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Report
Institut für Physiologie und Pathophysiologie und Interdisziplinäres Zentrum für Neurowissenschaften, Universität Heidelberg, D-69120 Heidelberg, Germany
Submitted 10 May 2003; accepted in final form 3 August 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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in the brain impair neuronal function. We studied the effects of on postsynaptic inhibition of cultured rat brain neurons using whole cell recording under nominally 
-free conditions. Application of shifted the reversal potentials for spontaneous inhibitory postsynaptic currents and currents elicited by dendritic GABA applications in a positive direction because [Cl–]i increased. The positive shift of the reversal potentials of GABA-induced Cl– currents was equal on equimolar elevation of 
or [K+]o, respectively. The -induced increase in [Cl–]i was reversed by an inhibitor of cation-anion cotransport, furosemide (0.1 mM), but not by bumetanide (0.01 mM) or by replacement of [Na+]o by Li+. We conclude that neuron-specific K-Cl cotransporter (KCC2) transports similar to K+. Despite this fact, the small increase of during metabolic encephalopathies will barely elevate [Cl–]i. However, an impairment of neuronal function may result because KCC2 provides a pathway to accumulate , and thereby, a continuous acid load to neurons. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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concentrations amount to 0.2 mM and can reach 0.5 mM during acute liver failure in humans (for review see Felipo and Butterworth 2002
). In experimental models of acute liver failure, comparable blood concentrations result in concentrations of 
5 mM in the rat brain (Swain et al. 1992). Hyperammonemia disrupts the glutamate-nitric-oxide cyclic GMP pathway (Hermenegildo et al. 1998), alters the phosphorylation of several proteins (Kosenko et al. 1994; Saez et al. 1999), suppresses oxidative metabolism (Jessy et al. 1991), and induces membrane depolarization (Raabe 1990). Furthermore, such concentrations impair synaptic transmission (for review see Szerb and Butterworth 1992). In crayfish neurons, induced a change in the reversal potentials of inhibitory postsynaptic potentials (IPSPs), which was related to an interaction with K-Cl cotransport (Aickin et al. 1982), but the underlying mechanism remained elusive. In cultured hippocampal neurons, long-term exposure induced [Cl–]i accumulation by upregulation of a 
exchanger (Irie et al. 1998).
, which constitutes more than 98% of ammonia at physiological pH, crosses many cell membranes via ion channels (Hille 1992; Marcaggi and Coles 2001) or membrane transporters (Bergeron et al. 2003; previously reviewed by Haas and Forbush 1998). Thus several pathways for uptake into glial cells are described, but a mechanism for its accumulation in neurons is not known (for review, see Marcaggi and Coles 2001). A neuron-specific K-Cl cotransporter (KCC2) (Payne et al. 1996) regulates intraneuronal Cl–. Here we show that KCC2 transports as well as K+. Uptake of through KCC2, therefore, could underlie its harmful effects on neurons.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Pregnant Wistar rats were anesthetized with ether and killed by decapitation. The removed embryos (E14) were placed in sterile ice-cold Gey's buffered salt solution containing (in mM) 137 NaCl, 5 KCl, 0.3 MgSO4, 1 NaH2PO4, 1.5 CaCl2, 2.7 NaHCO3, 0.2 KH2PO4, 1 MgCl2, and 5 glucose (pH 7.4) and immediately decapitated. Hippocampal or ventral midbrain tissue was mechanically dissociated and plated on a primary culture of glial cells from the corresponding area (see Jarolimek and Misgeld 1992). All experiments were approved by the Animal Care and Use Committees responsible for our institution and are in accordance with the European Communities Council directive regarding care and use of animals for experimental procedures.
Electrophysiological recordings
Whole cell voltage-clamp recordings on cultured hippocampal and midbrain neurons (40–80 days in culture) were performed (22–25°C) with an Axopatch 200 B (Axon Instruments, Union City, CA) patch-clamp amplifier. The composition of the extracellular solution was (in mM) 156 NaCl, 2 KCl, 2 CaCl2, 1 MgCl2, 15 glucose, and 10 HEPES, pH 7.3. When di- and monovalent ion concentrations were changed, osmolarity was compensated by adjusting the NaCl concentration. Addition of NH4Cl did not change the pH of the extracellular solution. Two types of pipette solutions were used to set the driving force for K-Cl cotransport as indicated in the results. High concentration of permeable anions contained ([hpA–]pip, in mM) 3.5 NaCl, 5 KCl, 130 K-glucuronate, 0.25 CaCl2, 0.5 MgCl2, 10 glucose, 10 HEPES, 5 EGTA, 5 5-N-(2,6-dimethylphenylcarbamoylmethyl)-triethylammonium bromide (QX 314-bromide), and 2 Mg-ATP, pH 7.3. Low concentration of permeable anions contained ([lpA–]pip, in mM) 140 K-glucuronate, 0.25 CaCl2, 10 glucose, 10 HEPES, 5 EGTA, 4 QX 314-bromide, and 2 Mg-ATP, pH 7.3, or when indicated, 4 QX 314-bromide was replaced by 4 KCl. NH4Cl (5 mM), when included in the pipette solution, replaced KCl. Solutions were applied by a multibarreled perfusion system allowing rapid exchange of the extracellular solution (5 s) around the neuron (for a more detailed description, see Jarolimek et al. 1999). All experiments were performed in the presence of 20 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 1 µM D-2-amino-4-methyl-5-phosphono-3-pentenoic acid (4-Me-Appa) or 10 µM 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP) to block fast glutamatergic synaptic currents. For GABA applications, 0.3 µM TTX was added. Drugs were from Sigma (Deisenhofen, Germany) except DNQX, TTX, and R-CPP (Tocris, Biotrend, Cologne, Germany).
Only recordings of cells with membrane potentials more negative than –40 mV and with access resistance <20 M
were accepted. All values were corrected for the liquid junction potential (–14 and –15.5 mV for [hpA–]pip and [lpA–]pip, respectively) (Jarolimek et al. 1999). Data were filtered at 1.3 kHz (4-pole Bessel filter) and acquired and analyzed with pCLAMP6 (Axon Instruments) and Igor Pro (WaveMetrics, Eugene, OR).
GABA (1 mM) was applied by pressure ejection (1–20 kPa; 20–40 ms) every 15 s from a pipette (<1 µm opening) to the dendrite of a neuron. The current (IGABA) was recorded at holding potentials (Vh) near its equilibrium potential (EGABA). Current amplitudes induced by two to three consecutive applications were averaged. The reversal potential of IGABA was determined by linear regression lines (r > 0.98, at least 3 points were used). Values are given as mean ± SE. Paired and unpaired Student's t-test was used (P < 0.05 was taken as significant difference).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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changes the reversal potential of inhibitory currents
Spontaneous synaptic currents were blocked by gabazine (10 µM, [hpA–]pip, 2 mM [K+]o, n = 8); therefore they were mediated by GABAA receptors (sIPSCs). Under our recording conditions, somatic [Cl–]i is set by the concentration in the recording pipette, while active Cl– outward transport lowers dendritic [Cl–]i. This Cl– gradient established by KCC2 leads to inward and outward sIPSCs at a certain range of holding potentials (Jarolimek et al. 1999). Adding 3 mM to 2 mM [K+]o or elevating [K+]o to 5 mM (Jarolimek et al. 1999) reduced amplitudes of outward and increased amplitudes of inward sIPSCs (n = 11; Fig. 1, A1 and A2). The effect of persisted on inclusion of 5 mM in the pipette solution, indicating that the transmembrane chemical potential of had no influence on Cl– uptake (n = 3). The frequency of sIPSCs was not altered by the addition of 3 mM . The effect of on sIPSCs was not due to intracellular pH changes (Thomas 1984), because neither out- nor inward sIPSCs (Fig. 1A3) were altered by trimethylamine (3 mM, n = 5). An elevation of divalent cations ([Mg2+]o, n = 6; [Ca2+]o, n = 4) by 3 mM also had no effect on the driving force of sIPSCs (Fig. 1B).
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or K+ induced the same changes in sIPSCs if added to 2 mM [K+]o. A comparable change in the driving force for sIPSCs would also result from blockade of K-Cl cotransport (Jarolimek et al. 1999). As indicated in METHODS, we used two types of pipette solutions to set the driving force for K-Cl cotransport, which is large if [Cl–]i is large compared with [Cl–]o x [K+]o/[K+]i. To exclude a blockade of K-Cl cotransport, we used [lpA–]pip and increased [K+]o to 5 mM to set the driving force for K-Cl cotransport near equilibrium. At a Vh at which outward sIPSCs prevailed (Fig. 2, A and Da), addition of 3 mM reversed the sIPSCs to inward currents (Fig. 2, B and Db). The cation-anion cotransport inhibitor furosemide (0.1 mM) re-reversed inward sIPSCs to outward sIPSCs (n = 7; Fig. 2, C, Dc and Dd). In 8 mM [K+]o, 3 mM enhanced (n = 6), but 0.1 mM furosemide reduced, inward sIPSCs (n = 5). Thus does not inhibit K-Cl cotransport but is cotransported with Cl–.
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KCC2 transports 
into cultured neurons
For a pharmacological characterization of uptake, we applied GABA focally to distal dendrites (Jarolimek et al. 1999; Kelsch et al. 2001). Adding 3 mM to 2 mM [K+]o as well as an elevation of [K+]o from 2 to 5 mM ([hpA–]pip) shifted EGABA in a positive direction (14.0 ± 1.6 and 14.0 ± 1.8 mV, respectively; n = 8; Fig. 3A). To reverse transport direction, we elevated to 8 mM recording with [lpA–]pip, which shifted EGABA (23.4 ± 1.7 mV, n = 11; Fig. 3, B and C) to the same degree as did adding 8 mM K+ (shift in EGABA, 23.6 ± 3.0 mV, n = 5, data not shown). Inhibition of cation-anion cotransport by 0.1 mM furosemide in 8 mM shifted EGABA back to a more negative value (–6.5 ± 0.6 mV, n = 11; Fig. 3, B and C). These data confirm that could replace [K+]o in its capability to reverse the direction of transport. To exclude that QX 314-bromide in the solution of the recording pipette (Jarolimek et al. 1999) had an influence on the uptake of , we replaced QX 314-bromide in the pipette solution by KCl. Neither EGABA in 2 mM [K+]o (–87.7 ± 2.1 mV, n = 11 vs. –88.6 ± 5.6 mV, n = 5, respectively) nor the shift in EGABA with 8 mM (22.2 ± 3.4 mV, n = 5; Fig. 3C) nor the inhibition of cation-anion cotransport with furosemide (0.1 mM, shift in EGABA –5.8 ± 0.8, n = 5; Fig. 3C) were different between recordings with or without Br–.
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may replace K+ at the binding/transporting site of NKCC1, a Na-K-2Cl cotransporter expressed ubiquitously (Delpire 2000). To test whether NKCC1 was involved in 
cotransport, we replaced most [Na+]o (143 of 156 mM) with Li+ ([lpA–]pip without Br–). Addition of 8 mM shifted EGABA by 21.1 ± 1.9 mV (n = 7), similar to control conditions (Fig. 3C). Furosemide (0.1 mM) shifted EGABA back to a more negative potential (–4.4 ± 1.2 mV, n = 5; Fig. 3C), indicating that was also able to reverse Cl– cotransport under a condition of reduced NKCC1 activity (Russell 2000). NKCC1 and KCC2 differ in their sensitivity to bumetanide and furosemide (IC50 of bumetanide = 0.1 vs. 55 µM; IC50 of furosemide = 3 vs. 25 µM, respectively) (Cabantchik and Greger 1992; Lauf 1984; Russell 2000). Therefore we compared the two drugs in their capacity to reduce 
Cl– transport. In 8 mM , 0.1 mM furosemide induced a significantly larger shift in EGABA than did 0.1 mM bumetanide (5.8 ± 1.0 vs. 3.0 ± 1.1 mV; n = 4, P < 0.05; Fig. 3D). Bumetanide (0.01 mM) had no effect (1.5 ± 0.9 mV, P = 0.18, n = 4; Fig. 3D). These results indicated that was transported by KCC2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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will result in an accumulation of 
by KCC2 with only a slight effect on [Cl–]i, and hence, the driving force for Cl–-dependent postsynaptic inhibition. Several studies report an impairment of inhibitory synaptic transmission by high either through altering transmitter release, altering the affinity of GABAA receptors, or elevating [Cl–]i (Basile 2002; Deisz and Lux 1982; Ha and Basile 1996; Irie et al. 1998). Our data show that KCC2 co-transports instead of K+ and thereby accumulates in neurons. This contributes to the difference between the moderate concentrations of found in plasma and cerebrospinal fluid (0.2–0.5 mM) and the rather high concentrations of (1–5 mM) in the brain during hyperammonemic encephalopathy (Swain et al. 1992; Szerb and Butterworth 1992). The higher sensitivity of the Cl– uptake to furosemide than to bumetanide and its [Na+]o independence indicated that flux through NKCC1 was not involved. The reduced sensitivity to of developing neurons (Audet and Butterworth 1998) may be explained by a delayed appearance of functional KCC2.
We measured uptake indirectly through changes in reversal potentials for GABAA receptor–mediated Cl– currents resulting from increases in [Cl–]i on elevation. In contrast to our acute experimental conditions in which we elevated to 3 or 8 mM, the increase in hyperammonemic encephalopathy is only minor (maximally to 0.5 mM). For the evaluation of changes in [Cl–]i in this situation, three possible pathways and concomitant equilibria for 
have to be considered: the permeability of the membrane for NH3, a passive conductance, and the transport by KCC2 (see Marcaggi and Coles 2001). Since NH3 is permeable across most cell membranes, it follows that
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is negligible in comparison to the pathway provided by KCC2. This allows us to calculate [Cl–]i at the equilibrium equation of KCC2 including and
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results in a re-decrease of [Cl–]i. As shown in this graph further, transport by KCC2 will result in a maintained intracellular acid load which may contribute to the harmful effects of on neuronal function. The degree of accumulation determines and will be determined by pHi, but the reduction of KCC2 activity by a reduced pHi (Bergeron et al. 2003) can slow down accumulation. In any case, only a small rise in [Cl–]i results from transport by KCC2 during hyperammonemia.
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DISCLOSURES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Present address of X. Liu: Department of Neurosurgery, Yale University, 333 Cedar Street, LSOG 228, New Haven, CT 06520-8082.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: U. Misgeld, Institut für Physiologie und Pathophysiologie, Universität Heidelberg Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany (E-mail: ulrich.misgeld{at}piol.uni-heidelberg.de).
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REFERENCES |
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), 5 mM [K+]o (
), and 2 mM K+ +3 mM 
). The positive shift in EGABA by 3 mM 
), EGABA recovered to the control value. C: intracellular Br– and extracellular Li+ had no influence on 
Cl– transport. There was no significant difference in the shift of EGABA induced by 8 mM 
[Cl–]i = 0.34 mM). In fact, the highest value for [Cl–]i would result if the pHi was identical to pHo, i.e., 
.