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Coupled and uncoupled proton movement by amino acid transport system N

2001; Springer Nature; Volume: 20; Issue: 24 Linguagem: Inglês

10.1093/emboj/20.24.7041

1460-2075

Farrukh A. Chaudhry, David Križaj, H. Peter Larsson, Richard J. Reimer, Christopher Wreden, Jon Storm‐Mathisen, David R. Copenhagen, Michael P. Kavanaugh, Robert H. Edwards,

Molecular Sensors and Ion Detection

Article17 December 2001free access Coupled and uncoupled proton movement by amino acid transport system N Farrukh A Chaudhry Farrukh A Chaudhry Departments of Neurology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Search for more papers by this author David Krizaj David Krizaj Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Search for more papers by this author Peter Larsson Peter Larsson Departments of Vollum Institute, Oregon Health Sciences University, OR, USA Search for more papers by this author Richard J Reimer Richard J Reimer Departments of Neurology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Search for more papers by this author Christopher Wreden Christopher Wreden Departments of Neurology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Search for more papers by this author Jon Storm-Mathisen Jon Storm-Mathisen Department of Anatomy, University of Oslo, Oslo, Norway Search for more papers by this author David Copenhagen David Copenhagen Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Search for more papers by this author Michael Kavanaugh Michael Kavanaugh Departments of Vollum Institute, Oregon Health Sciences University, OR, USA Search for more papers by this author Robert H Edwards Corresponding Author Robert H Edwards Departments of Neurology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Search for more papers by this author Farrukh A Chaudhry Farrukh A Chaudhry Departments of Neurology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Search for more papers by this author David Krizaj David Krizaj Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Search for more papers by this author Peter Larsson Peter Larsson Departments of Vollum Institute, Oregon Health Sciences University, OR, USA Search for more papers by this author Richard J Reimer Richard J Reimer Departments of Neurology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Search for more papers by this author Christopher Wreden Christopher Wreden Departments of Neurology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Search for more papers by this author Jon Storm-Mathisen Jon Storm-Mathisen Department of Anatomy, University of Oslo, Oslo, Norway Search for more papers by this author David Copenhagen David Copenhagen Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Search for more papers by this author Michael Kavanaugh Michael Kavanaugh Departments of Vollum Institute, Oregon Health Sciences University, OR, USA Search for more papers by this author Robert H Edwards Corresponding Author Robert H Edwards Departments of Neurology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA Search for more papers by this author Author Information Farrukh A Chaudhry1,2, David Krizaj2, Peter Larsson3, Richard J Reimer1,2, Christopher Wreden1,2, Jon Storm-Mathisen4, David Copenhagen2, Michael Kavanaugh3 and Robert H Edwards 1,2 1Departments of Neurology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA 2Departments of Physiology, Graduate Programs in Neuroscience, Cell Biology and Biomedical Sciences, UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA, 94143-0435 USA 3Departments of Vollum Institute, Oregon Health Sciences University, OR, USA 4Department of Anatomy, University of Oslo, Oslo, Norway *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:7041-7051https://doi.org/10.1093/emboj/20.24.7041 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The system N transporter SN1 has been proposed to mediate the efflux of glutamine from cells required to sustain the urea cycle and the glutamine–glutamate cycle that regenerates glutamate and γ-aminobutyric acid (GABA) for synaptic release. We now show that SN1 also mediates an ionic conductance activated by glutamine, and this conductance is selective for H+. Although SN1 couples amino acid uptake to H+ exchange, the glutamine-gated H+ conductance is not stoichiometrically coupled to transport. Protons thus permeate SN1 both coupled to and uncoupled from amino acid flux, providing novel mechanisms to regulate the transfer of glutamine between cells. Introduction The amino acid glutamine plays a crucial role in nitrogen metabolism. In multicellular organisms, glutamine serves as the principal nitrogen carrier between cells. Glutamine alone constitutes one-fifth of all the amino acids present in plasma and, at ∼0.5 mM, two-thirds of those present in the cerebrospinal fluid (McGale et al., 1977) and brain extracellular space (Hamberger and Nystrom, 1984). Produced by one group of cells and taken up by another, glutamine contributes to multiple physiological processes. In the liver, the release of glutamine from perivenous hepatocytes and the uptake of glutamine by periportal hepatocytes participate in ammonia detoxification and the urea cycle (Haussinger, 1990). In the kidney, glutamine metabolism promotes the secretion of acid (Bender, 1975). Glutamine transfer between distinct cell populations also occurs in the nervous system. Glutamine participates in a cycle that regenerates amino acid transmitters released during synaptic transmission. Re-uptake by the nerve terminal serves to recycle most classical transmitters as well as to terminate their signalling. However, cloned transporters for the principal excitatory transmitter glutamate generally do not appear at the nerve terminal (Rothstein et al., 1994; Chaudhry et al., 1995), requiring distinct mechanisms to recycle glutamate. After exocytotic release, glutamate is taken up by glia through glutamate transporters (Kanner, 1994; Seal and Amara, 1999). Glutamine synthetase expressed in glia then converts the accumulated glutamate into glutamine (Rothstein and Tabakoff, 1984). After transfer back to neurons, glutamine serves as the direct precursor for glutamate (Hamberger et al., 1979; Thanki et al., 1983). Supporting the importance of this glutamine–glutamate cycle, inhibition of the neuronal enzyme that converts glutamine to glutamate drastically reduces glutamate stores (Conti and Minelli, 1994) and exogenous glutamine can sustain synaptic activity in the absence of glutamate transport and glutamine synthetase activity (Barnett et al., 2000). Inhibition of glutamine synthetase also reduces the levels of γ-aminobutyric acid (GABA) in the nerve terminal (Pow and Robinson, 1994; Laake et al., 1995), consistent with the biosynthesis of GABA from glutamate by glutamic acid decarboxylase. The glutamine–glutamate cycle thus contributes to inhibitory as well as excitatory neurotransmission. In contrast to the well-characterized transporters involved in glutamate uptake by astrocytes, the mechanisms involved in glutamine transfer from astrocytes to neurons remain less well understood. In previous work, we proposed that the system N transporter SN1 mediates the efflux of glutamine from astrocytes required for the glutamine–glutamate cycle (Chaudhry et al., 1999). SN1 exhibits several properties that suggest a role in glutamine efflux. First, as predicted from classical studies of system N (Kilberg et al., 1980), SN1 preferentially recognizes glutamine. Secondly, astrocytes express SN1 activity (Nagaraja and Brookes, 1996) and SN1 localizes to the plasma membrane of astrocytic processes (Chaudhry et al., 1999). Thirdly, SN1 mediates glutamine efflux under physiological conditions (Chaudhry et al., 1999). We have shown previously that SN1 transports neutral amino acid and Na+ in exchange for H+ (Chaudhry et al., 1999). We have also observed that the flux mediated by SN1 reverses close to the prevailing extracellular glutamine concentration in the brain (∼0.5 mM); above that concentration, SN1 mediates uptake and, below that concentration, efflux. Since intracellular glutamine is in the low millimolar range, SN1 can thus generate only a shallow concentration gradient of amino acid across the plasma membrane. To account for the shallowness of this gradient, we have suggested that transport by SN1 may be electroneutral. Electroneutrality would eliminate the role of membrane potential as a driving force for Na+-dependent uptake by SN1. Transport by SN1 would then be driven primarily by the Na+ gradient across the plasma membrane, predicting a concentration gradient (in>out) for glutamine that is shallower than if membrane potential were also involved. Supporting this possibility, depolarization did not appear to influence amino acid uptake by SN1 (Chaudhry et al., 1999). Despite the proposed electroneutrality of transport, SN1 generates currents in Xenopus oocytes (Fei et al., 2000). To reconcile this observation with the apparent electroneutrality of SN1-mediated transport and its reversibility at physiological concentrations of extracellular glutamine, we have now characterized the associated currents. We report that although amino acid and Na+ activate the currents mediated by SN1, the currents are not stoichiometrically coupled to amino acid flux. In addition, the uncoupled conductance is selective for H+. Since transport by SN1 couples to H+ exchange, the uncoupled H+ conductance provides a mechanism to influence the ionic gradients that drive transport by system N. Results Transport by SN1 activates uncoupled currents Since the association of currents with SN1 has suggested that transport by SN1 is not electroneutral, we have used two-electrode voltage clamp in Xenopus oocytes to characterize these currents further. As previously reported (Fei et al., 2000), the addition of system N substrates glutamine, asparagine and histidine (1 mM) to oocytes expressing SN1 generates inward currents (Figure 1A). Uninjected oocytes produce no detectable currents in response to these amino acids (Figure 1B). Glutamine also produces inward currents with a potency similar to the Km for uptake (1–2 mM) (Figure 1C) (Kilberg et al., 1980; Chaudhry et al., 1999). In addition, the inward currents depend on Na+, tolerate substitution of Na+ by Li+ and do not depend on chloride (Figure 1F–H), as previously reported for transport mediated by system N (Kilberg et al., 1980; Chaudhry et al., 1999). Further, low external pH (pHo), which was shown previously to reduce uptake due to the H+ exchange coupling mechanism, also inhibits the currents (Figure 1D). The currents thus exhibit properties very similar to substrate flux, raising the possibility that they reflect electrogenic transport. Indeed, the system A transporters SA1 and 2 that are closely related to SN1 mediate electrogenic rather than electroneutral transport (Reimer et al., 2000; Sugawara et al., 2000; Varoqui et al., 2000; Yao et al., 2000). However, closer examination shows that the currents differ in certain respects from transport by SN1. Asparagine activates currents similar in size to those produced by glutamine (Figure 1A and E), but we have found previously that the same concentration of asparagine (1 mM) causes much smaller increases in intracellular pH (pHi) than 1 mM glutamine (Chaudhry et al., 1999). Unlabelled asparagine also inhibits the uptake of [3H]glutamine by SN1 much less strongly than glutamine (Fei et al., 2000). The currents thus differ from flux in their activation by different amino acid substrates. Figure 1.SN1 exhibits currents in Xenopus oocytes. (A) Xenopus oocytes injected with SN1 cRNA and held at −50 mV show inward currents on addition of glutamine (Q), asparagine (N), histidine (H) and alanine (A), but not glutamate (E) (each 1 mM) at pH 8. (B) The same amino acids (1 mM) produce no currents in uninjected oocytes held at −50 mV. (C) Increasing concentrations of glutamine (in mM) at pH 8 cause progressively larger inward currents, with half-maximal currents obtained at ∼2 mM glutamine. (D) Extracellular pH (pHo) influences the currents produced by 1 mM glutamine at −50 mV, with a maximum at pHo 8. It also influences the currents produced by 1 mM asparagine, with large currents at pHo 8 and 7 and smaller currents at pHo 9 and 6 (data not shown). Glutamine, asparagine and histidine generate currents in both Na+- (E) and Li+- (F) containing solutions. Other amino acids produce much smaller currents. In addition to the standard code for amino acids, m = MeAIB, c = cystine, g = GABA and t = taurine. The error bars indicate the SEM. (G) Current–voltage relationship of currents produced by 1 mM glutamine. The currents do not tolerate the replacement of Na+ by choline. (H) Substitution of chloride by gluconate has no effect on the glutamine-induced currents. Download figure Download PowerPoint To assess the relationship between currents and amino acid transport mediated by SN1, we determined the ratio of charge movement to amino acid uptake. Since uninjected oocytes exhibit more background uptake of glutamine than asparagine (Taylor et al., 1989), we used [3H]asparagine as a substrate. At pHo 7.5, the largest inward charge movement occurs at −60 mV, and the smallest at +30 mV (Figure 2A). At pHo 8.5, the charge movement is small at all potentials and does not correspond to substrate influx (Figure 2A). Currents induced by asparagine thus differ from those produced by glutamine, which fall off more steeply below pHo 8 than above (Figure 1D). Figure 2C shows the wide variation in charge/flux ratio at pHo 7.5, from ∼2 at −60 mV to essentially zero at +30 mV. At pHo 8.5, the charge/flux ratios all fall close to zero, supporting a dissociation between the currents and transport. Alternatively, the uptake of [3H]asparagine may reflect exchange for cytoplasmic amino acid rather than net flux. The electroneutrality of exchange would then predict low charge/flux ratios. However, charge movement by the closely related system A transporter SA1 is tightly coupled to flux (Figure 2B). In addition, we observed that glutamine produces outward currents at positive potentials (Figure 1G). Figure 2.Variable relationship of charge movement to substrate flux mediated by SN1. (A) In oocytes expressing SN1, the charge movement produced over 10 min by 250 μM [3H]asparagine at pH 7.5 (filled symbols) increases at more negative holding potentials. Oocytes held at −60 mV are indicated by triangles, −10 mV by squares and +30 mV by circles. At pHo 8.5 (open symbols), oocytes expressing SN1 exhibit smaller currents at all holding potentials. The currents produced by asparagine thus differ from those produced by glutamine, which fall off more steeply below pHo 8 than above (see Figure 1D). At both pHos, the charge movement caused by asparagine does not correspond to uptake. (B) In contrast, oocytes expressing the related system A transporter SA1 show charge movement that correlates with uptake at −60 mV (triangles), −10 mV (squares) and +30 mV (circles). (C) At pHo 7.5, the charge/flux ratio drops from ∼2 at −60 mV, to ∼1 at −10 and to ∼0 at +30 mV. At pHo 8.5, the currents are all low despite substantial flux, yielding very low charge/flux ratios (∼0.1) at all potentials. Error bars represent the SEM. Download figure Download PowerPoint If amino acid uptake involves the inward movement of positive charge, the addition of amino acid to the outside of the cell should not promote outward charge movement, even at depolarizing potentials. Thus, outward currents produced by substrate also argue against charge movement coupled to flux. These observations raise the possibility that SN1 mediates an uncoupled current analogous to the anion-selective uncoupled current associated with glutamate transporters (Wadiche et al., 1995; Sonders and Amara, 1996). Gating and permeation by protons To characterize the uncoupled conductance associated with SN1, we sought to identify the permeant ions. Glutamine activates progressively larger inward currents at polarized potentials and outward currents at depolarized potentials (Figure 3A). The negative reversal potential observed for these currents raised the possibility that they were carried by chloride, but substitution of external chloride by gluconate had no effect on the reversal potential (Figure 1H). Since the reversal potential for H+ is −20 to −30 mV at pHo 8, we also investigated permeation by H+. A reduction in pHo from 8 to 7 has two striking effects on the currents (Figure 3A). First, it shifts the reversal potential by +51.7 ± 2.5 mV (n = 6), as predicted by the Nernst equation for a H+-selective channel. Indeed, we have used the reversal potentials observed at pHo 8 and 7 to calculate a pHi of ∼7.5, consistent with previous reports using Xenopus oocytes (Webb and Nuccitelli, 1981). Thus, SN1 has the properties of a channel selective for H+. Figure 3.Protons gate and permeate the uncoupled SN1 conductance. (A and B) The analysis of induced currents (currents in the presence of amino acid from which currents in the absence of amino acid have been subtracted) shows that at pHo 8 (A, left), increasing concentrations of glutamine cause progressively larger inward currents under hyperpolarized conditions in oocytes expressing SN1 (96 mM Na+). However, glutamine also produces increasing outward currents under depolarized conditions, suggestive of a channel rather than a transporter. At pHo 7 (A, right), currents induced by glutamine in the same oocyte are reduced in magnitude. In addition, the reversal potential shifts from approximately −25 to approximately +30 mV, mean difference +51.7 ± 2.5 mV (n = 6 oocytes), as predicted by the Nernst equation for a H+-selective channel. (B) At pHo 8 (left), increasing Na+ concentrations activate progressively larger inward currents at hyperpolarized potentials and outward currents at depolarized potentials in the presence of 1 mM glutamine. Lowering the pHo to 7 (right) again shifts the reversal potential of the same oocyte by approximately +50 mV and reduces the size of the currents. However, the changes in Na+ do not influence reversal potential at either pHo, indicating that the conductance is relatively selective for H+. The left and right panels of (A) and (B) were derived from single oocytes, enabling direct comparison of the results at different pHos. Similar results were obtained in 2–6 experiments. (C) The Km of SN1 for Na+ does not vary with membrane potential. Values around the reversal potential were discarded due to the small size of the currents. In contrast, currents coupled to transport by SA1 exhibit a Km for Na+ that increases progressively with depolarization. The values indicate the mean ± SEM, n = 4 for each. Download figure Download PowerPoint Secondly, H+ modulates gating of the SN1 conductance. Even though increasing external H+ concentrations (lowering pHo) should increase the size of inward H+ currents through the uncoupled conductance due to the increased driving force, low pHo reduces their magnitude (Figure 3A and B). However, the H+ exchange mechanism for SN1 predicts that just as amino acid and Na+ activate the currents associated with SN1, low pHo should reduce the conductance. The reduced currents at low pHo are thus consistent with gating of the conductance by transport. To determine whether other cations also permeate the conductance associated with SN1, we varied the concentration of Na+ (Figure 3B). Similarly to glutamine, increasing Na+ concentrations activate outward currents at depolarizing potentials as well as inward currents at hyperpolarizing potentials. Unlike the changes in pHo, however, changes in external Na+ do not alter the reversal potential (Figure 3B), indicating that Na+, although required for transport by SN1, is relatively impermeant through the uncoupled conductance. The increase in current produced by Na+ is therefore attributable to increased activation of the uncoupled conductance rather than to permeation by Na+. External amino acid substrates are also required to gate the H+ conductance associated with SN1 (Figure 3A). In the absence of external substrate, the reversal potential of oocytes expressing SN1 does not change from pHo 5 to 8 (data not shown). Similarly to glutamine, increasing concentrations of asparagine activate inward currents at negative potentials and outward currents at positive potentials (Figure 4A). A reduction in pHo from 8 to 7 also shifts the reversal potential by +58.3 ± 1.7 mV (n = 6), consistent with an H+-selective channel. Further, the currents produced by asparagine can exceed those produced by glutamine. Unlike the currents produced by glutamine, however, low pHo does not affect the size of currents due to asparagine at physiological Na+ concentrations (Figure 4A). Like glutamine, asparagine requires Na+ to produce the currents (Figure 4B) but the currents evoked by asparagine at pHo 8 saturate at very low concentrations of Na+ 120 mM cation (Li+) (Figure 5B) whereas the induced currents saturate at 20 mM) than glutamine (Km 0.96 ± 0.21 mM). Despite the lower apparent affinity for asparagine, the Vmax for asparagine (>>4 nmol/min) also exceeds that for glutamine (4.04 ± 0.74 nmol/min). Representative experiments are shown in (A–C), and the kinetic values in (A) and (B) derive from three independent experiments. (B) In contrast to the very high sensitivity of asparagine currents to Na+ (Figure 4B), the uptake of 1 mM [3H]asparagine saturates only at very high concentrations of Li+ (Km >120 mM). (C) External pH influences the Km of glutamine uptake for Li+. The Vmax does not differ substantially at the two pHos, suggesting that H+ competes with Li+ for activation of transport as well as with Na+ to activate currents (see Table I for kinetic values, the number of independent experiments and experiments at additional pHos). (D) Depolarization does not affect the uptake of [3H]glutamine (left) or [3H]asparagine (right). Using Li+ at 60 mM for all conditions and pHo 8, replacement of 60 mM choline by 60 mM K+ does not reduce glutamine transport, even in the presence of the K+ ionophore valinomycin to ensure depolarization. The black bars represent the PS120 cells expressing SN1 and the open bars untransfected PS120 cells. The values indicate the mean ± SEM, n = 6 for glutamine and n = 3 for asparagine. Download figure Download PowerPoint We used the transport assay to characterize the interaction of H+ with SN1. An increase in external H+ concentration (decrease in pHo) lowers slightly the Vmax produced by saturating glutamine, from 4.37 ± 0.50 nmol/min at pHo 8 to 3.12 ± 0.65 nmol/min at pHo 7.5 (n = 3), and increases the Km modestly, from 1.12 ± 0.30 mM at pHo 8 to 3.31 ± 0.74 mM at pHo 7.5 (n = 3). When uptake is measured as a function of cation concentration, however, we find that a reduction in pHo from 8.5 to 7.0 progressively increases the Km for cation ∼6-fold without changing the Vmax >2-fold (Figure 5C, Table I), supporting a competition between Na+ and H+ to activate transport by SN1. Table 1. External pH influences the dependence of glutamine transport by SN1 on the cation pHo Km (mM) Vmax (nmol/min) n 8.5 28 ± 12 5.6 ± 0.7 3 8.0 73 ± 7 6.1 ± 0.6 4 7.5 >120 (269 ± 39) >2 (2.5 ± 0.5) 4 7.0 >120 (171 ± 59) >2 (3.0 ± 0.7) 3 [3H]glutamine uptake was measured as a function of Li+ concentration in PS120 cells expressing SN1, and the results fitted to Michaelis–Menten kinetics. The increasing Km (for Li+) with little change in Vmax (for saturating Li+ in 1 mM [3H]glutamine) at progressively