na or Li and the anion site loaded with PO4. Consequently, PO4, a substrate for the cotransporter, in a different mode could alter the rate of Na/Li countertransport and, consequently, the steady-state intracellular Li concentration in erythrocytes and neurons and could alter the therapeutic efficacy of Li.
A model is a framework for thinking about a set of data. The model we
propose for these data (Fig. 1)
is one with two pools of intrinsic membrane protein carriers (Co,
Ci) with transport-binding sites confined to either the outward
facing solution (Co) containing substrates (Na, Li, PO4)
or the inward facing solution (cytoplasmic; Ci) containing
differing concentrations of these substrates. Each of these two pools consists
of subpools of carriers, each with different degrees of loading of the
substrates: C (empty), Na-C, Li-C, Na-C-PO4,
Na2-C-PO4, Li2-C-PO4, or
Na,Li-C-PO4. The subpools on each side of the membrane are likely
to be in equilibrium with each other because the diffusion-limited
"on" rates of the substrates and the "off" rates (as
judged in conjunction with the apparent affinities, which are in the micro-
and millimolar range) will be rapid compared with the transport reaction rates
(reactions 14) that connect the outward and inward facing
pools of carriers. As proposed earlier
(26), the exchange transport
of only cations as either Na/Na, Li/Li, or Na/Li exchange in this model is
mediated by the carriers loaded with a single cation (reaction 2). In
this model there is none or very little transport (reaction 1) of the
empty carrier site, C, across the membrane compared with the rapid transport
rate of the singly loaded carrier forms. Consequently, there will be none or
very little net transport of the cations by the cycle of reactions
Co + Mo Mo - C
Mi -
C
Mi + Ci
Co + Mi
either from left to right or from right to left, where M is either one Na or
one Li. Rather, the first three of these reactions in this model may move
Mo to Mi and then take another Mi to
Mo and thus mediate exchange of Mo and Mi
(Na/Na, Li/Li, or Na/Li). The cotransport of Na and PO4 and the
cotransport of Li and PO4 are added to the exchanger model by
proposing an additional site for a cation (Na or Li) and a site for
PO4 (HPO42- or
H2PO4-) and proposing the additional slow
transport of Na2-C-PO4, Li2-C-PO4,
or Na,Li-C-PO4 when both cation sites and the PO4 site
are complexed with their substrate ions (reaction 4). The model
therefore has three transport reactions across the membrane: reaction
1, Co
Ci; reaction 2,
M-Co
M-Ci; and reaction
4,M2-Co-PO4
M2-Ci-PO4. The inclusion of reaction
3, M1-Co-PO4
M1-Ci-PO4, with a single cation with each
PO4 is for completeness, because it may occur in other cells.
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MATERIALS AND METHODS |
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Preparation of red blood cells. Blood was taken from one of us (R.
B. Gunn) and placed in a graduated cylinder that had been prerinsed with
heparin. The whole blood (30 ml) was then centrifuged at 12,000 g at
5°C for 10 min in a Sorvall RC-B5 centrifuge (DuPont Instruments-Sorvall,
Wilmington, DE). The blood plasma and the buffy coat were removed with the use
of an aspirator. The cells were washed three times in 30 ml of an
isosmotic (110 mM) MgCl2 solution. This involved adding the
ice-cold MgCl2 to the cells, stirring with a glass rod, and
centrifuging at 12,000 g for
1 min at 4°C and aspirating the
supernatant once again. This effectively results in fresh human red cells free
of plasma and nearly free of white cells at a 90% hematocrit in isotonic 110
mM MgCl2. In control experiments, no differences were found in the
32PO4 fluxes measured in fractions of blood cells that
were enriched in white cells and platelets or that were specifically depleted
in white cells and platelets. Thus all of the flux can be ascribed to the
erythrocytes.
The initial water and ion contents were measured after the washed cells were packed in nylon tubes (70 mm long, ID 3 mm) at 20,000 g for 10 min at 410°C. The extracellular space was previously shown to be 0.027 wt/wt under these conditions. The apparent percent water was then measured by drying a known weight of packed cells overnight at 95°C and weighing them once again. Either duplicate samples of packed cells were extracted with perchloric acid (PCA) or washed cells were lysed in distilled water and analyzed for ions.
Li influx measurements. Approximately 1 ml of washed (90% hematocrit) red cells was placed in 10 or 16 ml of media containing 10-4 M ouabain and differing concentrations of Li at 37°C, pH 7.40. Duplicate samples of the red cell suspension were removed at appropriate times, added to prelabeled 13-ml polystyrene test tubes (Starstedt, Newton, NC) containing 7 ml of ice-cold 110 mM MgCl2 to stop the influx reaction, and centrifuged at 3,000 g for 5 min. The supernatant was aspirated and discarded. The cell pellet was briefly vortexed, washed three more times in ice-cold isosmotic MgCl2, and finally lysed with 1.0 ml of distilled water. Each lysate was analyzed for hemoglobin (Hb) content and for Li (sometimes Na) using a model 3110 Perkin Elmer atomic absorption spectrometer in the emission mode. When Li and 32PO4 influxes were concurrently measured, aliquots of the supernatants of the PCA extracts (see PO4 influx measurements) were diluted (3- to 21-fold) with deionized, glass-distilled H2O and measured by atomic absorption using the Perkin Elmer HGA-600 furnace attachment to the atomic absorption spectrometer. PCA quenched the Li signal relative to the standards in H2O, so a PCA quench curve was prepared from measurements of a fixed Li concentration with increasing concentrations of PCA. The Li signals in diluted PCA extracts were corrected by using an apparent Ki = 0.386% PCA and the equation [Li] = Lisignal · (l + %PCAsample/0.386).
Li efflux measurements. Li loading of erythrocytes to 6.6 µmol Li/g Hb was achieved by incubating cells in a medium containing 45 mM LiCl, 105 mM NMDG, 5 mM D-glucose, and 20 mM HEPES, pH 7.40, for 2 h at 37°C. Li-loaded cells were placed in media of known different compositions, depending on the experiment in question, at 37°C, pH 7.40. Serial samples were taken into ice-cold test tubes at known time points and centrifuged in the cold at 3,000 g for 35 min. The supernatant was removed and analyzed for Li using the atomic absorption spectrometer.
PO4 influx measurements. One milliliter of washed, 90% hematocrit red cells was incubated in a solution containing radioactive phosphorous (32PO4) and specified concentrations of PO4 and cations at 37°C, pH 7.40, at a final hematocrit of 57%. Duplicate 1-ml samples of red cell suspension were removed, washed, and lysed using the same method as for Li influx measurements. Duplicate 50-µl samples of lysate were used to determine Hb (see Hb measurements). Lysate (500 µl) was then placed in 1.5-ml microcentrifuge tubes, and 250 µl of 7.2% PCA were added to denature the proteins. The tubes were vortexed and then centrifuged at 10,000 g for 23 min. The clear supernatant (350 µl) was then added to 3.0 ml of Optifluor cocktail in scintillation vials and counted in a Minmaxi Tri-carb 4000 series scintillation counter (United Technologies Packard, Downers Grove, IL). Each vial was counted for radioactivity for 10 min.
Specific activity measurements. Samples (1.4 ml) of the red cell suspension in 32PO4 were placed in labeled 1.5-ml microcentrifuge tubes and centrifuged for 23 min at 10,000 rpm. The supernatant (500 µl) was then transferred to another set of labeled microcentrifuge tubes, and 200 µl of 7% PCA were added to the tubes to denature the proteins. The tubes were vortexed and then centrifuged at 10,000 rpm for 23 min. The supernatant (10 µl) and 340 µl of distilled water were added to 3.0 ml of Optifluor cocktail in scintillation vials. Each vial was counted as before for radioactivity for 10 min together with the flux samples so that no correction for radioactive decay was needed.
Cation measurements. Li, Na, and K concentrations in solutions were determined using the atomic absorption spectrometer. The solution in question was diluted with deionized, glass-distilled water until the approximated cation concentration was <50 µM, and then the sample was measured against known standards at the following appropriate wavelengths: K at 766.5 nm, Na at 588 nm, and Li at 671 nm.
Hb measurements. Modified Drabkin's reagent (2 ml) (31) was added to 50 µl of cell lysate (a 41-fold dilution). This converts the Hb to the stable cyanomethemaglobin. The optical density of the mixture was measured using a Pharmacia LKB Novaspec II spectrometer at a 540-nm wavelength. The hemoglobin concentration of the 50-µl sample was calculated from the optical density (OD540) as OD540 · 1.465 · 41 = g Hb/liter lysate, which assumes a molecular extinction coefficient of 44 for the cyanomethemaglobin tetramer.
Extracellular PO4 measurements. Extracellular medium (100 µl) was removed and mixed with 100 µl of an oxidizing reagent and 1 ml of the dilution mix. The optical density of this solution was measured at 650 nm against standards prepared in the same manner. The dilution mix was composed of 100 ml of solution A, 33.3 ml of solution B, and 0.833 ml of solution C. Solution A was compounded of 47 mg of malachite green, 10 ml of 1 M HCl, and 90 ml of distilled water. Solution B was made up of 6.12 g of ammonium molybdate, 80 ml of 10 N HCl, and 120 ml of distilled water. Solution C consisted of 5 ml of distilled water and 0.1 ml of Tween 20 (7, 16).
Incubation solutions. Incubating solutions contained 20 mM HEPES buffer, 0.25 mM Na2- or choline2-DNDS, 5 mM dextrose, 0.1 mM ouabain, and varying LiCl, KCl, and NMDG concentrations always totaling 140 or 150 mM in a given experiment. This solution was titrated to a pH of 7.647.65 with 1 M KOH at room temperature to give a solution of pH 7.4 at 37°C at which the flux assays were performed.
Calculations and statistics. The values for optical density, ion concentration, specific activity, and sample radioactivity counts were entered in a Microsoft Excel spreadsheet. Initially, graphs were made of the tracer ion concentration inside the cells per gram of Hb as a function of time (Fig. 2). The calculated slopes of these graphs were the flux values in micromoles of ion transported per hour across cells with 1 kg of Hb. This amount of Hb corresponds to 2.8 x 1013 erythrocytes having 4.7 x 107 cm2 of surface area in this donor. The Li activation data were fit by a nonlinear least-squares procedure (Solver add-on to Excel; Microsoft, Redmond, WA) to the Hill equation: flux = a + Vmax · {b[Li]nH/(1 + b[Li]nH)}, where a is the constant background flux in the absence of Li, Vmax is the maximum flux when saturated by Li, b is the Hill constant, and nH is the Hill coefficient.
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The graphs contain error bars for the standard errors of the mean, but in most cases the graphical representation of the mean is quite larger than the error bars. Each experiment was performed at least twice with similar results.
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RESULTS |
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Because erythrocytes contain inorganic PO4 that might activate an unidentified 32PO4-PO4 exchange or because intracellular Na might be a necessary cofactor for 32PO4 influx, we measured Li activation of 32PO4 influx into Na-free and inorganic phosphate-free red cells prepared using the Nystatin technique (6). Although the 32PO4 flux into these cells (Fig. 4) was somewhat reduced compared with that in nondepleted cells (as reported previously in Ref. 28), external Li still activated the 32PO4 influx. The activation data have been fit with the Hill equation using nH = 1.58, but the fit of this sigmoidal line was not significantly better than the fit by a straight line as used in Fig. 3. As shown in Fig. 4, inset, 32PO4 influx was independent of extracellular Li concentration <20 mM. Additionally, when red cell metabolism was inhibited by pretreatment with 2 mM iodoacetamide for 30 min at 37°C, the 32PO4 influx was unaffected. Thus these data are similar to those for external Na activation of 32PO4 influx (28) in that neither internal Na, PO4, nor metabolism were required for the external Li activation of 32PO4 influx.
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To test the specificity of the alkali metals and other cations for activation of the 32PO4 influx, we incubated red blood cells in media containing 143 mM concentrations of LiCl, KCl, NaCl, NMDG-Cl, RbCl, CsCl, and choline chloride. Each medium also contained 0.3 mM PO4, 0.25 mM DNDS, and 5 mM D-glucose. As shown in Fig. 5, only Na and Li highly activated the 32PO4 influx. The maximum Li-activated 32PO4 influx was four- to fivefold higher than that in NMDG or K but was 1316% of the Na-activated influx. NH4Cl up to 50 mM added to NMDG-Cl medium did not activate 32PO4 influx (not shown).
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Li activation of Li influx. In the same cell samples, in concurrent measurements of Li and 32PO4 influx as a function of Liout, the Li influx increased hyperbolically (K1/2 = 9.1 mM) over 020 mM Liout, whereas the 32PO4 influx remained small and constant (Fig. 6). Thus the two fluxes appeared to be uncoordinated and independent of each other at low Li concentrations when a single site appeared sufficient to transport Li. Previously we showed that this Li influx was dependent on an equal efflux of Na (26). At 20 mM Liout and 1 mM PO4 out, the Liout/Nain exchange is 15- to 20-fold greater than the unstimulated 32PO4 influx.
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Na activation of 32PO4 influx. Shoemaker et al. (28) reported that the ratio of external PO4-activated 22Na influx to external Na-activated 32PO4 influx was 1.721.74 at pH 6.9, 7.4, and 7.75. Although this finding indicated that more than one Na was cotransported with PO4, the Na-activation curve of the 32PO4 influx was deemed hyperbolic (Hill coefficient: nH = 1) with no evidence of sigmoidicity. However, we recently reported that the Na activation of 32PO4 influx into both human erythrocytes and K562 cells, an erythroleukemic cell line, is sigmoidal with a Hill coefficient of 1.9 (30). The Naout-activation curve in Fig. 4 of Shoemaker et al. (28) shows that the flux at 40 mM Naout was half that measured at 140 mM Naout. This finding agrees with the K1/2 = 46 mM reported when sigmoidal activation was apparent (30). This sigmoidal activation by Naout combined with the stoichiometry of the fluxes demonstrated that two (or more) cation transport sites are on the external aspect of the Na-PO4 cotransporter. Because Liout also activates PO4 influx with some suggestion of cooperativity (Figs. 4 and 6), it seems reasonable that Na and Li compete for common activation sites on this transporter as they have been shown to on the Na/Li exchanger (23, 26).
Li activation of 32PO4 influx
in the presence of fixed Na concentrations. If Na and Li share a common
transporter as cosubstrates with PO4, there is no simple prediction
of how they should behave together. Although the Li-activated flux is only
1316% of the Na-activated flux at the same cation concentration, this
may be due to either a lower affinity for Li or a decreased transport rate for
the Li2-PO4 cotransporter complex. Because there are two
cation binding sites, Na/Li-PO4 cotransport may possibly occur. To
examine the relationship between Na and Li activation of
32PO4 influx, we measured the Li activation of the
32PO4 influx at three fixed Na concentrations (1, 10,
and 75 mM) by substituting the spectator cation, K, as shown in
Fig. 7. In general,
extracellular Li activated 32PO4 influx at each of the
Na concentrations and the increment of Li activation increased with an
increase in the Na concentration. Also, at any fixed Li concentration, the
addition of extracellular Na activated the 32PO4 flux
further, and the increment of Na activation increased with an increase in the
Li concentration. The increment of 32PO4 influx caused
by adding 70 mM external Li was greater in the presence of 75 mM Na (K
decreasing from 140 mM to 70 mM) (Fig.
7, 0.8 flux units per mM Li) than in the absence of Na (K
decreasing from 70 to 0 mM) (Fig.
3, 0.3 flux units per mM Li). Although the spectator cation, K,
was present at intermediate concentrations of Li, there was no K in the
solutions with 140 Li, 0 Na (Fig.
3), or 70 Li, 75 Na (Fig.
7). At 75 mM Li in the three data sets of
Fig. 7, K concentrations
decline 74 mM as one moves from the 1 mM Na line up to the 75 mM Na line.
Therefore, neither the decline in K nor the presence of other constant
components of the medium caused the enhanced Li-activated
32PO4 influx in the presence of Na. Thus both Na and Li
must be acting on the transporter at the same time to support
32PO4 influx.
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If Na and Li were activating 32PO4 influx on two independent transporters, the increment of flux by one cation would be independent of the concentration of the other cation. To test this hypothesis, we performed a separate set of experiments, one of which is shown in Table 2. We measured the activation of 32PO4 influx in 70 mM Na, 70 mM Li, or 70 Na and 70 Li using either K or NMDG as the spectator cation when needed to maintain osmolality and ionic strength. The 32PO4 influx in 70 mM Na plus 70 mM Li was 13 ± 3% greater than the sum of the 32PO4 flux in 70 mM Na and the 32PO4 flux in 70 mM Li when measured separately (Table 2). This result was independent of whether K or NMDG was the balancing cation. This coactivation was greater than that expected for two separate, parallel transport pathways.
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The coactivation in Fig. 7 and Table 2 shows that both Na and Li at intermediate concentrations mutually potentiate the 32PO4 flux. The additional activation by Li in the presence of Na suggested that both Na and Li can both be complexed with the carrier (C)-PO4 complex at the same time in a way to activate 32PO4 influx. Therefore, these results were consistent with at least two cation-binding sites on the cotransporter. Four possible mechanisms could explain the observed coactivation by Na and Li. First, the Na,Li-C-PO4 transport complex could have a higher affinity for PO4 than either the Na2-C-PO4 or Li2-C-PO4 complexes. Second, the Na,Li-C-PO4 complex could be more rapidly transported than either the Na2-C-PO4 or Li2-CPO4 complex. Third and fourth, the binding of one cation could greatly increase the affinity of the second cation site for only the other alkali metal cation with or without a higher affinity for PO4.
PO4 inhibition of Liout-activated ouabain-insensitive Na efflux. Because both the Na-PO4 cotransporter and Na/Na exchanger can utilize Li in the place of Na, they may have a common molecular basis. If the same transporter is used by both transport modes, then phosphate should interact with Na/Li exchanger as it does with the cotransport. Furthermore, because Na/Li and Na/Na exchangers are, in general, more rapid than cotransport, PO4 should inhibit the transport of Li and Na to the extent that it recruits transporters from carrying Na/Li exchange to carrying Na-PO4 or Li-PO4 cotransport.
Na efflux was measured in red blood cells preloaded with 22Na,
using the nigericin method
(26,
27), and incubated in a medium
containing 140 mM KCl and 10-5 mM ouabain
(Fig. 8). Li (30 mM) was then
added to the system. The added Li stimulated Na efflux by 32%. The joint
addition of 30 mM Li and 0.3 mM PO4 activated Na efflux, but not to
the extent of Li alone. PO4, therefore, inhibited Li-activated Na
efflux.
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PO4 inhibition of Li influx. Human
red blood cells were incubated in a 2 mM Li and 138 mM KCl solution, which
would be expected to activate Nain/Liout exchange to
57% of Vmax if
K
Li = 1.5 mM
(26). The addition of
K2HPO4/KH2PO4 ranging from 2 µM
to 0.32 mM inhibited Li uptake (Fig.
9). Under conditions where 2 mM Liout negligibly
activated PO4 influx (Figs.
3 and
4), PO4 inhibited
Nain/Liout exchange.
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PO4 inhibition of Li efflux. In vivo the active extrusion of Li from red cells is via Naout/Liin exchange. The experiments in Table 3 show that extracellular PO4 concentrations inhibited Li efflux only when Na was present in the extracellular solution. Thus only the Naout-activated portion of Li efflux was PO4 sensitive. Whereas an external cation was required for the measurement, it may not be required for external PO4 to bind to its external site. In contrast, phosphate increased Li efflux in the absence of extracellular sodium (<20 µM) and in the presence of either NMDG-Cl or KCl (not shown).
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Arsenate inhibition of Na-activated PO4 influx. Previous reports not withstanding (28), arsenate was found to inhibit Na-dependent PO4 influx in human red blood cells (Fig. 10). Assuming arsenate is a complete competitive inhibitor, Ki was 2.6 mM. In the absence of Li and Na or at low concentrations of Liout (<20 mM) or Naout (<10 mM; data not shown), the background PO4 influx (PO4 = 1 mM) of 3335 µmol PO4 · kg Hb-1 · h-1 was not inhibited by arsenate (Fig. 6).
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Arsenate inhibition of Li-activated PO4 influx. PO4 influx was measured in washed red cells incubated in media containing 125 mM LiCl, with and without 10 mM arsenate. The arsenate was found to inhibit 60% of Li-activated PO4 influx (Fig. 11). Similarly, 10 mM arsenate was found to inhibit Na-activated PO4 influx by 61% (Fig. 10). This agreement is in concert with the suggestion that both Li-activated and Na-activated PO4 fluxes are mediated by the same mechanism. The combination of phloretin (0.2 mM = 0.81 · Ki) and arsenate (10 mM = 3.8 · Ki) inhibited Li-activated PO4 influx by 89%, in agreement with a theoretical inhibition of 86% for these noncompetitive and competitive inhibitors in this system.
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Lack of arsenate inhibition of Na/Li exchange. When Liout concentration is <20 mM and 32PO4 influx is not activated, the Liout/Nain exchange was not inhibited by 5 mM arsenate (Fig. 6).
Phloretin inhibition. Phloretin, a noncompetitive inhibitor of carrier-mediated transport processes in erythrocytes and many other cells, has differential potencies for different transporters. The mechanism of phloretin's inhibition may be through its intercalation in the lipid bilayer and reduction of the intrinsic membrane dipole potential that results from the structured array of the phospholipid head groups (1, 12). This reduction in dipole potential then differentially alters the conformation of different transporters and their turnover rates. As shown in Fig. 12, the Li-activated and Na-activated 32PO4 influxes were inhibited by phloretin with the same apparent Ki (0.25 mM). These data are consistent with a common transport protein for Li-activated and Na-activated PO4 transport. Bumetanide (10 µM), an inhibitor of the Na-K-2Cl cotransporter, had no significant effect on the ouabain-insensitive, DNDS-insensitive PO4 influx (data not shown).
Phenomenological description of 32PO4 influx. The Hill equation used to fit the Naout dependence of the PO4 flux (30) is phenomenological and is not derived from first principles of chemical kinetics. However, the Michaelis-Menten equation is simply derived from a single site model of binding followed by transport or from more complex kinetic models that include the steps in the return of transporting conformations (13). The data for 32PO4 influx in this article can be approximately summarized by a phenomenological equation that combines the Hill equation to describe the Naout activation in the absence of Li (30) with a linear equation to describe Liout activation in the absence of Naout (Fig. 3), a term in Naout · Liout to describe the coactivation (Fig. 7), and a constant term for the background flux in the absence of both cations. This expression can be inserted into the Michaelis-Menten expression as an apparent maximal flux for extracellular PO4 activation by using K1/2 = 0.3 mM, which has been shown to be the same in Li or Na solutions. For fresh human erythrocytes in isoionic medium at 3637°C, pH 7.4, 20 mM HEPES, with ouabain and DNDS to block the Na pump and AE1, the 32PO4 influx (in µmol · kg Hb-1 · h-1) as a function of extracellular (mM) Na, Li, and total PO4 was described by 32PO4 influx = {[0.32[Na]o1.9/(1 + 0.00058[Na]o1.9)] + 0.64[Li]o + 0.012[Na]o[Li]o + 30} x [PO4]o/(0.3 + [PO4]o).
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DISCUSSION |
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Quite separately, all cells appear to have Na-PO4 cotransport into them that maintains cytoplasmic PO4 above its electrochemical equilibrium. The discovery that Li could substitute for Na on the erythrocyte isoform, but not on three of the other four known human isoforms (hPiT-2 has not been tested for Li transport), led us to determine that the erythrocyte isoform was probably hBNP1 (30), an isoform that is in neurons, glia, intestine, and testes (22), although other undiscovered isoforms that react with the hBNP1 antibody are not excluded. Clearly, hBNP1 could provide the missing link between erythrocyte Li transport and the neuronal effects of Li if hBNP1 is also the Na/Li exchanger. In this study, we have shown that Li-activated 32PO4 flux is mediated by the same mechanism as Na-activated 32PO4 flux. The evidence is that 1) both cations have similar cooperative activation of 32PO4 influx; 2) each cation potentiates the activation of PO4 flux by the other cation; and 3) the two activated PO4 fluxes have similar Ki values for arsenate and similar Ki values for phloretin. Second, in this study we have provided evidence that the Na/Li-PO4 cotransporter is the Na/Li exchanger by showing that PO4 concentrations in the range of the PO4 Km for cotransport is also an inhibitor of the Na/Li exchanger. Third, this article proposes a kinetic model to link these two transport functions to a single set of transmembrane transporters.
Na- or Li-activation of 32PO4
influx. Shoemaker et al.
(28) obtained PO4
influxes of 475 µmol · kg Hb-1 ·
h-1 in 140 mM NaCl and 1 mM external PO4,
compared with the 65 µmol · kg Hb-1 ·
h-1 fluxes we observed
(Fig. 3) in external media
containing 150 mM LiCl and 1 mM PO4. At these concentrations,
cations are 84% (Na) to 99% (Li) saturating the first or high-affinity site on
the Na/Li exchanger (26).
There is evidence for a second cation site on the cotransporter. Na activation
of 32PO4 influx into both K562 cells and human
erythrocytes is sigmoidal with Hill coefficients of 1.5 and 1.9, respectively
(30), and the ratio of
external PO4-activated 22Na influx to external
Na-activated 32PO4 is 1.72
(28). Thus at least two Na
ions are cotransported with a PO4. Li activation of
32PO4 flux is also sigmoidal, although this is not very
apparent in Figs. 3 and
4. The near absence of any Li
activation at low concentrations of Li is clearly shown in
Fig. 6 and
Fig. 4, inset.
Figure 6 shows that while Li
transport is increasing at low Li concentrations, it is unaccompanied by an
increase in 32PO4 transport. Only at higher Li (and Na)
concentrations is the cotransport activated. Therefore, at least two external
Li ions are needed to activate PO4 transport into the cell. The
second or low-affinity cation site is 50% saturated with Na at 46 mM
(K1/2) and with Li at 90100 mM.
Coactivation by Na and Li together. Coactivation by Li and Na was
shown in two kinds of experiments in Fig.
7 and Table 2. In
Fig. 7, Li activation not only
increased the 32PO4 influx at each fixed Na
concentration but increased it more at higher Na concentrations, even above
the KNa (46 mM; Ref.
30). At 75 mM
Naout, the 32PO4 influx increased 0.81 flux
units for each added millimolar concentration of Li. As shown in
Table 2, the
32PO4 flux into erythrocytes increased 0.46 [(182 -
150)/70] flux units per millimolar increase in Li concentration. Given the
scatter in the data at 75 mM Na in Fig.
7, these values are probably not different. Even the 0.46 value
from Table 2 exceeds the rate
of flux increase by Li at the two lower concentrations of Na shown in
Fig. 7. The data in
Table 2 quantitatively agree
with the data in Fig. 7 at the
two points of similarity; namely, the increment between 70 mM Na alone and 70
mM Na plus 70 mM Li was 4050 flux units in both, and the activation by
70 mM Li in the absence or near absence (1 mM) of Na was 1620 flux
units in both. The enhanced Li activation of 32PO4
influx caused by the addition of Na may be due to the different transport
rates of the different complexes with the carrier:
Na2-C-PO4, Li2-C-PO4, and
Na,Li-CPO4 (Fig. 1).
Figure 7 and
Table 3 seem to suggest that
Na,Li-C-PO4 is the most rapidly transported form, because at a
concentration of 75 mM Na, the first cation site should be 75% saturated and
Na should be more often bound than Li to the second cation site, yet Li
further activates the transport of PO4. One would anticipate that
because Li2-PO4 cotransport is only 1519% of the
rate of Na2-PO4 cotransport, the addition of Li to form
Li2-C-PO4 should only inhibit PO4 transport
by reducing the number of faster Na2-C-PO4 complexes.
However, the newly formed Na,Li-C-PO4 could enhance the
PO4 flux, as observed, if its transport rate were greater than that
of Na2-C-PO4. Together these data indicate that Na and
Li can both interact with the PO4 carrier at the same time. There
are at least two simple possibilities: Na and Li interact through competition
for the two cation transport sites, or Na and Li interact through modifier
(allosteric, regulatory) sites. The most parsimonious model is that of a
single cotransporter with two cation transport sites shared by the two
coactivating and two cotransported cations with the simple added assumption
that the heterogeneously cation-loaded transporter (Li,Na-C-PO4) is
more rapidly transported than either of the homogeneously cation-loaded
cotransporters (Na,Na-C-PO4 or Li,Li-C-PO4).
Arsenate inhibition. Arsenate is a structural homo-logue of PO4 and has been shown to inhibit Na-PO4 cotransport systems in renal brush border cells (17) and pig-kidney-derived LLC-PK cells (24). Arsenate, like PO4 and Cl, appears to be transported by AE1 and partially inhibits PO4 uptake in the absence of DNDS (28). We found that arsenate inhibited Na-activated PO4 influx with a Ki = 2.6 (Fig. 10). Arsenate also inhibited Li-activated PO4 influx (Fig. 11) with approximately the same potency. This suggests that the effect of arsenate on the Na/Li-PO4 cotransporter is independent of which cation (Na or Li) is activating the PO4 influx.
The arsenate inhibition of PO4 flux did not occur when Li- or
Na-activated PO4 flux (Liout 20 mM;
Fig. 4) was absent. Arsenate
also did not inhibit the Li-activated Li influx at low Liout
(Fig. 6). This suggests that
the arsenate inhibitory site is not present when the transporter is only
exchanging Liout for Nain but is present at higher
cation concentrations when Na2-PO4 or
Li2-PO4 cotransport occurs. If arsenate inhibition and
PO4 cotransport only occur at high cation concentrations, this
suggests that there is an ordered binding with PO4 (or arsenate)
last or, alternatively, the occupancy by Na or Li of both cation sites is a
prerequisite for the appearance of the anion binding/transport site. As
discussed below, this latter alternative is at variance with the
PO4 data if both PO4 and arsenate compete for a single
site.
PO4 inhibition of Na/Li exchange.
Three different aspects of the Na/Li exchange have been shown to be inhibited
by PO4: Liout-activated 22Na efflux
(Fig. 8), Li influx into normal
erythrocytes with Nain 25 mmol/kg Hb
(Fig. 9), and
Liin/Naout exchange
(Table 3). In these studies
extracellular PO4 concentrations in the range of the
Km (0.3 mM) for 32PO4 influx on the
Na/Li-PO4 cotransporter and in the range of normal plasma
PO4 concentrations (0.81.45 mM) inhibit the exchanger. In
Fig. 6, the
Km for Li influx is shifted from 1.5 mM
(26) in the absence of
PO4 to 9.1 mM in the presence of 1 mM PO4. This shift in
the apparent K
Li is consistent with a
Ki of 0.2 mM for PO4, which is not too
different from the Km = 0.3 mM for PO4
(28,
30).
These findings link the Na/Li exchange pathway to the extracellular PO4 concentration, which may be an important regulator of the exchange rate given that significant inhibition occurred at normal plasma PO4 concentrations. Previously noted changes in Na/Li exchange have been uncontrolled for plasma or intracellular PO4. The rate of Na/Li exchange increases during course of Li therapy (11, 19) and during the in vitro incubation of erythrocytes for several days with Li (8, 10). The rate of Na/Li-PO4 cotransport increases in K562 cells grown at lower PO4 concentrations (Toth A, Timmer RT, and Gunn RB, unpublished observations). The PO4 inhibition of Na/Li exchange indicates that there is a PO4-binding site on the exchanger with a Ki value in the range of the Km value for PO4 on the cotransporter. This may be coincidence but seems more likely to be the consequence of a single binding site on common pool of transporters that mediate Na-Li cotransport and Na/Li exchange. The inhibition of Na/Li exchange by PO4, especially that observed at low cation concentrations (Figs. 8 and 9), shows that PO4 inhibited under conditions when arsenate did not and shows that for PO4 binding, there is no prerequisite for two cations bound. Although arsenate inhibition could be modeled as a competitor of PO4, it may have more complex kinetics. It is, therefore, unclear from the present study why there are differences in PO4 and arsenate inhibition, and more particularly, it is unclear what the order of substrate addition is to the cotransporter except that the first cation can bind to the high-affinity site in the absence of PO4.
As a result of these experiments, we believe the Na-PO4 cotransporter mediates Na/Na exchange, Li/Li exchange, and Na/Li exchange. The transporter appears to be composed of three active sites. The first site has a high affinity for Li but can bind Na. The second is also a cation binding site able to bind either Na or Li, and the third accommodates PO4.
Model for Na/Li and Na/Na exchange on the Na/Li-PO4 cotransporter. The data from the separately studied Na/Na and Na/Li exchanger and the Na-PO4 cotransporter can be combined with the data of this study to formulate a model of Na/Li-PO4 cotransport with a Na/Na and Na/Li exchange mode (Fig. 1). This model can be quantitatively solved by using previously developed techniques (13). Na/Na or Na/Li exchange would be mediated by the transmembrane exchange of the singly loaded transporter through reaction 2, as indicated by arrow 2. After complexation with PO4 and a second cation, M (the order of additions is unknown, but one possibility is specified in Fig. 1), cotransport of 2 M+ and 1 PO4 would occur by reaction 4. The inhibition of the exchange reaction through arrow 2 by PO4 would be due to the mass action of the PO4 loading reaction. This reaction would increase the fraction of transporters in the M-C-PO4 and M2-C-PO4 conformations and thereby reduce the fraction in the M-C conformation and reduce the Mo/Mi exchange through reaction 2. The transport of the empty ion binding sites indicated by arrow 1 would allow net electrogenic Na and net electrogenic Li transport as well as net M2-CPO4 cotransport. The stoichiometry measurements for Na-PO4 cotransport (28) were all tracer measurements. Thus the extent to which the reaction through arrow 4 is accompanied by the return reaction through arrow 2 (1 net M ion transported per net PO4) or arrow 1 (2 net M ions transported per net PO4) is unknown. The relative rates are probably reaction 2 > reaction 4 > reaction 1 >> reaction 3 with relative rates of about 100:25:1:<0.1 in erythrocytes. Even under the special case of the heterogeneously loaded cotransporter shown on the right-hand side of Fig. 1 (where a Na and a Li are cotransported with the PO4), the relative rates would be the same as given above, but the heterogeneously loaded carrier in reaction 4 may be faster than reaction 4 when both cations are Na, which in turn appears to be faster than when both cations are Li. Reaction 3 is added for completeness. In erythrocytes there is no evidence for the coupling of one M with one PO4. Thus this reaction should be below the level of current experimental discrimination. However, in K562 cells there is suggestive evidence for the cotransport indicated by arrow 3 (see Fig. 6 in Ref. 30).
The equilibrium surface reactions on the outside can be crudely estimated
from the literature values
(26,
28).
Table 1 shows that the Na/Li
exchanger or the first cation site prefers Li by a factor of 17. The second
cation site has a low apparent affinity for Na [K1/2 = 46
mM for 32PO4 influx
(28,
30)] and an unknown but even
lower apparent affinity for Li, because the Li activation of
32PO4 fluxes are nearly linear up to 150 mM Li (Figs.
3 and
4). If only a single Li were
cotransported with PO4 on this common transporter, there should be
saturation of PO4 transport rate as a function of extracellular Li
activation with KLi out 1.5 mM. This was
not observed in erythrocytes. The activation by Li at higher concentrations
can be explained by requiring a Li bound to the second (low apparent affinity)
site, for the subsequent transport of PO4. The
Km for total external PO4 has only been
determined in nearly saturating Na and was 0.30 mM.
In summary, the previously published kinetics of Na-PO4 cotransport (28) and Na/Li exchange (26) as well as the data in this report can be modeled as resulting from the behavior of a single gene product in the erythrocyte membrane. This common transporter is most likely hBNP1, a Na-PO4 cotransporter also found in neurons and glia (30), or is a closely related but unknown protein. If so, serum or cytoplasmic PO4 concentrations could regulate Na/Li exchange and the intracellular activity of Li in a therapeutic setting.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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Present address of S. Elmariah: Department of Medicine, Hospital of the University of Pennsylvania, 3400 Spruce Street, 100 Centrex Building, Philadelphia, PA 19146.
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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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