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Kainate induces an intracellular Na⁺-activated K⁺ current in cultured embryonic rat hippocampal neurones

Qi-Ying Liu, Anne E. Schaffner and Jeffery L. Barker

Received 20 January 1998; accepted after revision 21 April 1998.

	ABSTRACT

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1. In embryonic rat hippocampal neurones cultured for < 3 days, kainate induced an inward current at negative potentials that recovered to baseline levels immediately upon termination of agonist application. However, in neurones cultured for longer, the kainate-induced current was often followed by a long-lasting inward current that slowly recovered to baseline levels. The amplitude of the delayed current (I_delay) triggered by kainate was positively related both to the duration of application at constant agonist concentration and to concentration at constant application duration.

2. I_delay could last for several minutes and was accompanied by a conductance increase, which closely paralleled current amplitude. Depression of the kainate-induced current response at receptor level with CNQX or at ionic level with Na⁺-free solution eliminated I_delay. However, when applied during I_delay neither CNQX nor Na⁺-free solution had any effect on I_delay. Li⁺ effected the same response as Na⁺ in mediating kainate-induced I_delay.

3. GABA-activated Cl^- current, which was associated with the same amount of inwardly directed charge flow at the same potential as that induced by kainate, did not trigger a long-lasting delayed current.

4. I_delay depended on the existence of extracellular K⁺ and its amplitude increased with the increase in K⁺ concentration. Neither applying Cl^-- or Ca²⁺-free solutions nor increasing intracellular Ca²⁺ buffering speed and capacity altered I_delay. Exposure to the specific K_Ca channel blockers apamin and charybdotoxin also failed to alter I_delay. However, I_delay could be blocked by Cs⁺, Ba²⁺ and high concentrations of 4-aminopyridine (4-AP) and TEA.

5. Inside-out excised patch-clamp recordings revealed that low density or highly clustered Na⁺-activated K⁺ channels were expressed in the cell bodies of cultured embryonic rat hippocampal neurones. These could be the elementary channels underlying I_delay.

	INTRODUCTION

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Excitatory synaptic neurotransmission in the mammalian central nervous system is mediated predominantly by non-NMDA-type glutamate receptor-channels, which are highly permeable to Na⁺ and K⁺ and less permeable to Ca²⁺ ions (Hatt et al. 1988; Colquhoun et al. 1992). When activated, these glutamate receptor-channels are typically associated with inward Na⁺ current. These channels are divided into kainate- and -amino-3-hydroxy-5-methylisoxazole (AMPA)- preferring types (Collingridge & Lester, 1989; Monaghan et al. 1989; Lodge, 1997), both of which have fast activation and desensitization kinetics when activated with their respective preferred agonists (Mayer & Vyklicky, 1989; Lerma et al. 1993). However, AMPA-preferring channels open persistently in the presence of kainate and AMPA-activated kainate-preferring channels desensitize slowly and to a lesser degree so as to induce a persistent influx of Na⁺ (Patneau et al. 1992; Spruston et al. 1995; Lodge, 1997).

K⁺ channels of enormous diversity have now been catalogued using electrophysiological, biophysical and molecular techniques. In contrast to voltage-gated K⁺ channels, other K⁺ channels are gated by intracellular second messengers, like G proteins and arachidonic acid, and ions, like Ca²⁺ and Na⁺. The existence of K⁺ channels gated by intracellular Na⁺ (K_Na) was first demonstrated in isolated guinea-pig ventricular myocytes (Kameyama et al. 1984). Since then, this class of K⁺ channels has been found in a wide variety of species and preparations, including neurones in crayfish (Hartung, 1985), Drosophila (Saito & Wu, 1991), snails (Partridge & Thomas, 1976), birds (Bader et al. 1985; Dryer et al. 1989; Haimann et al. 1990, 1992; Dryer, 1993) and mammals (Schwindt et al. 1989; Egan et al. 1992a,b). The physiological functions of this class of K⁺ channels have been difficult to define largely because their activation usually requires Na⁺ levels considerably higher than physiological intracellular Na⁺ concentrations ([Na⁺]_i). However, recent findings indicate that spontaneous oscillations in [Na⁺]_i (Rose & Ransom, 1997) could reach levels high enough to activate K_Na channels (Egan et al. 1992a; Koh et al. 1994). In certain pathological conditions such as anoxia and excessive activation of glutamate receptors, [Na⁺]_i can increase to as high as 60 mM (Friedman & Haddad, 1994; Kiedrowski et al. 1994; Pinelis et al. 1994; Khodorov et al. 1996). Furthermore, the increase in [Na⁺]_i induced by application of glutamate can last several minutes after the removal of glutamate (Kiedrowski et al. 1994). Here, we report that prolonged (tens of seconds) application of kainate can activate a K_Na current in embryonic rat hippocampal neurones which have differentiated for more than 3 days.

	METHODS

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Dissociation and culture of embryonic rat hippocampal neurones

Hippocampal neurones from day 19 rat embryos were dissociated and cultured on a monolayer of cortical astrocytes, as previously described (Liu et al. 1996). Embryos were obtained from pregnant mothers killed by inhalation of CO₂ followed by cervical dislocation. Embryos were removed by caesarian section. Fetuses and neonates were quickly decapitated with surgical scissors. All animal procedures were done in accordance with institutional guidelines for the anaesthetizing of fetuses and neonates and conformed to the Guide for the Care and Use of Laboratory Animals in the US.

Briefly, the astrocyte monolayer was prepared as follows. Cortices were removed from 3-day-old rat pups, cleaned of meninges and placed in 10 ml L-15 medium (Gibco) with 50 i.u. ml^-1 gentamicin. The tissues were triturated through a 5 ml pipette followed by mechanical dissociation. Cells were centrifuged at low speed and resuspended in plating medium consisting of Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10 % fetal calf serum (FCS) and 50 i.u. ml^-1 gentamicin, then plated at the equivalent of two brains per flask in 75 cm² flasks. The medium was changed completely after 72 h and twice weekly thereafter. When a confluent monolayer was present (after 1 week) the flasks were tightly capped and placed overnight on a rotary shaker at 180 r.p.m. at 37°C. The supernatant, containing microglia, loosely adherent oligodendrocyte type 2 astrocyte (O2A) progenitor cells and debris, was rapidly and completely removed after 12 h. Cultures were rinsed once with DMEM and incubated with a 1 : 50 dilution of A2B5 (cell surface GQ ganglioside) ascites in DMEM with 1 % FCS for 1 h at 37°C. After two rinses with DMEM-1 % FCS, the cultures were incubated for 1 h at 37°C with rabbit complement diluted 1 : 8 in DMEM-1 % FCS, rinsed twice again and then refed with plating medium. When needed, the astrocyte cultures were trypsinized and cells were transferred into 35 mm diameter culture dishes which had been pre-coated with low molecular weight (53 kDa) poly-D-lysine (PDL, Sigma). When the cells reached confluence they were exposed to 10 µM cytosine arabinoside in DMEM for 2 days and then maintained for 7 days in DMEM with 5 % FCS before being used.

Hippocampal tissue was dissected from rat embryos at day 19 of gestation, minced into small pieces, transferred into 5 ml Earle's balanced salt solution (EBSS) containing 20 U ml^-1 papain, 0·01 % DNase (both from Boehringer Mannheim), 0·5 mM EDTA and 1 mM L-cysteine and rocked in an incubator for 35-40 min at 37°C. Single neurones, obtained by triturating the tissue with a Pasteur pipette, were resuspended in EBSS with 1 mg ml^-1 trypsin inhibitor (TI) and 1 mg ml^-1 bovine serum albumin (BSA) and layered over 5 ml of EBSS with 10 mg ml^-1 TI and 10 mg ml^-1 BSA in a 15 ml plastic centrifuge tube. The gradient was spun at 80 g for 5 min, effectively removing dead cells and debris from the suspension. The cell pellet was resuspended in 90 % minimal essential medium (MEM; Gibco), 5 % FCS and 5 % horse serum (Biofluid, Rockville, MD, USA) and plated at a density of (3·5-4) × 10⁵ cells per dish on a monolayer of astrocytes in 35 mm plastic culture dishes. Cultures were kept at 37°C in a humidified atmosphere containing 10 % CO₂. The culture medium was changed to contain 95 % MEM and 5 % horse serum after 2 days and changed twice a week thereafter.

Current recording and analysis

All recordings were made in neurones cultured for between 1 day and 3 weeks. Prior to recording, dishes were removed from the incubator and the culture medium was completely replaced with Tyrode solution containing (mM): 145 NaCl, 5·4 KCl, 1·8 CaCl₂, 0·8 MgCl₂, 10 glucose, 10 Hepes-NaOH, pH 7·4 and 310 mosmol l^-1. Standard patch-clamp recordings (Hamill et al. 1981) were made with pipettes pulled in three stages from 1·5 mm o.d. glass capillary tubes (World Precision Instruments, Sarasota, FL, USA) with a computer-controlled pipette puller (BB-CH-PC, Mecanex SA, Switzerland). These pipettes had a resistance of 3-5 M when filled with internal solution composed of (mM): 145 CsCl, 2 MgCl₂, 0·1 CaCl₂, 1·1 EGTA, 5 Hepes, 5 K₂-ATP, 5 phosphocreatine (pH 7·2 and 290 mosmol l^-1). Whole-cell currents were recorded with a L/M EPC-7 patch-clamp amplifier (Medical Systems Corp., Greenvale, NY, USA) at a gain of 5 mV pA^-1. Series resistance was compensated for more than 70 %. Current signals were stored on videocassettes via a videocassette recorder and a VR-100 digital recorder (Instrutech, New York) for later off-line digitization with a Digidata 1200 (Axon Instruments) and analysis with pCLAMP v. 6.0 (Axon Instruments) on a Pentium-based personal computer. Whole-cell currents were also simultaneously recorded on a pen recorder (Gould Inc., Glen Burnie, MD, USA). For inside-out patch clamp recordings, pipettes were filled with a solution containing (mM): 145 KCl, 1·8 mM CaCl₂, 0·8 mM MgCl₂, 10 Hepes (pH 7·4 and 310 mosmol l^-1). The membrane patches were perfused with a solution containing (mM): 5·4 KCl, 5 Mg-ATP, 2 EGTA, 10 Hepes and either 145 NaCl or 145 N-methyl-D-glucamine (NMDG) chloride. All recordings were carried out at room temperature (22-25°C) on a Nikon inverted microscope. Cells were continuously superfused throughout recording with a perfusion system composed of a locally made perfusion controller and miniature electric solenoid valves (The Lee Co., Essex, CT, USA) that allows fast switching (< 200 ms complete solution exchange time) among different solutions (Liu et al. 1996). Nine inputs converge into a perfusion tube of about 300 µm i.d. and positioned 100-350 µm away from the recorded cell. The perfusion rate (0·3- 0·5 ml min^-1) was controlled by the air pressure applied to the solution reservoir. The start point of the delayed current (I_delay) was taken as, when judged under high time resolution, the point where the fast decaying phase of agonist washout changed to much slower decaying phase of I_delay. The amplitude of I_delay was measured as the difference between the beginning of I_delay and the steady-state value. The decaying phase of I_delay was best fitted with a single-exponential function (I = I_maxe^-t/; where I is the amplitude of I_delay at time t after the beginning of I_delay, Imax is the maximum current and is the time constant). The duration of I_delay was taken as the time from the beginning of I_delay to the time when the current decayed to the steady-state level.

Statistical tests

Data are given as means ± S.E.M. Student's two-tailed t test was used to assess significance. Differences were considered significant if P < 0·05 (indicated in figures by ) or P < 0·01 ( ).

	RESULTS

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Kainate induced a delayed inward current in cultured hippocampal neurones

At a holding potential of -80 mV, kainate induced inward currents (I_kainate) of variable magnitude in virtually all of the cultured embryonic rat hippocampal neurones tested in this study. Unlike AMPA- and glutamate-induced currents (not shown), I_kainate in these neurones exhibited little, if any, fading or desensitization, even during a minute-long application. With the fast perfusion system used in this study, I_kainate developed immediately and terminated immediately after switching back to control solution when recordings were carried out in neurones cultured for less than 3 days (Fig. 1A). In neurones cultured for more than 1 week, I_kainate was immediately followed after switching back to control solution by an inward current lasting for up to several minutes that resembled 'tail' currents following voltage steps and which slowly decayed to baseline. This occurred if the duration of the kainate application was at least several seconds (Fig. 1B). The delayed, slowly decaying current (I_delay) triggered by kainate was accompanied by an increase in membrane conductance, as revealed by current responses evoked by intermittent, brief hyperpolarizing commands, and the conductance changed in parallel with the relaxation of I_delay (Fig. 1C).

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Figure 1. Kainate induced an inwardly directed slowly decaying current (Idelay) in embryonic rat hippocampal neurones cultured for more than 1 week A neurone cultured for 3 days did not exhibit any residual trace of current following cessation of the kainate application (A) while another neurone grown in culture for 10 days exhibited significantly more kainate-elicited current, which was immediately followed by an inwardly directed, slowly decaying current (Idelay, B). The tail-like current was accompanied by an increase in membrane conductance as revealed by the current response to intermittent 10 mV hyperpolarizing commands in another neurone cultured for 12 days (C). Inset in C is a plot of Idelay amplitude () and the corresponding increase in membrane conductance () after termination of the kainate application. Membrane potential was clamped at -80 mV. KA, kainate. Here and in subsequent figures the dashed horizontal line shows the baseline holding current.

The amplitude and duration of I_delay were positively related to the duration of the kainate application (Fig. 2A and Ba). The relationship between I_delay amplitude and the duration of the kainate application could be well fitted with a single exponential function ( 14 s) (Fig. 2Ba). Moreover, I_delay induced by longer applications of kainate decayed more slowly than those induced by shorter applications (Fig. 2Bb). In fourteen neurones cultured for 12-15 days, the amplitude of I_delay induced by application of 50 µM kainate for 30 s averaged 212·9 ± 26·9 pA, while the mean duration of I_delay was 63·9 ± 8·3 s. Furthermore, I_delay required concentrations of kainate greater than 10 µM (Fig. 2Ca and Cb). I_delay was never induced by GABA (5 µM) which evoked an inward current carrying a similar amount of net charge (by the efflux of Cl^- ions) as the kainate-induced current in the same neurone (Fig. 3A and B). However, the long-lasting I_delay could also be induced with other glutamate agonists such as NMDA (50 µM, Fig. 3Cb) and AMPA (50 µM, data not shown), if the corresponding steady-state inward currents were intense enough.

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Figure 2. The amplitude and duration of Idelay depended on the duration of kainate application and kainate concentration A, the baseline before and immediately after kainate application is amplified to illustrate the duration-dependent appearance of Idelay. While 3 s application of kainate (not shown) did not induce Idelay, applications lasting longer than 6 s triggered Idelay with amplitude and duration increasing as the application duration increased. Arrowheads indicate the peak amplitudes of Idelay. Ba, plot of Idelay amplitude against duration of the kainate application, which can be best fitted with an exponential function (, 14·3 s). b, the time constants of Idelay (delay) were related to the duration of kainate application in a bimodal manner. C, in another neurone, the current evoked by 10 µM kainate (a) decayed to baseline level immediately after the termination of application but the current induced by 50 µM kainate (b) was followed by Idelay.

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Figure 3. The delayed current was not caused by non-specific inwardly directed charge movement A, inward charge movement evoked by 50 µM kainate was followed by Idelay. B, in the same cell, a comparable accumulation of inward charge movement (6·3 × 10-9 C compared with 6·2 × 10-9 C for kainate) evoked by 5 µM GABA was not followed by any detectable Idelay. In another neurone, currents induced by both kainate (50 µM, Ca) and NMDA (50 µM, Cb) were followed by Idelay. (Brief downward transients are synaptic-like GABAergic currents.)

I_delay depended on the Na⁺ influx during I_kainate

Under the present recording conditions, activation of non-NMDA receptors by kainate evoked an inward current carried primarily by Na⁺ ions at a holding potential of -80 mV. Kainate did not induce any observable I_delay in neurones cultured for less than 3 days, which had kainate-induced current responses of a lower amplitude, suggesting that I_delay requires a significant complement of kainate receptor-channels. Furthermore, if I_delay results indirectly from kainate-induced Na⁺ influx instead of being directly evoked by kainate, depression or elimination of kainate-induced current should decrease or abolish I_delay. We depressed kainate-evoked current by co-applying 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM) and kainate, then removing both (n = 4 neurones). No detectable I_delay developed under these conditions (Fig. 4Ab) in contrast to recordings made under control conditions in the same neurones (Fig. 4Aa). Similarly, replacement of extracellular Na⁺ with NMDG⁺ eliminated the majority of the kainate-induced current and the subsequent I_delay in three neurones tested (Fig. 4B a andBb). To further test whether I_delay is directly evoked by residual kainate activating membrane receptors, we applied CNQX coincident with termination of kainate application (Fig. 4Ac). No significant differences were detected in the amplitude of I_delay in the presence (185·8 ± 47·7 pA) or absence (186·5 ± 49·1 pA, n = 4; P > 0·05) of CNQX applied during I_delay, suggesting that I_delay was not due to the continued activation of kainate receptor-cation channels. Rather, the results reveal that I_delay evolves indirectly following activation of kainate receptor-cation channels.

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Figure 4. Idelay depended on Na+ influx A, under control conditions, application of 50 µM kainate to a neurone cultured for 14 days (a) induced a sizable inward current which was followed by Idelay; when kainate was applied with CNQX (10 µM, b) the majority of kainate-evoked current was blocked and Idelay disappeared; however, when CNQX was applied after termination of the kainate application (c) it had no effect on Idelay. B, in another neurone, suppressing the majority of kainate current (a) by replacing extracellular Na+ with non-permeable N-methyl-D-glucamine (b) prevented induction of Idelay. Ca and b, application of Na+-free saline after termination of the kainate application had little, if any effect on Idelay.

The induction of I_delay depended completely on an influx of Na⁺ occurring during, but not after, the kainate application since switching to a Na⁺-free solution upon termination of the kainate application had no obvious effects on I_delay (Fig. 4Ca and Cb). This indicates that I_delay did not itself involve Na⁺ as a charge carrier. The continued presence of I_delay after switching into a Na⁺-free solution also eliminates electrogenic Na⁺-Ca²⁺ exchange in the generation of the inwardly directed I_delay. Furthermore, ouabain (1 mM), a blocker of the electrogenic Na⁺-K⁺ pump, added to the control and to kainate-containing solutions did not have any observable effects on I_delay (data not shown).

Li⁺ effected the same response as Na⁺ in inducing I_delay

Li⁺ has been shown to effect the same response as Na⁺ in generating kainate-induced inward current (Murphy et al. 1987) but this is not the case in many other Na⁺-dependent processes such as activating the Na⁺ pump (Parker et al. 1996), Na⁺-Ca²⁺ exchange (Keele et al. 1997) and most K_Na channels (Dryer, 1994). To test whether Li⁺ could replace Na⁺ in activating I_delay, extracellular Na⁺ was exchanged for equimolar Li⁺. Under these conditions, kainate induced a comparable inward current followed by an I_delay similar in amplitude to that recorded in Na⁺-containing perfusion solution (Fig. 5). In three neurones cultured for 12 days, the mean amplitude of I_delay induced by 30 s application of 50 µM kainate was 138·7 ± 10·4 pA in the presence of Na⁺ and 134·7 ± 13·7 pA in the presence of Li⁺ (P > 0·05).

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Figure 5. Li+ effected the same response as Na+ in kainate-induced Idelay A, in control conditions with 145 mM extracellular Na+, application of 50 µM kainate evoked an inward current followed by Idelay in a neurone cultured for 10 days. B, in the presence of 145 mM Li+ (and 0 Na+), kainate evoked a slightly lower amplitude current response while Idelay remained comparable in amplitude and duration to that recorded under control (145 mM Na+) conditions.

Changes in intra- and extracellular Ca²⁺ and extracellular Cl^- concentrations did not affect I_delay

Several classes of Ca²⁺-activated Cl^- and K⁺ channels exist in rat hippocampal neurones in culture (Segal & Barker, 1986; Segal et al. 1987) and in slices (Lancaster et al. 1991; Wann & Richards, 1994). Kainate-activated channels in these neurones are partially permeable to Ca²⁺ (Donevan & Rogawski, 1995; Iino et al. 1996). To test whether I_delay is induced by Ca²⁺ influx during or after application of kainate, the solutions were made Ca²⁺ free. No obvious effects on the amplitude and time course of I_delay were detected in Ca²⁺-free solution in five neurones tested (Fig. 6A). Similarly, the amplitudes of I_delay were comparable when the pipette solution contained either 1·1 mM EGTA (254·8 ± 55·3 pA, n = 6) or 5 mM BAPTA (226·2 ± 58·8 pA, n = 5; P > 0·05), which has a higher and faster Ca²⁺-buffering capacity (Tsien, 1980). To further test whether K_Ca channels were activated and responsible for the observed I_delay, we applied kainate in the presence of 1 µM apamin (Fig. 6Bb) or 25 nM charybdotoxin (Fig. 6Bc). In six neurones cultured for 13 days, the mean amplitudes of kainate-induced I_delay were 171·0 ± 35·3, 181·7 ± 39·7 and 167·3 ± 40·0 pA in control, and in the presence of apamin and charybdotoxin, respectively. Hence, no significant differences were detected (P > 0·05) in I_delay amplitudes in the presence and absence of these K_Ca channel blockers.

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Figure 6. The induction and maintenance of Idelay did not depend on intracellular Ca2+ or extracellular Ca2+ and Cl- A, Ca2+-free solution applied after termination of the kainate application (b) or throughout the recording period (c) had no obvious effect on Idelay when compared with that recorded under control conditions (a). B, compared with control (a) Ca2+-activated K+ (KCa) channel blockers apamin (b) and charybdotoxin (c) had no effects on Idelay, indicating that small- and large-conductance KCa channels are not involved in the generation of Idelay. C, the amplitude and appearance of Idelay recorded in Cl--free solution applied immediately after termination of the kainate application or during the recording period were comparable to that recorded under control conditions.

At a holding potential of -80 mV and with chloride equilibrium potential (E_Cl) at 0 mV, Cl^- currents are also inwardly directed and could contribute to the kainate-induced I_delay. However, application of Cl^--free solution during and/or after the termination of kainate application had no clear effect on the properties of I_delay (Fig. 6C), suggesting that changes in Cl^- conductance were not involved in the induction or elaboration of I_delay.

I_delay depended on extracellular K⁺

Among other types of K⁺ channels, Na⁺-activated K⁺ channels are present in rat hippocampal neurones (Egan et al. 1992a; see also below). If I_delay is due to an increase in K⁺ conductance, its amplitude should change when extracellular K⁺ concentration is changed. We found that the typical inwardly directed I_delay induced in 5·4 mM K⁺ solution (Fig. 7Aa) was transformed into an outwardly directed current in the absence of extracellular K⁺ (Fig. 7Ab). Subsequent return to 5·4 mM K⁺ solution immediately led to the recovery of the familiar inwardly directed I_delay. In contrast, increasing the concentration of extracellular K⁺ increased the amplitude of I_delay dramatically (Fig. 7Ba). A short pulse of elevated K⁺ (15 mM) solution induced a large inwardly directed current when the K⁺ was delivered soon after the termination of the kainate application. After I_delay had decayed to baseline levels, similar pulses of 15 mM K⁺ evoked lower amplitude inward currents, which relaxed quickly and exhibited less obvious decay (Fig. 7Bb).

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Figure 7. Idelay depended on extracellular K+ A, in the presence of 5·4 mM potassium, kainate induced a sizable Idelay (a), which became outward when kainate was applied in K+o-free solution (b). Upon returning to K+-containing solution, an inwardly directed, slowly decaying Idelay appeared (b). B, in another neurone, the amplitude of Idelay was severalfold larger in 15 mM K+ than in 5·4 mM K+ solution (a). If kainate was applied in the presence of 5 mM Cs+, no observable Idelay could be detected (b). However, removal of Cs+ from the solution immediately revealed a delayed current. Brief applications of a 15 mM K+ solution induced a large inwardly directed, slowly decaying current shortly after removal of Cs+ but a noticeably smaller non-decaying inward current later. The non-decaying current probably reflects the contribution of the inward-rectifier K+ current.

We applied 1 s ramp protocols at the onset and conclusion of I_delay to characterize the conductance over a wide range of potentials (from -80 to +40 mV). We subtracted the current values recorded after I_delay had relaxed from those recorded at the onset to obtain I_delay values (Fig. 8). The reversal potential of I_delay (-21·3 ± 2·9 mV, n = 9) obtained from these ramp protocols was not significantly different from the theoretical potassium reversal potential (E_K) under these recording conditions (approximately -16 mV, P > 0·05), suggesting that the channels are permeable primarily to K⁺. There was a relatively linear I-V relationship over the -80 to 0 mV range. Outward currents in the positive potential range rectified strongly so that currents evoked at the most positive potentials were reduced by 40 % relative to those recorded at 0 mV. The slope conductance over the greater part of the potential range of inward current was 22 nS.

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Figure 8. Current-voltage relations of Idelay In neurones showing clear Idelay, 120 mV ramp protocols (1 s, -80 to +40 mV) were applied 1 s after the termination of kainate application. The baseline currents obtained before kainate application were subtracted and the putative Idelay plotted against membrane potentials. The chord conductance was about 130 pS. A negative slope appeared at positive membrane potentials. Data were obtained from 7 cells and were plotted as means ± S.E.M.

Organic and inorganic K⁺ channel blockers inhibited I_delay

Although I_delay was not sensitive to intracellular Cs⁺ since it could readily be induced with a pipette solution containing 145 mM Cs⁺, it was sensitive to extracellular Cs⁺. Low concentrations of extracellular Cs⁺ (5 mM) could completely and reversibly block I_delay when applied either immediately after the termination of kainate application (Fig. 9Ac) or during and after kainate application (Figs 7Bb, 9Ab and 9Bb). Subsequent switching back to Cs⁺-free solution resulted in an immediate unblock, revealing the decaying I_delay (Figs 7C and 9Bb). The mean amplitude of I_delay in twelve neurones cultured for 11-15 days was 182·2 ± 34·1 pA under control conditions and 3·7 ± 2·9 pA in the presence of 5 mM Cs⁺ (P < 0·01). I_delay could also be blocked by Ba²⁺, another inorganic K⁺ channel blocker (Fig. 9C). The mean amplitude of I_delay was 107·3 ± 9·9 pA and 0·6 ± 0·3 pA in control and in the presence of 5 mM Ba²⁺, respectively (n = 3, P < 0·01). We also tested two organic K⁺ channel blockers. At low concentrations (1 and 4 mM, respectively), 4-AP and TEA did not alter I_delay (data not shown). However, relatively high concentrations of 4-AP (10 mM) and TEA (20 mM) partially blocked I_delay (Fig. 10). In six neurones, I_delay values were 167·2 ± 29·5 pA, 67·3 ± 32·0 pA and 65·3 ± 19·1 pA in control and in the presence of 4-AP (P < 0·01) and TEA (P < 0·01), respectively.

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Figure 9. Extracellular Cs+ blocked kainate-induced Idelay A, Cs+ (5 mM) added to the extracellular solution blocked Idelay which is present under control conditions (a), when applied immediately after kainate (c) or during the whole kainate application-termination period (b). B, in another neurone, 50 µM kainate induced a delayed current response in the absence of 5 mM Cs+ (a) but not in the presence of Cs+ (b). Upon removal of Cs+, an inwardly directed, slowly decaying Idelay appears immediately (b). Another inorganic K+ channel blocker, Ba2+ (5 mM), also completely blocks Idelay when applied immediately after the termination of kainate application (C).

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Figure 10. Blocking of Idelay by TEA and 4-AP Kainate (50 µM) induced a sizable Idelay under control conditions (A) but not in the presence of 10 mM 4-AP (B) or 20 mM TEA (C) (vertical calibration, 100 pA). Note that an inwardly directed, slowly decaying Idelay appears upon removal of 4-AP and TEA (arrowheads). Insets show Idelay at higher gains (vertical calibration, 40 pA).

Direct activation of K⁺ conductance by Na⁺ at single-channel level

In order to detect the Na⁺-activated K⁺ current directly, inside-out excised patch-clamp recordings were made. Membrane patches were obtained from the soma of the cultured neurones and the membrane potential was clamped at 0 mV, the putative E_Cl. When the membrane patch was perfused with Na⁺-free solution no single-channel current was detected. However, when the perfusion solution was switched to one containing 145 mM Na⁺, single-channel currents with multi-conductance levels were recorded, which disappeared immediately after switching back to 0 Na⁺ (Fig. 11). An all-points amplitude histogram reveals two preferred levels of 1·3 and 3·3 pA (equivalent to about 15 pS and 39 pS in the presence of 5·4 mM [K⁺]_i and 145 mM [K⁺]_o, respectively). K_Na channels in these cultured embryonic rat hippocampal neurones are either very low in density or highly clustered because only two of thirty-five membrane patches (6 %) had Na⁺-activated channel currents.

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Figure 11. Na+-activated K+ single-channel currents in cultured embryonic rat hippocampal neurones A, Na+ (145 mM) reversibly induced elementary-sized, all-or-none transitions in current in an inside-out membrane patch excised from the soma of a neurone cultured for 14 days. At higher time resolution, two preferred current levels are evident (inset). B, all-points histogram shows two preferred current levels of 1·3 and 3·3 pA. The potential across the membrane patch was held at 0 mV, the equilibrium potential for Cl- under these recording conditions. The continuous line in the inset indicates the closed state of the channel and the dotted lines open states. Upward deflection represents inward (to the cytoplasmic side of the membrane) flow of currents.

Induction of Cs⁺-sensitive K⁺ conductance by high frequency activation of Na⁺ current

As a first attempt to explore the physiological relevance of I_delay, we tested whether repeated activation of voltage-dependent Na⁺ current could activate this current. Under control conditions, a train (20 Hz, 30 s) of depolarizing commands (to 0 mV from a holding potential of -80 mV) induced an inward, decaying current of 29 pA. In the presence of 10 mM Cs⁺, the same depolarizing command train induced a much smaller decaying current of 9 pA in the same neurone (Fig. 12). This indicates that Na⁺_i derived from Na⁺_o entry through voltage-dependent Na⁺ channels can activate a Cs⁺-sensitive current resembling I_delay.

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Figure 12. Induction of Idelay by high frequency activation of Na + current Membrane potential was clamped at -80 mV. A train of 12 ms voltage clamp steps to 0 mV was applied for 30 s at 20 Hz under control conditions (A) and in the presence of 10 mM Cs+ (B). Insets show Idelay at higher gain (left calibration).

	DISCUSSION

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Na⁺-activated K⁺ current in cultured embryonic rat hippocampal neurones

In this study we found that in embryonic rat hippocampal neurones cultured for more than 3 days, kainate-evoked currents were followed by an inwardly directed, slowly decaying I_delay which was accompanied by an increase in membrane conductance. The following evidence suggests that I_delay is due to an increase in K⁺ conductance. (1) The presence and amplitude of I_delay depended on the existence and concentration of extracellular K⁺ but not on any of the other major ions, including Ca²⁺, Na⁺ and Cl^-. (2) The reversal potential was close to E_K. (3) The current was blocked by inorganic (Cs⁺ and Ba²⁺) and organic (TEA and 4-AP) K⁺ channel blockers. (4) Electrogenic Na⁺-Ca²⁺ exchanger and Na⁺ pump activities were eliminated as mechanisms. (5) CNQX or Na⁺-free saline blocked both the kainate-evoked current and I_delay but did not alter I_delay when applied after its induction. (6) I_delay only appeared in neurones with large amplitude kainate-induced currents. Taken together, these results strongly suggest that I_delay is due to the activation of a separate conductance evoked by an increase in intracellular Na⁺ concentration ([Na⁺]_i) caused by Na⁺ influx through kainate receptor-cation channels rather than due to persistent activation of these channels by kainate remaining in equilibrium at the surface receptors. Possible involvement of both small- and large-conductance Ca²⁺-activated K⁺ channels was ruled out by the finding that neither apamin nor charybdotoxin affected I_delay. Therefore, we conclude that an increase in Na⁺_i caused by kainate-activated cation channels induces activation of Na⁺_i-dependent K⁺ channels in these cells. NMDA (Fig. 3), glutamate and AMPA (data not shown) also induced I_delay if appropriate concentrations were used to evoke a large-amplitude current response.

I_delay was not seen in neurones cultured for less than 3 days, probably because the expression of functional kainate receptors in the vicinity of [Na⁺]_i-dependent K⁺ channels was too low so that an elevation in [Na⁺]_i failed to activate detectable K⁺ conductance. Other developmentally regulated phenomena including few or no I_delay channels or low Na⁺ sensitivity of I_delay channels could also contribute to the lack of I_delay.

In inside-out, excised patch-clamp recordings, the holding potential was set at the Cl^- equilibrium potential, so eliminating Cl^- current. E_K was approximately +84 mV so that K⁺ current would flow inward. Elementary-sized transitions in patch current were not detected when the cytoplasmic face of the membrane was perfused with Na⁺-free saline. However, perfusion with 145 mM Na⁺ triggered inward currents, which disappeared immediately after stopping the perfusion of Na⁺ (Fig. 12). The inward direction of the Na⁺-triggered current indicates that Na⁺ is not the conducted ion since Na⁺ would flow outward under these recording conditions. We conclude from these results that cultured embryonic rat hippocampal neurones express Na⁺-activated K⁺ channels. However, these channels are either expressed at very low density or in a highly clustered pattern with only 6 % of thirty-five excised somal patches exhibiting unitary Na⁺-activated K⁺ currents. This is consistent with previous reports by Egan et al. (1992a). Low densities have also been reported in other preparations. For example, only nine of about 100 membrane patches from the soma of rat motoneurones contained K_Na channels (Safronov & Vogel, 1996).

The conductance of K_Na channels has been reported to vary almost 10-fold, from about 30 pS (Koh et al. 1994) to more than 200 pS (Kameyama et al. 1984). This may be due to the different origins of the cells and the intrinsic properties of K_Na channel expressed by different cell types. The conductance of K_Na channels is also influenced by the ion composition across the membrane. For example, the conductance of the K_Na channel in quail trigeminal ganglion neurones was 50 pS under physiological conditions but 174 pS when the intra- and extracellular potassium concentrations were 75 and 150 mM, respectively (Haimann et al. 1990). The two preferred amplitudes of the K_Na channel in our recordings were 1·3 and 3·3 pA at 0 mV, equivalent to about 15 and 39 pS in the presence of 5·4 mM K_i and 145 mM K_o, respectively. These conductances are considerably smaller than those recorded in heart myocytes (130-207 pS; Kameyama et al. 1984; Luk & Carmeliet, 1990) but close to those recorded under similar, and more physiological conditions in the nodal region of myelinated axons of Xenopus (34 pS; Koh et al. 1994), rat motoneurones (44·8 pS; Safronov & Vogel, 1996), quail trigeminal ganglion neurones (50 pS; Haimann et al. 1990) and chick brainstem neurones (50 pS; Dryer et al. 1989).

Kainate evokes Na⁺ influx that activates K_Na channels

In cultured, intact, rat hippocampal neurones, the steady-state, baseline [Na⁺]_i is 9 mM as measured with a Na⁺-sensitive fluorescent indicator dye and digital videomicroscopy (Pinelis et al. 1994; Rose & Ransom, 1997). However, spontaneous [Na⁺]_i transients could be as high as 20 mM (Rose & Ransom, 1997), a level high enough to activate K_Na channels in some cell types (Dryer et al. 1989; Haimann et al. 1990; Dale, 1993; Koh et al. 1994; Safronov & Vogel, 1996). Moreover, exposing cultured neurones to 50 µM glutamate could induce, through activation of both NMDA and non-NMDA receptors, an increase in intracellular Na⁺ to 60 mM that could last for more than 10 min after removal of glutamate (Kiedrowski et al. 1994; Pinelis et al. 1994). Furthermore, increases in intracellular Na⁺ resulting from brief depolarizing voltage commands can activate K_Na channels (Dryer et al. 1989; Haimann et al. 1990). Thus, it is not surprising that 50 µM kainate could cause elevated [Na⁺]_i and activate Na⁺-activated K⁺ channels.

Other properties and possible roles for K_Na channels in rat hippocampal neurones

We found that Li⁺ could replace Na⁺ both in carrying kainate-evoked current and in activating K_Na channels. This is consistent with the result obtained in isolated spinal neurones of Xenopus embryos (Dale, 1993) but in clear contrast to other reports where Li⁺ failed to activate K_Na (for a review see Dryer, 1994). In our experiments, I_delay was recorded with a pipette solution containing 145 mM CsCl. This suggests that K_Na channels in these cultured embryonic rat hippocampal neurones were not sensitive to intracellular Cs⁺. However, 5 mM extracellular Cs⁺ completely blocked I_delay. Similarly, 5 mM Ba²⁺ also completely blocked I_delay, consistent with the findings by Miyamoto et al. (1996). TEA and 4-AP were also capable of blocking I_delay but only at relatively high concentrations (20 mM and 10 mM, respectively). These results were consistent with those reported previously in other preparations (Dryer et al. 1988; Koh et al. 1994).

It is not yet clear what possible physiological roles these K_Na channels and their activation by glutamate or by a train of action potentials could play in the biology of hippocampal neurones. It seems unlikely that K_Na channels contribute significantly to the establishment and maintenance of resting membrane potential in these neurones because of the low resting levels of intracellular Na⁺ concentrations (< 10 mM; Pinelis et al. 1994; Rose & Ransom, 1997) and low concentrations of extracellular glutamate achieved by high-affinity glutamate transporters in the plasma membrane of neurones and surrounding glial cells (Robinson & Dowd, 1997; Takahashi et al. 1997). However, failure or reversal of these transporters during hypoxia or ischaemia may result in an elevated extracellular glutamate concentration and over-activation of glutamate receptors, both of which contribute to cellular damage (Choi, 1988; Takahashi et al. 1997). Other situations leading to intensive activation of glutamate receptors include the induction of long-term potentiation, a process widely considered to be a cellular correlate of associative memory, and epilepsy. Under these circumstances K_Na channels could be opened by increased [Na⁺]_i caused by Na⁺ influx through non-NMDA glutamate receptor-cation channels and high frequency action potential activity. Activation of these channels would be likely to hyperpolarize hyperexcited cells.

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Corresponding author

Q.-Y. Liu: Laboratory of Neurophysiology, Building 36/Room 2C02, NINDS, NIH, 9000 Rockville Pike, Bethesda, MD 20892, USA.

Email: liuqy{at}codon.nih.gov

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