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Originally published In Press as doi:10.1074/jbc.M509610200 on October 20, 2005

J. Biol. Chem., Vol. 280, Issue 50, 41404-41411, December 16, 2005
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Specific Enhancement of SK Channel Activity Selectively Potentiates the Afterhyperpolarizing Current IAHP and Modulates the Firing Properties of Hippocampal Pyramidal Neurons*

Paola Pedarzani{ddagger}1, Jaime E. McCutcheon{ddagger}2, Gregor Rogge{ddagger}3, Bo Skaaning Jensen§4, Palle Christophersen§, Charlotte Hougaard§, Dorte Strøbæk§, and Martin Stocker¶

From the {ddagger}Department of Physiology, University College London, Gower Street, London WC1E 6BT, United Kingdom, §NeuroSearch A/S, Pederstrupvej 93, DK-2750 Ballerup, Denmark, and Laboratory for Molecular Pharmacology, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom

Received for publication, August 31, 2005 , and in revised form, October 14, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SK channels are Ca2+-activated K+ channels that underlie after hyperpolarizing (AHP) currents and contribute to the shaping of the firing patterns and regulation of Ca2+ influx in a variety of neurons. The elucidation of SK channel function has recently benefited from the discovery of SK channel enhancers, the prototype of which is 1-EBIO. 1-EBIO exerts profound effects on neuronal excitability but displays a low potency and limited selectivity. This study reports the effects of DCEBIO, an intermediate conductance Ca2+-activated K+ channel modulator, and the effects of the recently identified potent SK channel enhancer NS309 on recombinant SK2 channels, neuronal apamin-sensitive AHP currents, and the excitability of CA1 neurons. NS309 and DCEBIO increased the amplitude and duration of the apamin-sensitive afterhyperpolarizing current without affecting the slow afterhyperpolarizing current in contrast to 1-EBIO. The potentiation by DCEBIO and NS309 was reversed by SK channel blockers. In current clamp experiments, NS309 enhanced the medium afterhyperpolarization (but not the slow afterhyperpolarization sAHP) and profoundly affected excitability by facilitating spike frequency adaptation in a frequency-independent manner. The potent and specific effect of NS309 on the excitability of CA1 pyramidal neurons makes this compound an ideal tool to assess the role of SK channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In hippocampal pyramidal neurons voltage-independent, Ca2+-activated K+ channels are responsible for the generation of two distinct afterhyperpolarizing currents, IAHP5 and sIAHP (1-4). IAHP is characterized by a time constant of decay of ~100 ms and by its sensitivity to the bee venom toxin, apamin, and to the scorpion toxins, scyllatoxin and tamapin (5-7). sIAHP is characterized by a slower time course (in the range of seconds), by its lack of sensitivity to apamin or any other classical K+channel blocker, and by its modulation by several neurotransmitters (1-3, 8). Based on their kinetic and pharmacological features and on the results obtained from genetically manipulated mice, SK channels mediate IAHP, whereas the molecular correlate for sIAHP is still unknown (2-4, 9, 10).

In addition to the use of selective blockers, an important contribution to the elucidation of the physiological role of SK and IK channels has arisen from the use of a small organic compound that enhances channel activity, the benzimidazolinone 1-EBIO (11-15). 1-EBIO enhances the activity of SK channels in the presence of the physiological activator, intracellular Ca2+, by increasing the apparent sensitivity of SK channels to Ca2+ (14). As a consequence, 1-EBIO increases the amplitude of SK-mediated AHP currents and their duration in a variety of neurons, leading to profound changes in neuronal activity and firing patterns (14, 16-18). Although 1-EBIO has been a useful tool to elucidate the function of SK channels in their native context, it has some important limitations. First, it affects not only the SK channels but also the as yet unidentified Ca2+-dependent K+ channels underlying sIAHP (14). Additionally, prolonged applications of 1-EBIO have been shown to lead to a decrease in Ca2+ currents in hippocampal neurons (14). Finally, and most importantly, 1-EBIO displays a relatively low potency (EC50 on SK channels ~700 µM) (14). These limitations of 1-EBIO have prompted the development of novel, more potent SK channel enhancers. DCE-BIO, a dichlorinated analogue of 1-EBIO, has been reported to enhance the activity of intermediate conductance Ca2+-activated K+ channels (IK channels) (19). Moreover, recently 6,7-dichloro-1H-indole-2,3-dione-3-oxime (NS309) (20) has been described as a more potent enhancer of the activity of recombinant SK and IK channels.

In the present study, we provide the first characterization of the effect of DCEBIO on recombinant SK channels and a quantification of the potency differences between DCEBIO, NS309, and 1-EBIO on recombinant SK2 channels, the predominant SK channel subtype in hippocampus. We have furthermore investigated the actions of DCEBIO and NS309 on the native SK channels mediating IAHP, on the distinct Ca2+-activated K+ current sIAHP, and on the firing behavior of CA1 pyramidal neurons in hippocampal slices.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Electrophysiology on Recombinant hSK2 and Kv7.2/7.3 Channels— HEK293 cells stably expressing human SK2 (20) or co-expressing Kv7.2 and Kv7.3 channel subunits (21) were plated on coverslips 12-24 h prior to the experiments. For each experiment, a coverslip was placed in a 15-µl perfusion chamber (flow rate ~1 ml/min). All experiments were performed at room temperature (20-22 °C) using borosilicate pipettes (resistance 2-3 megohms) controlled by a micromanipulator (Patch-Man, Eppendorf, Germany). The extracellular solution contained (in mM): 140 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, adjusted to pH 7.4 with KOH. The osmolarity of the extracellular solution was 285 mosM. For inside-out experiments on the hSK2 currents, three bath (intracellular) solutions were used and denoted as 0.01, 0.2, and 10 to indicated their calculated free [Ca2+] in µM. Free concentrations of Ca2+ and Mg2+ were calculated using EqCal software (Biosoft, United Kingdom) and verified by a Ca2+-selective electrode (World Precision Instruments). The solutions containing 0.01 and 0.2 µM free [Ca2+] included (in mM): 1 free Mg2+, 10 EGTA, 10 HEPES, and 123 KCl or 110 KCl, respectively. The solution containing 10 µM free [Ca2+] included (in mM): 1 free Mg2+, 1 EGTA, 9 NTA, 10 HEPES, and 120 KCl. Adjustment of pH with KOH resulted in a final [K+] of 154 mM. The osmolarity of intracellular solutions was ~280 mosM. Kv7.2/7.3 currents were measured in whole-cell experiments with an intracellular solution consisting of (in mM): 120 KCl, 5.374 CaCl2, 1.75 MgCl2, 10 HEPES, 4 Na-ATP, 0.4 GTP (pH 7.2 with KOH). In hSK2 experiments, linear voltage ramps (-80 to +80 mV, 200 ms duration) were applied every 5 s from a holding potential of 0 mV. The Kv7.2/7.3 channels were activated by a 1-s step to -30 mV (close to the voltage of half-maximal activation), and the deactivation was followed for 1 s at -60 mV (close to the activation threshold) before stepping to the holding potential of -90 mV. The protocol was applied every 5 or 10 s.

Electrophysiology on Brain Slices—350 µm thick transverse hippocampal slices were prepared from Sprague-Dawley rats (20-23 days old) with a vibratome (VT 1000S, Leica, Germany) and subsequently incubated in a humidified interface chamber at room temperature for ≥1 h. Tight seal whole-cell voltage clamp recordings were obtained from 58 CA1 pyramidal neurons using the "blind method" (22). Patch electrodes (4-6 megohms) were filled with an intracellular solution containing (in mM): 135 potassium gluconate, 10 KCl, 10 HEPES, 2 Na2-ATP, 0.4 Na3-GTP, 1 MgCl2 (osmolarity 280-300 mosM, pH 7.2-7.3, with KOH). 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (8CPT-cAMP; 50 µM) was included to suppress sIAHP when the apamin-sensitive IAHP was measured in isolation. Recordings were performed in a submerged recording chamber with a constant flow of artificial cerebrospinal fluid (2 ml/min) at room temperature. Artificial cerebrospinal fluid contained (in mM): 125 NaCl, 1.25 KCl, 2.5 CaCl2, 1.5 MgCl2, 1.25 KH2PO4, 25 NaHCO3, 16 d-glucose, and was bubbled with carbogen (95% O2/5% CO2).

All neurons included in this study had a resting membrane potential below -55 mV (-63 ± 1 mV) and an input resistance of 195 ± 10 megohms. Neurons were voltage-clamped at -50 mV, and 100-ms-long depolarizing pulses to +10 mV were delivered every 30 s to elicit robust and reliable Ca2+ action currents in the presence of 0.5 µM tetrodotoxin and 1 mM tetraethylammonium, leading to the activation of the Ca2+-dependent IAHP and sIAHP (see Refs. 5, 23, and 24). The amplitude of IAHP and sIAHP were estimated 50-80 ms and 1 s after the end of the depolarizing pulse, respectively. In current clamp recordings, tetrodotoxin and tetraethylammonium were omitted, and action potentials were elicited by current injections from the resting membrane potential. Only cells with a stable resting potential throughout the current clamp protocols (±1 mV) were included in the analysis of the current clamp data. Series resistance (range 15-25 megohms) was monitored at regular intervals throughout the recording. All recordings included in this study presented minimal variations (≤10%) of the series resistance and of the amplitude and duration of the Ca2+ action current, well within the limits needed to maintain a stable amplitude of IAHP and sIAHP under control conditions. Data are reported without corrections for liquid junction potentials.

Data Acquisition and Analysis—Data were acquired using a patch clamp EPC9 amplifier (HEKA, Lambrecht, Germany) filtered at 0.25-1 kHz, sampled at 1-4 kHz, and stored on a Macintosh G4 or Power PC. Analysis was made using the Pulse and Pulsefit (HEKA, Lambrecht, Germany), Igor Pro (Wave Metrics), SigmaPlot (SPSS, Inc.), InStat (Graphpad), and Excel (Microsoft) software. Values are presented as mean ± S.E. For statistical analysis, the Student's t test was used, and differences were considered statistically significant when p < 0.05. Concentration-response relationships were fitted to the Hill equation I/Imax = [E]n/([E]n + (EC50)n) to obtain EC50 values and Hill-coefficients (n). [E] is the concentration of the enhancer.

Pharmacology on Cells and Brain Slices—Drugs were applied in the bath solution. NS309, DCEBIO, 1-EBIO, and retigabine were dissolved in Me2SO as 500-1000-fold concentrated stock solutions and stored at -20 °C, diluted prior to use, and bath-applied in the perfusing artificial cerebrospinal fluid. All controls were performed in Me2SO at the same final concentration as during NS309, DCEBIO, or 1-EBIO application (0.005-0.3%). NS309 was synthesized at NeuroSearch A/S6; DCEBIO was from Tocris (Bristol, UK) or synthesized at NeuroSearch A/S; 1-EBIO was from Sigma-Aldrich; retigabine (N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester) was synthesized at NeuroSearch; tetraethylammonium, potassium gluconate, Na2-ATP, Na3-GTP, 8CPT-cAMP, and dimethyl sulfoxide (Me2SO) were obtained from Sigma; tetrodotoxin was from Alomone Laboratories (Jerusalem, Israel); noradrenaline and d-tubocurarine were from RBI (Natick, New Jersey); apamin was from Latoxan (Rosans, France); all other salts and chemicals were obtained from Merck or Sigma.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DCEBIO and NS309, SK Channel Enhancers More Potent than 1-EBIO—DCEBIO and NS309 have been shown to modulate recombinant IK and SK channels, respectively, at lower concentrations than required for 1-EBIO. To compare their relative potency on recombinant SK channels, we have first characterized the effect of DCEBIO on SK2 channels, in view of the essential role played by the SK2 (KCa2.2) subunits in mediating IAHP in hippocampal pyramidal neurons (5, 7, 9). We then compared the effect of DCEBIO to that of NS309 and 1-EBIO on SK2 channels. The three enhancers augment SK channel activity by increasing the apparent sensitivity to Ca2+, and concentration-response experiments were therefore performed in the inside-out configuration, which allows full control of the free [Ca2+]. Activation curves yielded an EC50 of 0.42 µM for Ca2+ (n = 8) (data not shown) with maximal activation obtained at 10 µM free Ca2+. The concentration-response curves for the three enhancers were performed at 200 nM free Ca2+, which activated 5% (0.048 ± 0.009) of the maximal SK current. Fig. 1A shows the control currents (Ctrl) as well as the currents obtained in the presence of the enhancers upon application of voltage ramps from -80 to +80 mV. For all compounds, potentiation was concentration-dependent but not voltage-dependent. The traces shown in Fig. 1A were obtained from experiments similar to the one shown in Fig. 1B, where the current at -75 mV is depicted as a function of time. The inside-out patch was exposed first to 0.01 µM Ca2+ to determine the background current level and subsequently to 10 µM Ca2+ to define the maximal current, which was used to normalize the currents. The concentration-response for DCEBIO was then determined at the subthreshold Ca2+ concentration of 200 nM, with control of maximal and background currents at the end of the experiment (Fig. 1B). The currents measured at the steady-state level of activation were plotted as a function of the DCEBIO concentration as illustrated in Fig. 1C, together with the values obtained for 1-EBIO and NS309 in similar experiments. Fig. 1C underscores the higher potency on SK2-mediated currents of DCEBIO (EC50 = 27 µM; n = 1.4) and even more remarkably of NS309 (EC50 = 0.62 µM; n = 1.4) when compared with 1-EBIO (EC50 = 453 µM; n = 1.6). Furthermore, all three compounds were found to have an efficacy of 100% with respect to saturating [Ca2+].



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  		FIGURE 1.
NS309, DCEBIO, and 1-EBIO potentiate recombinant SK2 channel activity but do not affect Kv7.2/7.3 currents. A, current measured from inside-out patches obtained from HEK293 cells stably expressing hSK2 upon application of 200-ms-long voltage ramps (-80 to +80 mV). Each current-voltage plot shows a control trace and two traces obtained in the presence of NS309 (left panel), DCEBIO (middle panel), or 1-EBIO (right panel) at the concentrations (µM) indicated at the traces. The free [Ca2+] in the bath/intracellular solution was 200 nM for all traces. B, hSK2 current at -75 mV obtained from voltage ramps (A) depicted as a function of time. The inside-out patch was exposed to a [Ca2+]i of 0.01, 0.2, or 10 µM as indicated. In the presence of 200 nM free [Ca2+]i, the patch was exposed to increasing DCEBIO concentrations (0.1-300 µM) as indicated by the bars. C, concentration-response curves for NS309, DCEBIO, and 1-EBIO in the presence of 200 nM Ca2+. Currents were normalized with respect to the effect of 10 µM Ca2+, and data points represent mean ± S.E. of three experiments. The solid lines are the fit of data to the Hill equation yielding the following EC50 and Hill coefficients, respectively: NS309, 0.62 µM and 1.4; DCE-BIO, 27 µM and 1.4; 1-EBIO, 453 µM and 1.6. D, bar diagram summarizing the lack of effect of 10 µM NS309 (105 ± 3%; n = 6), 100 µM DCEBIO (91 ± 9%; n = 5), and 1 mM 1-EBIO (93 ± 3%; n = 6) on Kv7.2/7.3 currents. The effects (measured at the end of the step to -30 mV) were normalized to the control current, with no effect equal to 100%. E, whole-cell currents measured from a HEK293 cell co-expressing Kv7.2/7.3 channel subunits. Each panel shows the current just before (Control) and after perfusion with DCEBIO (100 µM), retigabine (3 µM), 1-EBIO (1 mM), or NS309 (10 µM). Kv7.2/7.3 currents were activated by a 1-s-long step to -30 mV, and deactivation was followed by a subsequent step to -60 mV. The holding potential was -90 mV, and the protocol was applied every 10 s. F, Kv7.2/7.3 current at -60 mV (circles) and -30 mV (squares) obtained from voltage steps as in D, depicted as a function of time. The cell was exposed to the compounds during the periods indicated by the bars. 0.2% Me2SO was tested as a vehicle for the 1 mM 1-EBIO application.

 



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  		FIGURE 2.
Effect of DCEBIO on neuronal AHP currents. A,IAHP and sIAHP were measured in CA1 pyramidal neurons in response to depolarizing pulses activating Ca2+ influx through voltage-gated Ca2+ channels. IAHP was enhanced by application of DCEBIO (100 µM). The enhanced IAHP was blocked by the SK channel blocker d-tubocurarine (curare, 100 µM), whereas the remaining, unaffected sIAHP was inhibited by noradrenaline (1 µM). B, superimposition of IAHP and sIAHP traces, before (Control, gray) and after (DCEBIO, black) application of 100 µM DCEBIO, emphasizing the effect of this compound on the amplitude and time course of deactivation of IAHP. C, bar diagram summarizing the effect of 100 µM DCEBIO on the IAHP and sIAHP peak amplitudes (ampl.) and total integral of the two AHP currents in seven cells. * indicates statistical significance (p < 0.05).

 
Kv7 channels underlie IM, a current contributing to the generation of the mAHP in hippocampal neurons (26-28). We have therefore tested the SK channel enhancers on recombinant Kv7.2/7.3 channels. Fig. 1, D-F, illustrates the lack of effect of DCEBIO (100 µM), NS309 (10 µM), and 1-EBIO (1 mM) on these channels at the highest concentrations used for recordings in brain slices in this study. Fig. 1E shows the Kv7.2/7.3 currents activated by a 1-s-long step to -30 mV before and after the addition of the SK enhancers, as well as the reference Kv7 activator retigabine (3 µM). The time course in Fig. 1F depicts the current at the end of the step to -30 mV (open circles) and at the end of the step to -60 mV (open boxes). The bar diagram in Fig. 1D summarizes the effects from 5-6 experiments and shows that neither DCEBIO nor NS309 nor 1-EBIO significantly affect the Kv7.2/7.3 current, even at the highest concentrations tested.

DC-EBIO and NS309 Selectively Increase IAHP but Not sIAHP in Hippocampal Pyramidal Neurons—When tested on CA1 pyramidal neurons in acute hippocampal slices, DCEBIO potentiated the SK-mediated Ca2+-activated K+ current IAHP without affecting the sIAHP at all concentrations tested (10 and 50 µM; n = 4) (data not shown) (100 µM; n = 7) (Fig. 2). In particular, 100 µM DCEBIO increased the amplitude of the SK-mediated IAHP by 195 ± 40% and prolonged its duration by 319 ± 42%. sIAHP co-exists with IAHP in CA1 pyramidal neurons but is mediated by channels clearly distinct from the SK channels underlying IAHP and of as yet unknown molecular identity (2-5,9). Neither the sIAHP amplitude (118 ± 10%) nor its time constant of decay (112 ± 5%) were significantly increased by DCEBIO (Fig. 2). The remarkable potentiation of IAHP led to an increase of the charge transfer, estimated as the integral of the two AHP currents (IAHP + sIAHP), by 154 ± 16% (n = 7) (Fig. 2C). The specificity of the DCEBIO effect on neuronal SK channels was confirmed by the full block of the enhanced IAHP upon application of the SK channel blocker d-tubocurarine (curare, 100 µM) (Fig. 2A). sIAHP was instead identified by its suppression by noradrenaline (1 µM) (Fig. 2A), known to inhibit this current by activating the cAMP/protein kinase A pathway in hippocampal neurons (23, 29). These results demonstrate that DCEBIO is a more potent enhancer of both recombinant and neuronal SK currents when compared with 1-EBIO.

The rest of our study focuses on NS309, which displays an enhanced potency on recombinant SK2 channels compared with both 1-EBIO and DCEBIO (Fig. 1C). A crucial question is how effective and selective this compound is on SK channels in their native, neuronal environment. When tested on IAHP and sIAHP in CA1 pyramidal neurons in hippocampal slices, 10 µM NS309 induced a marked increase of IAHP amplitude with respect to the control currents recorded prior to application of the compound (Fig. 3A). IAHP was measured in isolation, upon inhibition of sIAHP by the cAMP analogue 8CPT-cAMP (50 µM). The relative increase in amplitude of the apamin-sensitive IAHP was 182 ± 22% (n = 8) (Fig. 3, A and C). NS309 had an even more prominent effect on the time constant of deactivation of IAHP, which was slowed by ~6-fold, changing from 119 ± 19 ms to 654 ± 77 ms after NS309 application (n = 8). As a consequence, the charge transfer of IAHP measured as the integral of the current was increased by almost 10-fold (949 ± 165%; n = 8) (Fig. 3, A-C). By comparison, the application of the same concentration of DCEBIO (10 µM) resulted in an increase of the IAHP amplitude by 158 ± 20% and in an increase of its decay time constant by 149 ± 20% (n = 4) (data not shown). NS309 was applied for several minutes to yield a maximal and stable potentiation of IAHP, as illustrated by the time course of action of this drug (Fig. 3B). The augmentation of IAHP by NS309 was only scarcely reversible, even after prolonged wash out periods (data not shown). The application of NS309 (10 µM) did not affect the input resistance of the neurons (n = 18).

At a lower concentration (1 µM), NS309 had qualitatively similar but slightly less pronounced effects on both amplitude and charge transfer of IAHP. Thus, IAHP amplitude was increased to 139 ± 10% (n = 3) (Fig. 3, D and F). Similar to what was previously observed at higher concentrations, 1 µM NS309 slowed the deactivation of IAHP by ~4-fold, increasing its time constant of decay ({tau}) from 86 ± 9 ms to 361 ± 39 ms (n = 3). As a consequence, the IAHP charge transfer increased by ~5-fold (500 ± 18%; n = 3) (Fig. 3, D-F). Also at 1 µM, the action of NS309 developed slowly (Fig. 3E). The effect of NS309 can be entirely ascribed to an enhancement in the activity of the SK channels underlying IAHP, as the NS309-enhanced current was fully blocked by the SK channel blockers d-tubocurarine (d-TC, curare; 200 µM; n = 8) (Fig. 3, A and B) and apamin (25 nM; n = 3) (Fig. 3, D and E).

Next, to investigate the effects of NS309 (10 µM) on the apamin-insensitive AHP current sIAHP, we applied the compound together with 25 nM apamin to block IAHP. NS309 affected neither the amplitude (96 ± 13%; p = 0.63; n = 3) (Fig. 4) nor the charge transfer (108 ± 15%; p = 0.67; n = 3; Fig. 4, A and C) of the sIAHP once it had reached steady state. Furthermore, after NS309 application, sIAHP was fully inhibited by 2.5 µM noradrenaline (Fig. 4, A and B). This result underscores the selective nature of NS309 as an SK channel enhancer in contrast to 1-EBIO, which increased both IAHP and sIAHP in CA1 pyramidal neurons (14). Thus, we can conclude that NS309 is a selective enhancer of the SK channels mediating IAHP in hippocampal neurons and more potent than both 1-EBIO and DCEBIO.



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  		FIGURE 3.
NS309 enhances the apamin-sensitive IAHP in CA1 pyramidal neurons. A and D,IAHP measured in isolation in the presence of 50 µM 8CPT-cAMP (left panels in A and D) is potentiated by the application of 10 µM NS309 (A) and even by 1 µM NS309 (D). The potentiated IAHP is fully blocked by 200 µM d-tubocurarine (A; Curare)or25 nM apamin (D). The right panels in A and D show a superimposition of scaled IAHP traces before (Control, black) and after (NS309, gray) application of 10 µM (A) and 1 µM NS309 (D), emphasizing the effect of this compound on the time course of deactivation of IAHP. B and E, time course of action of NS309 (10 µM in B; 1 µM in E) on the IAHP shown as charge transfer and estimated from the integral of each current trace. IAHP was elicited every 30 s. NS309 was applied until the potentiated IAHP reached a stable amplitude (ampl.) and area. The application of NS309 was followed by d-tubocurarine (d-TC; 200 µM) in B and apamin (25 nM) in E, which completely blocked the enhanced IAHP. In B, points were omitted after IAHP reached steady-state level to allow for testing of the access resistance. The diagrams in B and E are from the same representative cells shown in A and D. C and F, bar diagrams summarizing the effects of 10 µM NS309 (C) and 1 µM NS309 (F) on the IAHP peak amplitude and charge transfer in eight (C) and three cells (F).

 
NS309 Alters the Firing Pattern of Hippocampal Pyramidal Neurons— The selective potentiation of IAHP, without any effect on sIAHP, produced by NS309 prompted us to use this compound to test the specific impact of SK channel enhancement on the firing properties of hippocampal neurons. This was investigated in current clamp recordings performed in the absence and presence of NS309 (10 µM). Under control conditions, depolarizing current pulses elicited trains of action potentials characterized by early and late spike frequency adaptation (Fig. 5A, left panel). Application of NS309 decreased the firing frequency of all CA1 pyramidal neurons tested. As shown in Fig. 5A, middle panel, in the presence of NS309, most cells fired only 3-4 action potentials in a burst-like fashion followed by a prolonged silent phase. This marked effect of NS309 was fully counteracted upon application of the SK channel blocker apamin (25 nM) (Fig. 5A, right panel). In the same cells, the effect on the firing pattern coincided with a pronounced enhancement of the medium AHP (Fig. 5B, mAHP), whereas the slow AHP (Fig. 5B, sAHP) was not affected (n = 3), in accordance with the lack of effect of NS309 on sIAHP observed in voltage clamp recordings (Fig. 4).

The NS309-mediated specific enhancement of IAHP amplitude and duration, which affects in turn the medium and late phases of spike frequency adaptation, reveals a prominent effect of SK channels on neuronal excitability and signal encoding properties.

Does the Potentiation of IAHP by NS309 Depend on Frequency?—The slow time course of action of NS309, with time constants in the range of 6-8 min to reach the maximal effect on IAHP, raised the question as to whether the effect of this compound on IAHP is frequency-dependent. To test this hypothesis, we designed three experimental paradigms. In the first paradigm, short current injections (5 ms long) of sufficient intensity to elicit action potentials were applied in trains of eight at a frequency of 10 Hz (Fig. 6A)(n = 5) or 33 Hz (Fig. 6B)(n = 5) in current clamp experiments. This stimulation pattern mimics the physiological input from bursting CA3 neurons to CA1 neurons (30, 31). Upon application of NS309 (10 µM), the mAHP following the single action potentials and the trains of spikes were increased both in amplitude and duration (Fig. 6, A and B, right panels). The increase in mAHP amplitude was not significantly different at 10 Hz compared with 33 Hz (Fig. 6B, left panel inset). However, in two of the cells stimulated at 33 Hz, the increase of mAHP after the application of NS309 was large enough to prevent firing in response to some of the current injections within the train (not shown).



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  		FIGURE 4.
NS309 does not affect the apamin-insensitive sIAHP in CA1 pyramidal neurons. A,sIAHP, measured in the presence of apamin (25 nM; left panel) to block IAHP, is not affected by the application of 10 µM NS309. 2.5 µM noradrenaline completely inhibited sIAHP. The right panel shows a superimposition of sIAHP traces before (black) and after (gray) the application of 10 µM NS309, emphasizing the lack of effect of this compound on the peak amplitude and time course of sIAHP. B, lack of effect of NS309 (10 µM) on the peak amplitude (ampl.) of sIAHP plotted against time. NS309 was applied for 18 min after the sIAHP amplitude had stabilized and produced no effect on the sIAHP amplitude. The subsequent application of noradrenaline (NA; 2.5µM) induced a complete suppression of sIAHP. The time course is from the same representative cell shown in A. C, bar diagram showing that 10 µM NS309 had no effect on the sIAHP peak amplitude and charge transfer.

 
In the second paradigm, depolarizing steps (100 ms long to +10 mV) were delivered in the presence of tetrodotoxin and tetraethylammonium to elicit Ca2+ action currents in voltage clamp every 30 s (0.033 Hz; n = 8) (Fig. 6, C-E) or 6 s (0.167 Hz; n = 5) (Fig. 6, F-H). Fig. 6, C and F, shows that at both frequencies, NS309 (10 µM) caused a substantial increase in the amplitude and duration of IAHP and that the enhanced current was fully suppressed by the application of curare (Fig. 6, C and D, d-TC) (200 µM) or apamin (50 nM) Fig. 6, F and G). Neither the enhancement of IAHP amplitude (Fig. 6E) nor its charge transfer (Fig. 6H) nor the time course of potentiation of IAHP by NS309 (Fig. 6I) were significantly different at 0.033 Hz compared with 0.167 Hz.

Finally, the third paradigm comprised trains of eight short depolarizing steps (5 ms to +10 mV) delivered either at 10 or 33 Hz in voltage clamp from a holding potential of -40 and -50 mV to mimic the stimulation pattern provided by bursts of action potentials but in voltage clamp recordings. Upon NS309 (10 µM) application, neither the time course of IAHP potentiation (Fig. 6J), nor the enhancement in amplitude and charge transfer of IAHP (not shown) were significantly different at the two frequencies tested. In conclusion, our experimental evidence does not support a frequency-dependent component in the action of NS309 on IAHP in CA1 pyramidal neurons.



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  		FIGURE 5.
NS309 reduces the firing frequency of CA1 pyramidal neurons. A, trains of action potentials elicited by 800 ms current injections (260 pA) from the resting membrane potential (-57 mV; no steady current injected) before and after the application of 10 µM NS309. In this representative cell, the number of action potentials elicited by the same current injection was decreased in the presence of NS309, and the late phase of spike frequency adaptation was enhanced. These effects were reversed upon application of apamin (25 nM; right panel), which produced an additional small increase in the number and initial frequency of the action potentials in the train, as previously reported (5). NS309 did not affect the resting membrane potential or the input resistance of the cell. Similar results were obtained in three cells. B, afterhyperpolarizations (medium AHP, mAHP; slow AHP, sAHP) following a burst of action potentials triggered by a 200 ms current injection (500 pA) before and after the application of 10 µM NS309. NS309 enhanced the mAHP (superimposed traces in right panel) without affecting the sAHP. The effect of NS309 on mAHP was fully counteracted upon application of apamin (25 nM), in agreement with the results obtained on the underlying currents (IAHP and sIAHP) in voltage clamp recordings. Action potentials were truncated for better resolution of the after hyperpolarizations following the bursts. Similar results were obtained in three cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results obtained in this study show that, in accordance with their effects on recombinant channels, DCEBIO and even more prominently NS309 are potent and specific enhancers of the activity of neuronal SK channels. In particular, the SK-mediated IAHP was increased in amplitude and even more remarkably in duration by the application of concentrations as low as 1 µM NS309 (Fig. 3). NS309 did not influence the currents mediated by recombinant BK channels Kv7.4 (KCNQ4) (20) or Kv7.2/7.3 channels (this study) expressed in HEK293 cells. In particular, the lack of effect of NS309 on Kv7.2/7.3 channels supports the hypothesis that the enhancement of the mAHP observed in this study (Fig. 5B) is not because of an increase of IM, a voltage-dependent current contributing to the generation of the mAHP (26), but solely of IAHP. The selectivity of the effects of DCEBIO and NS309 on neuronal SK channels is further supported by the full suppression of the enhanced IAHP and mAHP by the SK channel blockers apamin and d-tubocurarine (Figs. 2, 3, 5, and 6). These results are in good agreement with the reported enhancement of recombinant SK channels by NS309, whereby the enhanced currents were completely blocked by apamin in a heterologous expression system (20). Additionally, we have not observed any effect of DCEBIO and NS309 on the amplitude or time course of sIAHP in CA1 neurons (Figs. 2 and 4). These results further strengthen the notion that sIAHP is mediated by a conductance clearly distinct from the SK channels, which are unlikely to contribute to its generation, as supported by differences in their kinetics, pharmacology, distribution, and by recent data obtained from genetically modified animals missing specific SK channel subunits (1-4, 9, 10). NS309 has been reported to block recombinant hERG channels with a Ki value of 1.3 µM (20), and hERG channels have been suggested to play a role in spike frequency adaptation in dorsal root ganglion-neuroblastoma hybrid cells (25). However, the blocking of hERG channels by NS309 would produce an effect opposite of what we observed on the firing pattern of CA1 neurons. Additionally, the changes in the firing pattern observed after the application of NS309 were fully reversed by the specific SK channel blocker apamin (Fig. 5), making it unlikely for conductances other than SK to contribute to the observed NS309 effects. Finally, even at high concentrations (10 µM), NS309 did not change the membrane resistance of the CA1 neurons (n = 18) (data not shown).



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  		FIGURE 6.
The action of NS309 is not frequency-dependent in CA1 neurons. A and B, action potentials elicited by trains of 5-ms-long current injections delivered at 10 (A) and 33 (B) Hz before and after the application of 10 µM NS309. NS309 increases the afterhyperpolarization following the single action potential and the trains. The enhancement of the AHPs following single action potentials and the trains is better illustrated in the right panels, showing the two last action potentials in the trains (truncated) and the AHPs on an expanded voltage and time scale. The traces in gray are before and those in black after NS309 application. The relative increase of AHP was not significantly different at 10 and 33 Hz, as shown in the inset in B (left panel), summarizing the results obtained in five (10 Hz) and three cells (33 Hz). C and F,IAHP traces obtained in response to 100-ms-long depolarizing pulses to +10 mV (holding potential, -50 mV) delivered every 30 s (C; 0.033 Hz) or 6 s (F; 0.167 Hz). Three superimposed traces are shown, illustrating the change in amplitude and time course of IAHP before (black, Control) and after the application of 10 µM NS309 (dark gray, NS309). The third set of traces (light gray) shows that, at both frequencies, the enhanced IAHP was fully blocked by d-tubocurarine (C; d-TC, 200 µM) or apamin (F; 50 nM). D and G, time course of action of NS309 (10 µM) on the IAHP charge transfer estimated from the area under each current trace. IAHP was elicited every 30 s in D and every 6 s in G. The application of NS309 was followed by d-tubocurarine (d-TC; 200 µM)in D and by apamin (Apa; 50 nM) in G, which completely blocked the enhanced IAHP. The plots are from the same representative cells shown in C (plot D) and in F (plot G). E and H, bar diagrams summarizing the effects of 10 µM NS309 on the IAHP peak amplitude (E) and charge transfer (H) in eight and five cells stimulated at 0.033 and 0.167 Hz, respectively. No significant differences were observed in the potentiation of IAHP at these two frequencies. I, the time course of the potentiating action of 10 µM NS309 (see, for example, plots D and G) on the IAHP peak amplitude (left bars) and charge transfer (right bars) were fitted with exponential functions, and the corresponding time constants were obtained for seven cells stimulated at 0.033 Hz and four cells at 0.167 Hz (same protocol as in C and F). The time required by NS309 to have a full effect on IAHP was not significantly different at the two stimulation frequencies. J, the time course of the potentiating action of 10µM NS309 on the IAHP peak amplitude (left bars) and charge transfer (right bars) were fitted with exponential functions, and the corresponding time constants were obtained. IAHP was elicited using a sequence of eight short (5 ms) pulses to +10 mV (holding potential = -50 mV) delivered in four cells at 10 Hz and five cells at 33 Hz. The time required by NS309 to have a full effect on IAHP was not significantly different at the two stimulation frequencies.

 
When compared with the first SK channel enhancer tested on neurons, 1-EBIO (14), DCEBIO and NS309 were more potent, as expected from the 17-fold difference in the potency of 1-EBIO and DCEBIO, and the 731-fold difference of 1-EBIO and NS309 on recombinant SK2 channels reported in this study. Additionally, both DCEBIO and NS309 were more selective, as they enhanced IAHP without affecting sIAHP, whereas 1-EBIO produced a small but significant enhancement also of sIAHP (14). It could be argued that the increased selectivity of DCEBIO and NS309 on IAHP is because of their higher potency on SK channels, allowing us to use lower concentrations of these compounds on hippocampal neurons compared with 1-EBIO. However, when 1-EBIO and DCEBIO were tested at the same concentration (100 µM) on CA1 pyramidal neurons, 1-EBIO caused a significant increase of sIAHP amplitude (1.3-fold, n = 6),7 whereas DCEBIO did not affect sIAHP (n = 7) (Fig. 2C). Nonetheless, we cannot exclude that, at concentrations higher than those tested in this study, DCEBIO and NS309 could affect also sIAHP. Finally, we have observed a difference between the actions of NS309 and of 1-EBIO and DCEBIO on IAHP, in that NS309 has a more pronounced effect on the time course rather than the peak amplitude of the current when compared with 1-EBIO and DCEBIO. Although all three enhancers act in a Ca2+-dependent manner, further experiments are needed to clarify whether the pronounced change in the time course of IAHP is the result of a direct effect of NS309 on the SK single channel kinetics or of changes in the dynamics of intracellular Ca2+ in CA1 neurons. NS309 is approximately equipotent on the different SK channel subtypes (homomeric hSK1, hSK2, and hSK3 channels, as well as rat SK2 and SK3 channels) expressed in HEK293 cells (20). NS309 therefore does not provide indications on the subunit composition of the native hippocampal SK channels, which are likely to be SK2 homomeric channels or heteromers, including the SK2 subunit (5, 7, 9).

The time course of action of NS309 on IAHP in CA1 neurons is ~10-fold slower than that of 1-EBIO, whereas the effect of both compounds on recombinant SK2 channels in inside-out patches are very fast (comparable with DCEBIO as shown in Fig. 1). The results of our experiments using different stimulation frequencies to elicit IAHP (Fig. 6) argue against a use dependence of the NS309 effect on IAHP. The slow time course of action of NS309 when compared with 1-EBIO might instead be due to a slower penetration of NS309 through the slice.

The potent and specific effect of NS309 on the excitability and firing behavior of CA1 pyramidal neurons through the enhancement of SK channel activity makes this compound a better tool than 1-EBIO and DCEBIO to assess the role of these channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability.


   FOOTNOTES
 
* This work was supported by a Career Establishment grant from the United Kingdom Medical Research Council (to P. P.) and a Wellcome Trust Senior Research fellowship (to M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

2 A four-year Wellcome Ph.D. Programme in Neuroscience predoctoral fellow. Present address: Dept. of Anatomy and Developmental Biology, University College London, Gower St., London WC1E 6BT, UK. Back

3 Supported by the Studienstiftung des Deutschen Volkes. Back

4 Present address: Arpida A/S, Vesterbrogade 188, DK-1800 Frederiksberg, Denmark. Back

1 To whom correspondence should be addressed: Dept. of Physiology, University College London, Gower St., London WC1E 6BT, UK. Tel.: 44-20-7679-7744; Fax: 44-20-7383-7005; E-mail: P.Pedarzani{at}ucl.ac.uk.

5 The abbreviations used are: IAHP, apamin-sensitive afterhyperpolarizing current; AHP, afterhyperpolarization; sIAHP, slow afterhyperpolarizing current; sAHP, slow AHP; mAHP, medium AHP; DCEBIO, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; 1-EBIO, 1-ethyl-2-benzimidazolinone; NS309, 6,7-dichloro-1H-indole-2,3-dione-3-oxime; cAMP, cyclic AMP; 8CPT-cAMP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate; SK channel, small conductance Ca2+-activated K+ channel; IK channel, intermediate conductance Ca2+-activated K+ channel; HEK, human embryonic kidney. Back

6 Jensen, B. S., Jørgensen, T. D., Ahring, P. K., Christophersen, P., Strøbaek, D., Teuber, L., and Olesen, S. P. (1999) NeuroSearch A/S, assignee, International Patent Application WO 00/33834. Back

7 G. Rogge and P. Pedarzani, unpublished observation. Back



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 RESULTS
 DISCUSSION
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