Reversibility and Cation Selectivity of the K+-Clminus Cotransport in Rat Central Neurons

Yasuhiro Kakazu,1,2 Soko Uchida,1 Takashi Nakagawa,2 Norio Akaike,1 and Junichi Nabekura1

 1Cellular and System Physiology and  2Otorhinolaryngology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kakazu, Yasuhiro, Soko Uchida, Takashi Nakagawa, Norio Akaike, and Junichi Nabekura. Reversibility and Cation Selectivity of the K+-Clminus Cotransport in Rat Central Neurons. J. Neurophysiol. 84: 281-288, 2000. The reversibility and cation selectivity of the K+-Cl- cotransporter (KCC), which normally extrudes Cl- out of neurons, was investigated in dissociated lateral superior olive neurons of rats using the gramicidin perforated patch technique. Intracellular Cl- activity (alpha [Cl-]i) was maintained well below electrochemical equilibrium as determined from the extracellular Cl- activity and the holding potential, where the pipette and external solutions contained 150 mM K+ ([K+]pipette) and 5 mM K+ ([K+]o), respectively. Extracellular application of 1 mM furosemide or elevated [K+]o increased alpha [Cl-]i. When the pipette solution contained 150 mM Cs+ ([Cs+]pipette), alpha [Cl-]i increased to a value higher than the passive alpha [Cl-]i. An increase of alpha [Cl-]i with the [Cs+]pipette was not due to the simple blockade of net KCC by the intracellular Cs+ since alpha [Cl-]i, with the pipette solution containing 75 mM Cs+ and 75 mM K+, reached a value between those obtained using the [K+]pipette and the [Cs+]pipette. The higher-than-passive alpha [Cl-]i with the [Cs+]pipette was reduced by 1 mM furosemide, but not by 20 µM bumetanide or Na+-free external solution, indicating that the accumulation of [Cl-]i in the [Cs+]pipette was mediated by a KCC operating in a reversed mode rather than by Na+-dependent, bumetanide-sensitive mechanisms. Replacement of K+ in the pipette solution with either Li+ or Na+ mimicked the effect of Cs+ on alpha [Cl-]i. On the other hand, Rb+ mimicked K+ in the pipette solution. These results indicate that K+ and Rb+, but not Cs+, Li+, or Na+, can act as substrates of KCC in LSO neurons.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The K+-Cl- cotransporter (KCC) is one of the cation/chloride cotransporters (CCC) present in the plasma membrane. The KCC can carry K+ and Cl- in the same direction electroneutrally with a stoichiometry of 1:1 (for review, see Alvarez-Leefmans 1990; Kaila 1994; Lauf and Adragna 1996; Payne 1997). The thermodynamic driving force for net KCC is derived from the sum of the chemical potential differences for K+ and Cl-. Normally the KCC, driven in a net outward direction by the K+ gradient produced by Na+-K+ pump (Alvarez-Leefmans 1990), pumps Cl- out of the cell. The Cl- extrusion results in the maintenance of a steady state low intracellular Cl- concentration ([Cl-]i), thus producing a Cl- electrochemical gradient that favors Cl- influx into the cell through GABA- or glycine-operated Cl- channels. KCC2, a subtype of KCC, is expressed in the brain with an exclusively neuronal location (Payne et al. 1996; Rivera et al. 1999; Williams et al. 1999). This KCC2 most likely plays a major role in Cl- homeostasis in mature neurons (Kakazu et al. 1999; Payne 1997; Rivera et al. 1999; Thompson and Gähwiler 1989). Until now, the kinetics of CCCs have been evaluated by measuring radioactive Rb+ fluxes (86Rb+) instead of the physiologically relevant K+ fluxes (Gillen et al. 1996; Payne 1997). The detailed properties and kinetics of the KCC have been extensively studied by this method in red blood cells (Gibson et al. 1998; Kelley and Dunham 1996; Lauf and Adragna 1996). Although this flux technique is appropriate to examine the basic functions of the cotransporters in an aggregation of cells of one type, it is difficult to evaluate directly the ionic equilibrium potentials and to monitor the transport activities in a single neuron. KCC2 has also been suggested to function as a buffer carrying excess extracellular K+ ([K+]o) into the cell under conditions of elevated [K+]o (Payne 1997). To explain this bidirectionality of the net fluxes mediated by the KCC and its functional consequences in a single neuron, the direct demonstration of the reversibility of KCC net fluxes in native central neurons is necessary (Kaila 1997; Thompson and Gähwiler 1989).

In addition, it has been reported that EGABA and EIPSP recorded with Cs+ filled microelectrodes are more depolarized than those with K+ filled electrodes (Thompson and Gähwiler 1989), which also suggests that intracellular cations, such as Cs+, affect neuronal Cl- regulation (Kakazu et al. 1999). However, the effects of various intracellular cations on [Cl-]i regulation, i.e., CCC, have not been elucidated in detail in neurons.

In the present study, we employed the gramicidin perforated patch recording technique on acutely dissociated lateral superior olive (LSO) neurons to examine the mechanisms of [Cl-]i regulation. The gramicidin technique applied on LSO neurons is particularly well suited to study the KCC for two reasons. First, only monovalent cations, but not Cl- ions, are permeant through the gramicidin pore, thus allowing electrical recording without a direct influence of Cl- in the pipette solution on [Cl-]i (Akaike 1997). With the gramicidin technique, the effects of various monovalent cations perfused from the pipette solution on native [Cl-]i regulation could be investigated by observing the recovery of [Cl-]i after its alteration induced by the opening of glycine-operated Cl- channels. Second, KCC is well developed and plays a major role in [Cl-]i regulation in mature rat LSO neurons (Kakazu et al. 1999). In this paper, we demonstrate the reversibility of the KCC and its cation selectivity. Our results also strongly argue against using Cs+ as an internal cation in perforated patch experiments designed to examine neuronal Cl- regulation.


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METHODS
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Acutely dissociated LSO neurons

The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of our institution. The mechanical dissociation technique of CNS neurons was employed as described previously (Rhee et al. 1999). In brief, Wistar rats aged postnatal day 13-15 (P13-15, either sex) were decapitated under anesthesia with pentobarbitone sodium (i.p., 55 mg/kg), and 300 µm transverse slices of the brain including the LSO were prepared with a microslicer (VT-1000S, Leica, Nussloch, Germany). The slices were incubated for 1 to 2 h in the incubation solution containing (in mM): 124 NaCl, 5 KCl,1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 10 glucose, and 24 NaHCO3. The pH was 7.4 oxygenated with 95% O2-5% CO2 gas mixture at room temperature (22-26°C). Thereafter, the hemi-slice divided into the right and left LSO side was transferred into a Petri dish and a fire-polished glass pipette was lightly applied to the surface of the LSO identified under a binocular microscope (Nikon, SMZ-1, Japan). The pipette was vibrated horizontally at 5-10 Hz for about 2 min, and then the slice was removed. The mechanically dissociated LSO neurons, prepared without using any enzyme, adhered to the bottom of the dish within 30 min. Neuronal somata with intact morphological features, such as the proximal dendritic processes, were used in the present experiments.

Solutions

The standard external solution used for electrical recordings contained (in mM): 150 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES), 10 glucose. For Na+-free experiments, NaCl in the standard external solution was replaced with N-methyl-D-glucosamine (NMDG)-OH, which was first dissolved in water and titrated with HCl at pH 7.0 ± 0.1. The Cl- activities of the extracellular solutions (alpha [Cl-]o) measured with Cl--sensitive electrodes using a liquid ion exchanger (WPI IE-170, WPI) according to the method reported by Komune et al. (1993) were 114.5, 120, and 122 mM in the standard solution, 20 mM [K+]o and 30 mM [K+]o, respectively. Since alpha [Cl-]o in the Na+-free solution (94.2 mM) was different from that in the standard solution (114.5 mM), we prepared a modified standard solution containing (in mM): 110 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose, 80 mannitol (alpha [Cl-]o; 95.6 mM). The pH in all external solutions were adjusted to 7.4 with tris(hydroxymethyl)aminomethane (Tris)-OH. Osmolarities in all external solutions were confirmed to be similar (317-326 mOsm/kg) by osmometric measurements (OM802, Vogel, Germany). The patch pipette solution for gramicidin perforated patch recording contained (in mM): 150 KCl and 10 HEPES. For Cs+, Li+, Na+, and Rb+ containing pipette solutions, KCl in the pipette solution was simply replaced with an equimolar CsCl, LiCl, NaCl, and RbCl, respectively. All pipette solutions were buffered to pH 7.2 with Tris-OH. Gramicidin was first dissolved in methanol to prepare a stock solution of 10 mg/ml and then diluted to a final concentration of 100 µg/ml in the pipette solutions. The gramicidin-containing solution was prepared just before the experiment.

Electrophysiological measurements

Ionic currents and voltages were measured with a patch clamp amplifier (EPC-7, List-Electronic, Germany), low-pass filtered at 1 kHz (FV-665, NF Electronic Instruments, Japan), monitored on an oscilloscope (HS-5100A, Iwatsu, Japan), and recorded on both a pen recorder (Recti-Horiz-8K21, Nihondenki San-ei, Japan) and a digital FM tape recorder (RD-120TE, TEAC, Japan). In ramp experiments, 3 × 10-7 M tetrodotoxin (TTX) and 10-5 M LaCl3 were added to the extracellular solutions. To measure the reversal potential of the glycine induced current (Egly), ramp voltage steps from 0 to -100 mV of 2-s duration were applied before and during glycine by using a function generator (MacLab, AD Instruments, Australia). Data were also simultaneously collected using computer software (Scope v3.6, AD Instruments, Australia). Patch pipettes were constructed of glass capillary tubes with an outer diameter of 1.5 mm prepared using a vertical puller (PB-7, Narishige Scientific Instruments, Japan). The tip resistance of the electrodes was 4-8 MOmega . The junction potential between the patch pipettes and bath solution was nulled before giga ohm (GOmega ) seal formation. After establishing contact with the cell surface, a GOmega seal was established by applying gentle suction to the patch pipette interior. After the cell attached configuration had been attained, the patch pipette potential was held at -50 mV, and -10 mV hyperpolarizing step pulses of 300 ms duration were periodically delivered to monitor the access resistance. In the gramicidin perforated patch recording, the access resistance reached a steady level <30 MOmega within 40 min after making the GOmega seal. In the conventional whole-cell configuration after rupturing gramicidin perforated patch membrane by applying greater negative pressure to the pipette interior, the reversal potential of Egly moved to around 0 mV (+4.7 mV was obtained as ECl from the Nernst equation using the Cl- activity in the standard solution and 150 mM KCl pipette solution). Thus the difference in Egly values between gramicidin-perforated patch and conventional whole cell recordings is a good indicator for monitoring the stability of the recording configuration. All experiments were carried out at room temperature (22-26°C).

Drugs

Rapid "square-wave" change of external solution was performed with "Y-tube" method described previously (Nakagawa et al. 1990). Using this method, the external solution could be completely exchanged within 20 ms. Drugs used in the experiments were gramicidin D, bumetanide (Sigma Chemical, St. Louis, MO), glycine (Kanto, Tokyo), furosemide (Tokyo Kasei, Tokyo), and TTX (Sankyo, Tokyo). The final dimethyl sulfoxide (DMSO) concentration in the experiments did not exceed 0.1%.

Statistical analysis

Data were expressed as mean ± SE. Differences between groups were analyzed for statistical significance using the Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Regulation of intracellular Cl- by K+-Cl- cotransporter in LSO neuron

Under voltage clamp conditions at a holding potential (VH) of -50 mV, repetitive applications of 3 × 10-5 M glycine at an interval of 3 min elicited outward currents with constant amplitude in all LSO neurons examined. These experiments used a pipette filled with 150 mM K+ ([K+]pipette) and a standard extracellular solution containing 5 mM K+ ([K+]o, see Fig. 3A, bottom trace). Since the reversal potential of the glycine responses (Egly) can be taken to be ECl in our nominally bicarbonate-free in vitro system, the intracellular Cl- activity (alpha [Cl-]i) was directly calculated from the extracellular Cl- activity (alpha [Cl-]o, 114.5 mM) and Egly (ECl = 58 log alpha [Cl-]i, -119.4 mV). Thus to monitor the net activity of the neuronal Cl- regulatory system, which directly influences alpha [Cl-]i, we measured Egly in the present study. Assuming that no Cl- regulatory mechanism existed, an increase of alpha [Cl-]i by passive Cl- influx through glycine channels should degrade the electrochemical gradient. However, glycine at the intervals of 3 min induced a constant outward current and the cell alpha [Cl-]i stayed constant at 5.1 ± 0.2 to 11.2 ± 0.4 mM (n = 6, see Fig. 3B), significantly lower than passive alpha [Cl-]i (15.7 mM at a VH of -50 mV). Therefore, a functional Cl- extrusion mechanism must exist in the LSO neurons.

Effect of furosemide and bumetanide

In the presence of 1 mM furosemide, the outward glycine-induced current (Igly) gradually decreased in amplitude (Fig. 1A, top trace) and alpha [Cl-]i, determined from Egly (data not shown), gradually increased toward passive alpha [Cl-]i (Fig. 1B). Following washout of furosemide, the amplitude of Igly and alpha [Cl-]i recovered almost to those values before the application of the drug. The value of alpha [Cl-]i obtained at the third glycine application in furosemide (13.5 ± 1.8 mM) was significantly higher than that just before furosemide (9.3 ± 1.7 mM, n = 3, P < 0.05, paired t-test). Furosemide at the concentration employed here inhibits not only KCC, but also the Na+-K+-Cl- cotransporter (NKCC; Gillen et al. 1996), which drives Cl- inward and accumulates [Cl-]i (Kaila 1994). However, bumetanide, a potent blocker of NKCC (Gillen et al. 1996; Payne 1997), at a concentration of 20 µM did not alter the amplitude of outward Igly and alpha [Cl-]i (Fig. 1A, bottom trace, and Fig. 1C, n = 3). It is possible for the NKCC in the LSO neurons to have a slow rate constant for the inhibition and thus require more time. However, the outward Igly and alpha [Cl-]i were little changed even after about 25 min of incubation (Fig. 1C). These results suggest that the NKCC does not seem to be responsible for Cl- regulation in the LSO neurons.



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Fig. 1. Effects of furosemide and bumetamide on Igly and [Cl-]i in rat LSO neurons. A: in the presence of 1 mM furosemide (shaded bar), the amplitude of 3 × 10-5 M glycine (closed bars)-induced current (Igly) gradually became smaller at a holding potential (VH) of -50 mV (top). However, Igly was not affected by 20 µM bumetanide (dotted bar, bottom). Glycine was applied at an interval of 3 min. B: intracellular Cl- activity (alpha [Cl-]i) were plotted as a function of time before, during, and after the furosemide (closed circles). Dashed line indicates a passive alpha [Cl-]i (15.7 mM) calculated from VH (-50 mV) and active extracellular Cl- concentration (alpha [Cl-]o; 114.5 mM). Data are the mean ± SE of 3 LSO neurons at P13-15. *, significant difference in alpha [Cl-]i compared with those just before (-2 min) and during (7 min) the furosemide (P < 0.05, paired t-test). C: alpha [Cl-]i were plotted as a function of time just before and after the bumetamide for over 20 min (open diamonds). Data are the mean ± SE of 3 neurons.

[Cl-]i regulation by the K+ chemical gradient

An increase of extracellular K+ concentration ([K+]o) from 5 to 20 mM gradually reduced the amplitude of outward Igly (Fig. 2A, top trace) and increased alpha [Cl-]i close to the calculated passive alpha [Cl-]i at a VH of -50 mV (Fig. 2B, filled circles with asterisk; 15.6 ± 0.4 mM, n = 4). After the return of [K+]o from 20 to 5 mM, Igly and alpha [Cl-]i gradually recovered to the level prior to 20 mM [K+]o. The ideal value of alpha [Cl-]i in 20 mM [K+]o from the chemical potentials alone (see DISCUSSION) is 16 mM, similar to the passive alpha [Cl-]i. Then, we next tried 30 mM [K+]o to clarify whether the actual alpha [Cl-]i was determined by VH or merely by the chemical potentials. A change of [K+]o from 5 to 30 mM reversed the polarity of Igly from outward to inward (Fig. 2A, bottom trace); the actual alpha [Cl-]i attained a higher value (19.0 ± 1.3 mM, n = 3) than the passive alpha [Cl-]i (Fig. 2B, open squares). For experiments in which the [K+]pipette was used, [K+]o increased alpha [Cl-]i in a concentration-dependent manner (Fig. 2C). These results indicate that [K+]o-dependent [Cl-]i regulation plays a major role in Cl- extrusion in LSO neurons. Together with the results from the furosemide and bumetanide experiments, we conclude that the KCC is a likely candidate for the mediation of Cl- extrusion and the maintenance of an alpha [Cl-]i lower than the passive alpha [Cl-]i in the LSO neurons.



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Fig. 2. Effect of [K+]o on Igly and [Cl-]i in the [K+]pipette A: an increase of extracellular K+ concentration ([K+]o) from 5 to 20 (top) and 30 mM (bottom) induced an abrupt inward shift of the base current level. Twenty millimeters of [K+]o gradually decreased the amplitude of outward Igly. In the case of 30 mM [K+]o, the polarity of Igly was reversed to be inward. In both cases, the baseline current levels and outward Iglys recovered after the return of [K+]o to 5 mM. B: alpha [Cl-]i was plotted as a function of time before, during and after 20 mM (closed circles) and 30 mM [K+]o (open square). Note that an increase of [K+]o to 30 mM increased alpha [Cl-]i exceeding a passive alpha [Cl-]i at a VH of -50 mV (dashed line). Data are the mean ± SE of 4 and 3 neurons in the 20 and 30 mM [K+]o, respectively. *, **, significant difference between alpha [Cl-]is just before (-1 min) and during (11 min) the 20 and 30 mM [K+]o (P < 0.01, paired t-test). C: alpha [Cl-]i was plotted as a function of [K+]o. VH was -50 mV and pipette solution contained 150 mM K+.

To examine the effect of intracellular K+ concentration ([K+]i) on [Cl-]i regulation, the 150 mM K+ in the [K+]pipette was replaced with 150 mM Cs+ ([Cs+]pipette). Just after the access resistance dropped to <30 MOmega and became stable, as confirmed by rectangular pulses from -50 to -60 mV of 300 ms duration, we started to measure Igly and Egly. For the [K+]pipette, the amplitude of outward Igly at a VH of -50 mV was almost constant throughout the experiment (Fig. 3A, bottom trace, n = 6). On the other hand, the amplitude of outward Igly gradually decreased when recording with the [Cs+]pipette, and finally Igly reversed to an inward current (Fig. 3A, top trace). Although alpha [Cl-]i in the [Cs+]pipette at the first application of glycine was almost equal to that in the [K+]pipette (Fig. 3B, arrow), alpha [Cl-]i in measurements with the [Cs+]pipette gradually increased and reached a significantly higher value than that seen with the [K+]pipette (Fig. 3B, **; 18.3 ± 1.0 mM, n = 4). We also used a pipette solution containing 75 mM K+ and 75 mM Cs+ ([K+ and Cs+]pipette). Although the initial alpha [Cl-]i in [K+ and Cs+]pipette were similar to that in the [K+]pipette, alpha [Cl-]i in experiments with the [K+ and Cs+]pipette also gradually increased, similar to that seen with the [Cs+]pipette. However, alpha [Cl-]i finally attained a value between those measured with the two types of the pipette after several applications of glycine (Fig. 3B, *; 10.8 ± 0.1 mM, n = 3). These results indicate that the KCC activity depends on [K+]i and that Cs+ cannot substitute for K+ to drive KCC.



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Fig. 3. Effects of [K+]i on Igly and [Cl-]i. A: replacement of 150 mM K+ in the pipette solution with 150 mM Cs+ ([Cs+]pipette) gradually decreased the outward Igly in amplitude and finally reversed the direction of Igly to be inward (top). In contrast, the pipette solution containing 150 mM K+ ([K+]pipette) maintained constant amplitude of outward Iglys throughout the experiment (bottom). B: alpha [Cl-]is were plotted as a function of time after the first application of glycine when recording with the [Cs+]pipette (closed triangles), the pipette solution with 75 mM K+ and 75 mM Cs+ ([K+ and Cs+]pipette, open circles) and the [K+]pipette (closed circles). Note that alpha [Cl-]is in [Cs+]pipette and [K+ and Cs+]pipette were almost equal to that seen with the [K+]pipette at the first application of glycine (arrow), but gradually increased during repetitive glycine applications. alpha [Cl-]i in the [Cs+]pipette reached a higher value than the passive alpha [Cl-]i at a VH of -50 mV (dashed line). Each symbol and bar represents the mean ± SE in 3-6 neurons. *, **, significant differences in alpha [Cl-]i between the first (0 min) and the 8th application of glycine (21 min) in recordings with the [Cs+]pipette and the [K+ and Cs+]pipette (P < 0.01, paired t-test). n.s.: no significant changes in alpha [Cl-]i for the [K+]pipette throughout the experiment (P > 0.05, paired t-test). VH was -50 mV.

Reversibility of KCC

The data obtained from the experiments changing extra- and intracellular K+ concentrations implies the possible reversibility of the KCC. The outward Igly in [K+ and Cs+]pipette in 5 mM [K+]o was reversed to an inward direction in 20 mM [K+]o (Fig. 4A). In addition, alpha [Cl-]i in experiments with the [K+ and Cs+]pipette in 20 mM [K+]o attained a value over passive alpha [Cl-]i (Fig. 4B), unlike the case with the [K+]pipette and 20 mM [K+]o (Fig. 3A). This finding suggests that the net direction of KCC is determined by the K+ chemical gradient across the membrane. To demonstrate the involvement of KCC operating in the reverse mode as the cause for the elevated alpha [Cl-]i in recording with [Cs+]pipette as compared with the passive alpha [Cl-]i at a VH of -50 mV (Fig. 3B), furosemide was applied and the inward Igly in [Cs+]pipette was monitored. In the presence of 1 mM furosemide, the amplitude of inward Igly and alpha [Cl-]i gradually decreased for the [Cs+]pipette (Fig. 5A, top trace, and B, n = 6). In contrast, 20 µM bumetanide did not affect the inward Igly or the alpha [Cl-]i even after an incubation period of over 20 min (Fig. 5A, middle trace, and C, n = 3). In addition, treatment with Na+-free extracellular solution in [Cs+]pipette to block NKCC activity by depriving Na+ substrate from both intra- and extracellular sides did not change the inward Igly or the alpha [Cl-]i (Fig. 5A, bottom trace, and D, n = 3). Together with the results of Fig. 1, these results indicate that: 1) the decrease of [K+]i, by replacing K+ with Cs+ in the pipette solution, reversed the direction of KCC, thus resulting in an accumulation of Cl- in the cell and the maintenance of alpha [Cl-]i higher than passive alpha [Cl-]i; and 2) the NKCC appears to have little effect on [Cl-]i homeostasis in our acutely dissociated LSO neuron at postnatal day 13-15, as previously demonstrated (Kakazu et al. 1999).



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Fig. 4. Effect of an increase of [K+]o on Igly and [Cl-]i in the [K+ and Cs+]pipette A: an increase of [K+]o from 5 to 20 mM changed the direction of Igly from the outward to the inward when using the [K+ and Cs+]pipette. VH was -50 mV. B: alpha [Cl-]i was plotted as a function of time before, during, and after 20 mM [K+]o. Note that an increase of [K+]o to 20 mM increased alpha [Cl-]i exceeding the passive alpha [Cl-]i at a VH of -50 mV (dashed line) unlike in the case with the [K+]pipette (Fig. 2).



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Fig. 5. Cl- accumulation resulting from inwardly driving the KCC. A: in the presence of 1 mM furosemide, the inward Igly in [Cs+]pipette gradually decreased in amplitude (top). Twenty micrometers of bumetanide (middle) and extracellular Na+-free solution (0 mM [Na+]o, bottom) had no effect on the inward Igly in [Cs+]pipette. The bar indicating each glycine application was omitted. B-D: alpha [Cl-]i measured using the [Cs+]pipette were plotted as a function of time before, during, and after the applications of furosemide (B; closed triangles), bumetanide (C; open diamonds), and 0 mM [Na+]o (D; open circles). In furosemide, alpha [Cl-]i gradually decreased toward the passive alpha [Cl-]i (dashed line). However, bumetanide and 0 mM [Na+]o did not affect alpha [Cl-]i. VH was -50 mV. Data are the mean ± SE of 3-6 neurons. Note that the value of passive alpha [Cl-]i in D was 13.1 mM, different from other experiments (see METHODS).

Selectivity of intracellular monovalent cations on KCC function

We also investigated the selectivity of intracellular monovalent cations other than K+ and Cs+ on KCC function. As in the case with the [Cs+]pipette, the amplitudes of outward Iglys gradually decreased and finally reversed their polarities when using a pipette solution containing 150 mM Li+ ([Li+]pipette) or 150 mM Na+ ([Na+]pipette) (Fig. 6A, top and middle traces). In addition, the time courses of the increases of alpha [Cl-]i when using the [Li+]pipette or the [Na+]pipette (Fig. 6B) were also similar to those in recordings with the [Cs+]pipette (Fig. 3B). The initial value of alpha [Cl-]i for the [Li+]pipette (10.2 ± 0.3 mM, n = 3, Fig. 6B, arrow) measured at the first application of glycine was somewhat higher than those seen with the [Cs+]pipette (8.5 ± 0.8 mM, n = 4, Fig. 6B, arrow) or the [Na+]pipette (8.5 ± 1.9 mM, n = 3, Fig. 6B, arrow). However, there were no statistically significant differences between two of three initial values (P > 0.1, unpaired t-test). alpha [Cl-]is in the [Li+]pipette, [Na+]pipette, and [Cs+]pipette measured at the eighth glycine applications were 18.8 ± 1.0, 17.4 ± 1.2, and 18.3 ± 1.0 mM, respectively. Significant difference was observed in alpha [Cl-]is between the first and the eighth glycine applications in each pipette solution (P < 0.01, paired t-test). There was no significant difference between each two of three values obtained at the eighth glycine application (P > 0.1, unpaired t-test). On the other hand, in the pipette solution containing 150 mM Rb+ ([Rb+]pipette), Igly, and alpha [Cl-]i at the initial values were well maintained, similar to those in [K+]pipette (Fig. 6A, bottom trace, and B). There was no significant difference in alpha [Cl-]is at the eighth glycine application between [K+]pipette (8.0 ± 0.9 mM, n = 6, Fig. 3B) and [Rb+]pipette (9.4 ± 1.3 mM, n = 3, Fig. 6B, P > 0.1, unpaired t-test). These results suggest that 1) neither intracellular Li+, Na+, nor Cs+ can substitute for K+ in the activation of the KCC; and 2) the potency of Rb+ to drive KCC is similar to that of K+.



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Fig. 6. Effects of various intracellular monovalent cations on Igly and [Cl-]i. A: substitution of 150 mM K+ in the pipette solution either with 150 mM Li+ ([Li+]pipette) and 150 mM Na+ ([Na+]pipette), the amplitude of outward Igly gradually decreased and finally reversed their polarities to the inward. In contrast, the pipette solution containing 150 mM Rb+ ([Rb+]pipette) maintained the outward Igly in amplitude. Glycine was applied at an interval of 3 min. B: alpha [Cl-]is recorded using each pipette solution was plotted as a function of time after the first application of glycine. alpha [Cl-]is measured with the [Li+]pipette (open triangles), the [Na+]pipette (closed circles), and the [Rb+]pipette (open circles) obtained at the first application of glycine were similar to each other (arrow). Note the alpha [Cl-]is observed with the [Na+]pipette and the [Li+]pipette finally exceeded a passive alpha [Cl-]i at a VH of -50 mV (dashed line). Each symbol and bar represents the mean ± SE of 3 neurons. *, significant differences between alpha [Cl-]is at the first (0 min) and the 8th application of glycine (21 min) in [Li+]pipette and [Na+]pipette (P < 0.01, paired t-test). n.s.: no significant changes of alpha [Cl-]i in [Rb+]pipette (P > 0.05, paired t-test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

An inwardly directed Cl- electrochemical gradient drives Cl- influx through ligand-gated Cl- channels located in postsynaptic membranes and thus induces a hyperpolarization in the majority of mature CNS neurons. In the absence of active Cl- transport, [Cl-]i will be passively distributed according to the [Cl-]o and the resting membrane potential, preventing any net Cl- flux following Cl- channels activation (Alvarez-Leefmans 1990; Zhang 1991). Under these conditions, only shunting inhibition would be effective (Funabiki et al. 1998). In the present study, a constant outward Igly and alpha [Cl-]i below electrochemical equilibrium were maintained in the LSO neurons (Fig. 3A, bottom trace, and B), indicating the presence of an active Cl- extrusion mechanism, which works during the "resting interval" when the glycine-gated channels are closed. This Cl- extrusion mechanism was apparently mediated by KCC, because furosemide, but not bumetanide, inhibited the Cl- extrusion (Fig. 1), because a reduction of K+ chemical gradient by manipulating [K+]i and/or [K+]o increased alpha [Cl-]i (Figs. 2-4), and last, because the extrusion mechanism operated normally in sodium-free media (Fig. 5).

Reversibility of KCC direction

The KCC carries K+ and Cl- in the same direction in an electroneutral fashion (Alvarez-Leefmans 1990; Lauf and Adragna 1996). In recent studies, KCC2, a subtype of KCC, has been identified exclusively in the brain (Payne et al. 1996; Williams et al. 1999) and more specifically, KCC2 showed a specific neuronal location with a central role in Cl- extrusion as demonstrated in hippocampal pyramidal neurons (Rivera et al. 1999). Its net transport activity is inhibited with furosemide at a concentration of 1 mM (Gillen et al. 1996; Payne 1997) and depends on the chemical gradients of K+ and Cl- (Payne 1997). Under physiological conditions, the difference between [K+]i and [K+]o generated by Na+-K+ pump produces a net outward driving force on the KCC, resulting in a net Cl- extrusion along with K+ (Alvarez-Leefmans 1990). In the present study, the polarity of Igly was reversed and the alpha [Cl-]i was in excess of the passive alpha [Cl-]i (as determined from the repetitive application of glycine) in both 30 mM [K+]o (Fig. 2) and when making recordings with the [Cs+]pipette (Fig. 3). These results are most likely due to the reversed driving force on the net transport of the KCC induced by an increase of [K+]o and/or a decrease of [K+]i. In both cases, Cl- would accumulate in the cell. Conditions where [K+]o increases include an increase of the rate of neuronal action potentials during which delayed K+ channels are activated (Hounsgaard and Nicholson 1993), a large glutamate release opening nonselective cation channels and activating the excitatory amino acid transporter (Levy et al. 1998; Szatkowski and Attwell 1994), a high-frequency stimulation of GABAergic inputs (Kaila et al. 1997), or application of a large concentration of exogenous GABA (Barolet and Morris 1991). Furthermore, [K+]o reaches approximately 60 mM during ischemia (Hansen 1985), which reduces intracellular ATP, resulting in an opening of ATP-sensitive K+ channels and deactivating the Na+-K+ ATPase (Koyama et al. 1999), and nearly 100 mM in some forms of epileptic discharge (Avoli et al. 1996).

The driving force for net KCC (Delta µKCC) is the sum of the chemical potential differences of K+ (Delta µK) and Cl- (Delta µCl). When net KCC reaches thermodynamic equilibrium, Delta µKCC is zero (i.e., Delta µKCC = Delta µK + Delta µCl = 0). As Delta µK = RT ln [K+]i/[K+]o and Delta µCl = RT ln [Cl-]i/[Cl-]o, the forces driving KCC simplify to [K+]i × [Cl-]i = [K+]o × [Cl-]o. In the case of [K+]i = 150 mM and [K+]o = 5, 20, and 30 mM, the ideal alpha [Cl-]i are calculated to be 3.8, 16, and 24.4 mM, respectively. Actually, alpha [Cl]i in 20 mM [K+]o solution (*; 15.6 ± 0.43 mM, Fig. 2B) was very close to the ideal value calculated above (16 mM). We also confirmed the actual alpha [Cl-]i values scarcely changed even after the eighth glycine application (~21 min) in 20 mM [K+]o solution. Thus, it could be concluded that alpha [Cl-]i obtained after the fourth glycine application in 20 mM [K+]o solution nearly reaches a stable state, suggesting that the thermodynamic driving force for KCC is zero. However, in 30 mM [K+]o solution, alpha [Cl-]i after the fourth glycine application (19.4 ± 1.3 mM) was different from the value calculated (24.4 mM). Also the 3.8 mM theoretical alpha [Cl-]i in 5 mM [K+]o was actually found to be 5-11 mM. One possible reason for these discrepancies might be the difficulties in the maintenance of the higher or the lower [Cl-]i compared with the passive [Cl-]i (i.e., from ECl) for the membranes having appreciable conductance. Also, other Cl- transport pathways might be in the system under study.

Several mechanisms could explain the net increase of [Cl-]i induced by the replacement of [K+]i with [Cs+]i. The chloride actually overshoots the expected passive alpha [Cl-]i, whereas simple blockade of the transporter should result in the passive distribution. Therefore, Cs+ does not seem to directly block the KCC function. From the observation of [Cs+ and K+]pipette, one possibility is that the Cs+ binds with very low affinity to the same transport site as K+. Thus, replacement of [K+]i with [Cs+]i will drive most of the transporters into an inwardly facing state in the transport cycle. This would greatly reduce the ability of KCC to mediate net flux. Another possibility is that the depletion of [K+]i could prevent KCC function directly. We could not distinguish between these possibilities in our experimental protocols.

A decrement in the K+ chemical gradient would result in a change of net fluxes from the KCC (Kaila et al. 1997; Payne 1997). In this paper, we directly demonstrated the reversibility of KCC transport in function in native mammalian neuron. We note that alpha [Cl-]i in the [Cs+]pipette should be infinite using the above formula. However, the experimental values of alpha [Cl-]i in [Cs+]pipette was about 17-23 mM. This discrepancy might be explained by residual [K+]i remaining even in the [Cs+]pipette condition. K+ might enter the cell on the KCC faster than it can diffuse up the gramicidin pipette. At some steady state value of alpha [Cl-]i in [Cs+]pipette, [K+]i would presumably be constant, but unknown. In any case, intracellular perfusion of Cs+ by the [Cs+]pipette might not exclude [K+]i completely, so that the driving force of the KCC would, in fact, be finite. Also, other Cl- regulators might be present on the membrane. It is conceivable that accumulated Cl- may escape from the inside of the cell by other Cl- regulators including leak pathways.

Cation selectivity of KCC function

Since only monovalent cations, but not Cl-, can pass the gramicidin pore (Akaike 1997), this method allowed the internal dialysis of various monovalent cations simply by adding them to the pipette solution (Figs. 3 and 6). The [Rb+]pipette did not increase alpha [Cl-]i, suggesting that Rb+ could substitute for K+ for KCC function (Fig. 6), as well as for the Na+-K+ pump (Munakata et al. 1997) and K+ channel (Jansen et al. 1998). In contrast, internal perfusion of Cs+, Li+, or Na+ decreased the amplitude of the initially outward Igly and finally reversed its polarity to inward (Figs. 3 and 6). The effects on the alpha [Cl-]i observed using a [Cs+]pipette, [Li+]pipette, or [Na+]pipette are probably based on the same mechanism, because of the similarity of the time courses of alpha [Cl-]i increase (Figs. 3 and 6). Moreover, our studies, together with the report that EGABA and EIPSP recorded with Cs+ filled microelectrodes are more depolarized than those with K+ filled electrodes (Thompson and Gähwiler 1989), strongly suggest that one should not perfuse intracellularly with monovalent cations other than K+ or Rb+ to examine native neuronal Cl- regulation.

There is a possibility that an increase of [Na+]i resulting from diffusion from the [Na+]pipette might affect some other [Cl-]i regulating transporters, such as the Na+-dependent HCO3--Cl- exchanger or the Na+-(K+)-Cl- cotransporter (N(K)CC) (Kaila 1994). In the present experiment, however, we employed the standard extracellular solution buffered with HEPES, in the nominal absence HCO3-, and thus Na+-dependent HCO3--Cl- exchanger should have an insignificant role. The N(K)CC transports in an electroneutral fashion. Its net direction is determined by the chemical gradients of Na+, (K+), and Cl-, and stoichiometry of the transporter (Haas 1994; Kaila 1994). Intracellular perfusion of Na+ could be expected to drive the N(K)CC in a net outward direction and thus to expel intracellular Cl-. However, our results showed an increase of [Cl-]i when using the [Na+]pipette (Fig. 6). In addition, bumetanide and an extracellular solution lacking Na+ did not affect the Igly or alpha [Cl-]i for either the [K+]pipette or the [Cs+]pipette (Figs. 1 and 5). These results suggest that the Na+-dependent [Cl-]i regulators, including N(K)CC, seem to be insignificant in [Cl-]i regulation in LSO preparation. Thus LSO neurons examined in the present study would seem to be suitable to study KCC function in native CNS neurons because of its constitutive expression of KCC and lack of NKCC (Figs. 1 and 5).

The LSO initially integrates the auditory information from the two ears. The probability of action potentials in the LSO is determined by the time differences of arrival from the ipsilateral (glutamatergic) and contralateral (glycinergic) inputs. This process, which encodes interaural intensity differences, supplies the cues to localize sound in space (Koyano and Ohmori 1996; Sanes and Rubel 1988). Therefore, the maintenance of the chloride gradient mediated by the KCC, with its consequent hyperpolarizing IPSP as well as shunting inhibition (Funabiki et al. 1998), is likely to be very important for LSO neurons in auditory information processing.


    ACKNOWLEDGMENTS

We thank Prof. Kai Kaila and M. Brodwick for discussions and helpful comments on this manuscript.

Grant-in-Aids (Nos. 1170240 and 11670044 to J. Nabekura, and Nos. 10470009 and 10044301 to N. Akaike) from the Ministry of Education, Science and Culture, Japan, supported this work.


    FOOTNOTES

Address for reprint requests: J. Nabekura, Cellular and System Physiology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan (E-mail: nabekura{at}mailserver.med.kyushu-u.ac.jp).

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.

Received 13 September 1999; accepted in final form 30 March 2000.


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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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