Control of Electrical Activity in Central Neurons by Modulating the Gating of Small Conductance Ca2+-activated K+ Channels*

Paola PedarzaniDagger , Johannes Mosbacher§, Andre Rivard, Lorenzo A. CingolaniDagger , Dominik Oliver||, Martin StockerDagger **, John P. Adelman, and Bernd Fakler||DaggerDagger

From the Dagger  Max-Planck Institut für Experimentelle Medizin, 37075 Göttingen, Germany, § Novartis Pharma AG, TA Nervous System, CH 4002 Basel, Switzerland,  Vollum Institute, Oregon Health Science University, Portland, Oregon 97201, and || Department of Physiology II, University of Tübingen, 72074 Tübingen, Germany

Received for publication, November 2, 2000, and in revised form, December 22, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In most central neurons, action potentials are followed by an afterhyperpolarization (AHP) that controls firing pattern and excitability. The medium and slow components of the AHP have been ascribed to the activation of small conductance Ca2+-activated potassium (SK) channels. Cloned SK channels are heteromeric complexes of SK alpha -subunits and calmodulin. The channels are activated by Ca2+ binding to calmodulin that induces conformational changes resulting in channel opening, and channel deactivation is the reverse process brought about by dissociation of Ca2+ from calmodulin. Here we show that SK channel gating is effectively modulated by 1-ethyl-2-benzimidazolinone (EBIO). Application of EBIO to cloned SK channels shifts the Ca2+ concentration-response relation into the lower nanomolar range and slows channel deactivation by almost 10-fold. In hippocampal CA1 neurons, EBIO increased both the medium and slow AHP, strongly reducing electrical activity. Moreover, EBIO suppressed the hyperexcitability induced by low Mg2+ in cultured cortical neurons. These results underscore the importance of SK channels for shaping the electrical response patterns of central neurons and suggest that modulating SK channel gating is a potent mechanism for controlling excitability in the central nervous system.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In many central nervous system neurons, action potentials (APs)1 are followed by a prolonged afterhyperpolarization (AHP) of the membrane potential that controls excitability and firing pattern (1, 2). Two classes of AHPs and the underlying Ca2+-activated currents may be distinguished based on their time course and pharmacological properties. IAHP underlies part of the medium AHP (mAHP) following single or repetitive APs, is sensitive to the bee venom toxin apamin, and exhibits fast rise and decay (time constants approx 100 ms). sIAHP mediates the slow AHP that follows trains of APs, is apamin-insensitive, has much slower kinetics (range of seconds), and is modulated by several neurotransmitters (1). The mAHP controls the tonic firing frequency of neurons, whereas the slow AHP is responsible for spike frequency adaptation, a prominent reduction in the firing frequency in the late phase of responses to prolonged depolarizing stimuli (1).

The Ca2+-activated currents underlying different phases of the AHP have been ascribed to the activation of small conductance Ca2+-activated potassium (SK) channels (1, 3). SK channels are potassium-selective and voltage-independent and are activated by an increase in intracellular Ca2+ ([Ca2+]i) such as occurs during an AP. Three highly homologous mammalian SK channels (SK1, SK2, and SK3) have been cloned (4), as well as a related channel with an intermediate conductance (IK1; Refs. 5-7). Whereas IK channels are predominantly found in epithelial and blood cells (8-11), SK channels are widely expressed in the central nervous system (4) with high levels of expression in the regions presenting prominent AHP currents such as neocortex (SK1 and SK2), monoaminergic neurons (SK3), and CA1-3 layers in the hippocampus (SK1 and SK2) (12). As for their native counterparts, these channels are gated by [Ca2+]i in the submicromolar range independent of the transmembrane voltage. At steady state, [Ca2+]i of 0.3 - 0.5 µM leads to half-maximal activation of the channels, whereas saturation is observed with [Ca2+]i of around 10 µM (13-15).

Analysis of Ca2+ gating showed that SK and IK channels use calmodulin (CaM) constitutively associated with the C terminus of the SK/IK alpha -subunit as a high-affinity Ca2+ sensor (13, 15). Activation of the channels occurs by Ca2+ binding to at least one of the N-terminal EF hands of CaM in each of the four SK alpha -subunits, which, by the subsequent conformational changes in CaMs and the SK C termini, leads to channel opening. Channel deactivation is the reverse process and occurs upon dissociation of Ca2+ from CaM (14, 15). When modeled, Ca2+ gating of SK channels is adequately described by a multiple-state model analogous to that used for voltage-dependent gating in Shaker K+ channels (14, 16, 17). In this model, the rate-limiting transitions between closed stated are controlled by Ca2+ and strongly depend on [Ca2+]i, the open-state transition occurs rapidly and is independent of [Ca2+]i (14, 18, 19).

Recently, it was shown that the compound 1-ethyl-2-benzimidazolinone (EBIO) activates IK channels in colonic epithelia as well as in transfected cultured cells when applied extracellularly under physiological conditions (10, 20-22). Pedersen et al. (21) showed an EBIO-induced shift in the Ca2+ concentration-response relationship of IK1 currents such that [Ca2+]i of about 30 nM was enough to elicit robust channel activity. The molecular mechanism behind this shift in Ca2+ sensitivity remained unclear.

Here we investigated the molecular mechanism underlying the EBIO-mediated activation of cloned SK channels at subthreshold Ca2+ concentrations and tested the effects of EBIO on the excitability and firing pattern of hippocampal neurons and cortical neuronal networks. The results suggest that EBIO affects the interaction between channel alpha  subunits and CaM and that modulating SK channel activity is an effective mechanism for tuning excitability in central neurons.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Molecular Biology and Electrophysiology on Cloned SK Channels

In vitro mRNA synthesis and oocyte injections were performed as described previously (23). Giant patch recordings were made 3-7 days after injection. Pipettes made from thick-walled borosilicate glass had resistances of approx 0.3 megohms when filled with 120 mM KOH, 10 mM HEPES, and 0.5 mM CaCl2 (pH adjusted to 7.2 with methanesulfonic acid) or with 115 mM NaOH, 5 mM KOH, 10 mM HEPES, and 0.5 mM CaCl2 (pH adjusted to 7.2 with methanesulfonic acid). Inside-out patches were superfused with an intracellular solution containing 119 mM KOH, 1 mM KCl, 10 mM HEPES, and 1 mM EGTA (pH adjusted to 7.2 with methanesulfonic acid); all chemicals used were of the highest grade; Millipore water was treated with Chelex100 (Bio-Rad, Hercules, CA) before solution preparation. The amount of CaCl2 required to yield the concentrations indicated was calculated as described by Fabiato (24) and added to the EGTA solution under pH meter control; thereafter, pH was readjusted to 7.2 with KOH.

EBIO (Sigma (St. Louis, MO) and Tocris Cookson, Bristol, UK) was added to the intracellular solution to yield the final concentrations indicated. Rapid exchange of Ca2+ was achieved using a piezo-driven application system (25); the time constant of solution exchange with this system was 0.5 ms. Data analysis and fitting were performed with IgorPro (WaveMetrics, Lake Oswego, OR) on a Macintosh PowerPC; for the Ca2+ concentration responses in symmetrical and asymmetrical K+, currents recorded at -80 mV and -120 mV were used, respectively. All data are presented as mean ± S.D. of n experiments.

Electrophysiology on Hippocampal Neurons

Slice Preparation-- Transverse hippocampal slices (300 µM thick) were prepared from Wistar rats (23-28 days old) with a vibratome (VT 1000S Leica) and subsequently incubated in a humidified interface chamber at room temperature for >= 1 h.

Electrophysiology-- Tight-seal whole-cell voltage-clamp recordings were obtained from 28 CA1 pyramidal neurons using the "blind method" (55). Patch electrodes (4-7 megohms) were filled with an intracellular solution containing 135 mM potassium gluconate, 10 mM KCl, 10 mM HEPES, 2 mM Na2-ATP, 0.4 mM Na3-GTP, and 1 mM MgCl2 (osmolarity, 280-300 mosmol; pH 7.2-7.3 with KOH). 8-(4-Chlorophenylthio)adenosine 3',5'-cyclic monophosphate (8CPT-cAMP; 50 µM) was included to measure the apamin-sensitive IAHP in isolation. All neurons included in this study had a resting membrane potential below -55 mV (-61 ± 1 mV; n = 19) and an input resistance of 215 ± 5 megaohms (n = 19). Recordings were performed in a submerged recording chamber with a constant flow of artificial cerebrospinal fluid (2 ml/min) at room temperature. Drugs were applied in the bath solution. Artificial cerebrospinal fluid contained 125 mM NaCl, 1.25 mM KCl, 2.5 mM CaCl2, 1.5 mM MgCl2, 1.25 mM KH2PO4, 25 mM NaHCO3, and 16 mM D-glucose and was bubbled with carbogen (95% O2/5% CO2). EBIO was dissolved in Me2SO and stored at -20 °C as a 0.4 M stock solution, diluted before use, and bath-applied in the perfusing artificial cerebrospinal fluid. All controls were performed in Me2SO at the same final concentration used during EBIO application (0.25%), and no significant effects of Me2SO were detected. Neurons were voltage-clamped at -50 mV, and 100-ms-long depolarizing pulses to +10 mV were delivered every 30 s. These pulses led to unclamped Ca2+ action currents sufficient to activate IAHP and sIAHP. Series resistance was monitored at regular intervals throughout the recording, and only recordings with stable series resistances <= 25 megohms were included in this study. No series resistance compensation and no corrections for liquid junction potentials were made. Only cells with a stable resting potential throughout the current-clamp protocols (±1 mV) were included in the 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 PowerPC. Analysis was performed using the programs Pulse and Pulsefit (HEKA), Igor Pro 3.01 (Wave Metrics), and Excel (Microsoft). Values are presented as mean ± S.E. For statistical analysis, the Student's t test was used, and differences were considered statistically significant if p <=  0.05.

Chemicals-- Tetraethylammonium, potassium gluconate, Na2-ATP, Na3-GTP, 8CPT-cAMP, and Me2SO were obtained from Sigma; tetrodotoxin was obtained from Alomone Laboratories (Jerusalem, Israel); noradrenaline was obtained from RBI (Natick, MA); apamin was obtained from Latoxan (Rosans, France); samples of EBIO were obtained from Sigma and from Tocris Cookson; and all other salts and chemicals were obtained from Merck.

Fluorescence Plate Reader Experiments on Neuronal Networks

Primary cultures of cortical neurons were prepared from embryonic day 17 Harlan Sprague-Dawley rats as described by Wang and Gruenstein (26). Cells were incubated at 37 °C in 5% CO2 for 7-10 days. About 15 min before the experiments, the culture medium was removed, and cells were loaded with 2 µM fluo-4 (Molecular Probes) in Hanks' balanced salt solution (Life Technologies, Inc.) supplemented with 10 mM HEPES (pH adjusted to 7.4). After loading, cells were washed twice in Hanks' balanced salt solution + 2 mM Ca2+ without Mg2+ and then transferred into a fluorescence plate reader I fluorescence image plate reader (Molecular Devices). Fluo-4 fluorescence from approx 5000 cells (on an area of approx 10) was measured at a sampling rate of 0.5 Hz, drug application (20 µl of a 5-fold concentrated stock in 80 µl of Hanks' balanced salt solution + 2 mM Ca2+ without Mg2+) was performed within 1 s simultaneously in 96 wells during measurement. Fluorescence oscillations were analyzed by determining (i) the number of peaks above threshold and (ii) the area under curve (AUC) after baseline subtraction during intervals before and after drug application.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of SK Channels at Subthreshold Ca2+ by EBIO-- The effect of EBIO on cloned SK channels was investigated in giant inside-out patches excised from Xenopus oocytes expressing either hSK1 (SK1) or rSK2 (SK2) channels.

Fig. 1 illustrates the response of SK2 channels to cytoplasmic application of EBIO at various [Ca2+]i; SK currents were recorded under symmetrical K+ conditions at a membrane potential of -80 mV (intermittently stepped to 50 mV for 50 ms every 1 s). EBIO induced robust activation of SK2 channels at Ca2+ concentrations as low as 50 and 100 nM, which, in the absence of the benzimidazolinone, did not elicit any SK currents (Fig. 1A). In the absence of Ca2+, however, EBIO failed to activate SK currents, similar to reports on IK channels (21). Moreover, EBIO did not increase the amplitude of SK currents when applied at a saturating [Ca2+]i of 10 µM (Fig. 1A).


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Fig. 1.   EBIO shifts the Ca2+ concentration-response relationship of SK channels. A, currents recorded from inside-out patches with heterologously expressed SK2 channels at a [Ca2+]i of 0, 0.05, 0.1, and 10 µM in the absence and presence of 2 mM EBIO; dotted line represents zero current. Inset, the voltage protocol used in this experiment. B and C, Ca2+ concentration-response curves for SK2 (B) and SK1 (C) channels determined in the absence and presence of 2 mM EBIO under symmetrical () and asymmetrical (black-square) K+ conditions. Symmetrical K+ is 120 mM K+ on either side of the membrane; asymmetrical K+ is 5 mM K+ on the external side and 120 mM K+ on the internal side. Data points represent the mean ± S.D. of five experiments. Lines represent fit of a logistic function to the data; values for EC50 and Hill coefficient for symmetrical K+ conditions were 68.7 nM and 5.6 (SK2 with EBIO), 480 nM and 3.8 (SK2 without EBIO), 67.1 nM and 6.3 (SK1 with EBIO), and 440 nM and 4.2 (SK1 without EBIO). Values for EC50 and Hill coefficient for asymmetrical K+ conditions were 69.4 nM and 5.9 (SK2 with EBIO), 480 nM and 4.0 (SK2 without EBIO), 67.3 nM and 6.0 (SK1 with EBIO), and 456 nM and 3.8 (SK1 without EBIO).

The results suggest that EBIO shifts the apparent Ca2+ sensitivity of SK channels. This was quantified in Ca2+ concentration-response relationships determined for SK1 and SK2 currents in the absence and presence of EBIO under symmetrical and physiological K+ conditions. As shown in Fig. 1, B and C, EBIO induced a leftward shift of the concentration-response curve by about 7-fold for either SK subtype, with [Ca2+]i required for half-maximal activation (EC50) of SK1 and SK2 channels of 81.6 ± 31.4 nM (n = 6) and 69.4 ± 3.2 nM (n = 5), respectively. This shift in the Ca2+ concentration-response curve was independent of the extracellular K+ concentration (120 or 5 mM; Fig. 1, B and C). Additionally, the steepness of the concentration-response curves obtained in the presence of EBIO appeared slightly increased with respect to the controls (Hill coefficients were 4.0 ± 1.0 and 5.6 ± 0.5 for SK2 channels in the absence and presence of EBIO; the respective values for SK1 were 4.4 ± 1.0 and 6.6 ± 1.1).

EBIO Affects the Gating Mechanism of SK Channels-- The observations that EBIO was ineffective either in the absence of [Ca2+]i or with saturating [Ca2+]i suggested that the compound may operate by interacting with the gating apparatus of SK channels rather than working as a classical channel opener requiring Ca2+ as a "cofactor." Therefore, the kinetics of channel gating were examined in "fast application" experiments where activation and deactivation kinetics of Ca2+ gating can be monitored separately (15, 25).

Fig. 2A shows SK2 currents measured in response to fast application and removal of a saturating [Ca2+]i of 10 µM to inside-out patches in the absence (gray trace) and presence of EBIO. Whereas the time course of channel activation was not affected by EBIO at this [Ca2+]i, the current decay upon removal of Ca2+ was markedly slowed down by the benzimidazolinone (Fig. 2A, top panel). When fitted with monoexponentials, the respective time constants for activation (tau on) and deactivation (tau off) were 4.4 ± 0.5 ms (n = 8) and 294.2 ± 21.9 ms in the presence of EBIO, whereas control values were 4.4 ± 1.1 ms (n = 7) and 39.4 ± 4.1 ms. EBIO prolonged channel deactivation by about 7.5-fold (Fig. 2B), and the increase of tau off was only observed when EBIO was present after removal of Ca2+; washout of EBIO together with Ca2+ resulted in a current decay identical to that recorded in controls (Fig. 2A, middle panel; tau off = 40.2 ± 5.8 ms; n = 4).


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Fig. 2.   EBIO affects Ca2+-gating of SK channels. A, top panel, SK2 currents measured in response to fast application of 10 µM Ca2+ to inside-out patches with SK2 channels in the absence (gray trace) and presence of 2 mM EBIO (black trace). Currents were scaled to the inward maximum; application of Ca2+ and EBIO is indicated by horizontal bars. Middle panel, experiment as described in A, but with EBIO present only in the Ca2+-containing solution. Bottom panel, experiment as described in A, but with 0.5 µM Ca2+; current responses in the presence (black trace) and absence (gray trace) of EBIO were scaled to their maximum right before the washout of Ca2+. B, relative change in tau on and tau off as obtained from monoexponential fits to the activation and deactivation time course of currents as described in A at the Ca2+ concentrations indicated. Bars represent mean ± SD of seven experiments. C, SK2-mediated current response as described in A, recorded in the presence of 2 mM EBIO at the Ca2+ concentrations indicated; scaling and Ca2+ application were as indicated.

To test whether EBIO may also affect channel activation, fast applications were done with [Ca2+]i of 0.5 µM, a value close to the EC50 for SK channels (Fig. 1, B and C). Under these conditions, application of EBIO changed both tau on and tau off; tau off increased by about 6.8-fold, whereas tau on decreased by a factor of 1.5 (Fig. 2A, bottom panel and Fig. 2B). Thus, the predominant effect of EBIO was on channel deactivation, whereas the activation kinetics were only slightly affected.

The EBIO-induced increase of tau off was independent of channel activation (Fig. 2C). In the presence of EBIO, either 50 or 100 nM Ca2+ activated currents that decayed with essentially the same tau off of about 300 ms (283.2 ± 7.6 and 303.9 ± 13.7 at 50 and 100 nM, respectively). In contrast, channel activation strongly depended on [Ca2+]i; tau on exhibited values of 608.3 ± 49.3 and 83.6 ± 7.3 ms (n = 8) for 50 and 100 nM Ca2+, respectively (Fig. 2C).

These results suggested that EBIO modulates the interaction between the SK channel alpha -subunit and Ca2+-bound CaM (Ca2+-CaM). This was further tested in experiments probing the influence of the SK alpha -subunit and CaM on the EBIO effect.

In a first set of experiments, EBIO was tested on SK2 channels coexpressed with either wild-type CaM or mutant CaMs in which Ca2+ binding was largely impaired in both N-terminal EF hands (Mut CaM1, 2 and Mut CaM1-4, (14, 15)). Fig. 3A shows that whereas EBIO effectively activated SK channels at [Ca2+]i of 50 nM when coexpressed with wild-type CaM, little or no current induction was observed for SK channel coexpression with Mut CaM1,2 or Mut CaM1-4 (data not shown). The small increase in current is most likely due to the fraction of SK2 channels coassembled with endogenous wild-type Xenopus CaM (15); the amplitude of this current was 0.02 ± 0.01 (n = 7) of that obtained by coexpression with wild-type CaM.


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Fig. 3.   EBIO effect on Ca2+ gating of SK channels requires Ca2+-CaM and the C terminus of the SK alpha -subunit. A, currents recorded from SK2 channels coexpressed with the CaM molecules indicated. In Mut CaM1,2, the first aspartate in both N-terminal EF hands was replaced by alanine, which largely impairs Ca2+ binding. B, EBIO concentration-response curve determined for SK2 and IK1 channels (top panel) and for SK2-IK1C-term chimeric channels (bottom panel) in the presence of 0.1 µM Ca2+. Data points represent mean ± S.D. of 6 experiments; lines are the results of logistic fits to the data, and values for EC50 and Hill coefficient were 28.4 µM and 2.9 for IK1, 654.2 µM and 2.5 for SK2, and 22.3 µM and 1.7 for SK2-IK1C-term.

The second set of experiments probed the SK C terminus, the domain where CaM interacts with the channel alpha -subunit, as a candidate site for EBIO action (13, 15). SK and IK channels share the basic Ca2+ gating mechanism mediated by CaM and exhibit similar Ca2+ concentration-response relations (13). However, the primary sequences of the C termini, including the CaM binding domains, show considerable variability (4-7). Therefore, concentration-response relations for EBIO-induced channel activation in the presence of a constant [Ca2+]i of 100 nM were determined for both channel types. As shown in Fig. 3B, the concentration-response relationships for IK1 and SK2 differed in their EC50 by more than 20-fold with values of 28.4 and 654.2 µM for IK1 and SK2, respectively. Moreover, when the C terminus of IK1 was exchanged into the SK2 subunit (SK2-IK1C-term), the resulting chimeric channel showed an affinity for EBIO very similar to that obtained for IK1 (EC50 for SK2-IK1C-term was 22.3 µM; Fig. 3B).

Taken together with the effects on kinetics, the results shown are consistent with EBIO modulating the Ca2+ gating of SK channels by stabilizing the interaction between Ca2+-CaM and the SK alpha -subunit.

EBIO Alters AHP Currents and Excitability in Hippocampal Neurons-- In hippocampal pyramidal neurons, voltage-independent, Ca2+-activated K+ channels are responsible for the generation of two distinct phases of AHP, the mAHP and the slow AHP. According to their distribution and pharmacological properties, SK channels likely underlie at least part of the mAHP in hippocampal neurons (27).

Given the observed effects of EBIO on the Ca2+ gating of cloned SK channels, the compound was tested on the Ca2+-activated K+ currents underlying mAHP (IAHP) and slow AHP (sIAHP) in CA1 pyramidal neurons in acute hippocampal slices. In whole-cell voltage-clamp experiments, currents were elicited by a standard protocol (27) in the presence of tetrodotoxin (0.5 µM) and tetraethylammonium (1 mM) to block Na+ channels and Ca2+- and voltage-dependent K+ (BK) channels, respectively. As shown in Fig. 4A, EBIO induced a marked increase of the IAHP amplitude with respect to the controls recorded under steady-state conditions before application of the compound. The relative increase of the IAHP was 3.5 ± 0.4 (n = 5) (Fig. 4D). Moreover, the enhanced IAHP was fully suppressed by apamin applied to the neurons in the presence of the benzimidazolinone (Fig. 4B).


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Fig. 4.   EBIO increases medium and slow AHP currents in hippocampal CA1 neurons. A, whole-cell recording of Ca2+-activated K+ currents in CA1 pyramidal neurons as a response to a 100-ms depolarizing pulse to +10 mV in the absence (left panel) and presence (middle panel) of 1 mM EBIO; the right panel is an overlay of the currents from the left and middle panels. Time and current scaling were as indicated. B, recordings as described in A in the absence (left panel) and presence (middle panel) of EBIO but with 8CPT-cAMP (50 µM) present in the patch pipette to suppress sIAHP; the right panel is a recording after the addition of apamin (50 nM) to the bath medium. C, recordings as described in A, but after suppression of IAHP by the addition of 50 nM apamin to the bath medium. D, relative increase in amplitude of IAHP and sIAHP in CA1 neurons by EBIO; bars represent mean ± S.E. of five experiments. E and F, EBIO increased tau decay of the IAHP, whereas the time-to-peak interval (tpeak) was unchanged. In E, IAHP was measured with 8CPT-cAMP (50 µM) present in the patch pipette to suppress sIAHP. Bars in F are mean ± S.E. of six experiments.

EBIO also increased the apamin-insensitive sIAHP. When applied together with 50 nM apamin to block IAHP (27), EBIO induced an increase of the remaining sIAHP by a factor of 1.5 ± 0.1 with respect to the control (Fig. 4, C and D; n = 4). The EBIO-enhanced current was fully inhibited by noradrenaline at 1 µM (data not shown), identifying it as the sIAHP (28, 29).

The effect of EBIO on the time course of IAHP was examined in isolation by inhibiting sIAHP by including the cAMP analogue 8CPT-cAMP in the patch pipette (27, 29, 30). Consistent with the effect of EBIO on activation and deactivation kinetics of cloned SK channels (Fig. 2), the time constant of decay of the IAHP (tau  decay) was increased by the benzimidazolinone. The values for tau  decay were 90.5 ± 7.5 and 214.5 ± 9.8 ms (n = 6) in the absence and presence of EBIO, respectively (Fig. 4, E and F). In contrast, the rise time (time-to-peak) of the current remained essentially unchanged (Fig. 4, E and F).

Ca2+ influx during the depolarizing pulse used to elicit the AHP currents was qualitatively examined by monitoring the partially clamped Ca2+ current. When the EBIO effect on AHP currents was maximal (2-3 min after starting EBIO application), the Ca2+ current was not obviously altered (data not shown); prolonged applications of EBIO (>10 min), however, caused a reversible reduction of the inward Ca2+ current.

The effects of EBIO on AHP currents suggested that it might profoundly affect neuronal excitability and signal encoding; an increase of the IAHP and the sIAHP would be expected to slow down the neuronal firing rate and enhance slow spike frequency adaptation. This was investigated in current clamp recordings performed in the absence and presence of EBIO. Under control conditions, long (0.8-1-s) depolarizing current pulses elicited trains of APs characterized by early and late spike frequency adaptation (Fig. 5A, left panel). Application of EBIO dramatically decreased the firing frequency of all CA1 pyramidal neurons tested (n = 5). As shown in Fig. 5A (middle panel), in the presence of EBIO, most cells only fired one or two APs right upon current injection and remained silent thereafter (Fig. 5A, middle panel; n = 5). This marked effect of EBIO was fully reversible (Fig. 5A, right panel) and was independent of the amplitudes of the injected current (Fig. 5B). When the same set of experiments was performed with only the apamin-sensitive IAHP present (sIAHP suppressed by 8CPT-cAMP), tonic firing was observed instead of slow spike frequency adaptation (Fig. 5C, left panel; see also Ref. 27). As expected, in the presence of EBIO, all neurons tested fired substantially less APs spaced by prolonged, regular interspike intervals when compared with control conditions (Fig. 5C, middle panel; n = 5). This effect was fully reversible (Fig. 5C, right panel) and consistently observed in response to injected currents of different amplitudes (Fig. 5D). In the same cells, EBIO produced a reversible slight hyperpolarization of the resting membrane potential (approx  -2 mV) but did not significantly affect AP duration.


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Fig. 5.   EBIO reduces electrical activity of CA1 neurons. A, APs elicited in CA1 neurons by 0.8-1-s current injections (see "Materials and Methods") before application of EBIO (-EBIO), during application of 1 mM EBIO (+EBIO), and after washout of the compound (wash). B, number of APs recorded in experiments as described in A, plotted against the amplitude of the injected current; conditions were as indicated. C, APs were recorded as described in A, but after suppression of sIAHP by intracellular application of 8CPT-cAMP (50 µM). D, number of APs recorded in experiments as described in C and plotted as described in B.

The results from CA1 pyramidal neurons demonstrate a prominent effect of EBIO on neuronal excitability and signal encoding properties mediated by the enhancement of the IAHP and the sIAHP amplitudes and the IAHP duration.

EBIO Suppresses Electrical Activity in Hyperexcitable Cortical Neuronal Networks-- The effect of EBIO on neuronal activity was further investigated in a network model derived from dissociated cortical neurons (26). Neocortical neurons were recently shown to express SK1 and SK2 subunits (12).

Fig. 6A illustrates synchronized cytoplasmic Ca2+ oscillations elicited by bursts of APs (26) and monitored by a fluorescence image plate reader (fluorescence plate reader, see "Materials and Methods"). These oscillations were elicited by removal of Mg2+ from the bath medium, a paradigm for the induction of epileptiform activity (31, 32). The oscillations occurred at rates of 0.2-0.3 Hz under control conditions (Fig. 6A); addition of the divalent back to the extracellular fluid stopped the oscillations (data not shown; Ref. 26).


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Fig. 6.   EBIO extinguishes oscillations in a network of dissociated cortical neurons in hyperexcitable conditions. A, network oscillations observed in primary cultures of cortical neurons as fluo-4 fluorescence (see "Materials and Methods") in the absence of EBIO; oscillations were elicited by withdrawal of Mg2+ from the bath medium. Time scaling was as indicated; application of 10 nM apamin is represented by the horizontal bar. B, extinction of network activity by EBIO (0.6 mM) and partial recovery upon addition of apamin (10 nM). C, analysis of network oscillations as recorded in A and B at the conditions indicated; for the control experiments, EBIO was omitted from the application solutions. Bars represent the mean ± S.D. of area under curve (AUC) obtained from eight cultures.

Application of EBIO at concentrations >0.2 mM led to a rapid cessation of the electrical activity that lasted as long as the compound was present. This effect of EBIO was due at least in part to activation of SK channels because the addition of 10 nM apamin to the bath medium induced a partial recovery of neuronal activity (Fig. 6, B and C). At this concentration, apamin completely blocks cloned SK2 channels, the SK subtype most likely underlying the apamin-sensitive IAHP in cortical neurons (12, 33).

These results emphasize the importance of SK channels for shaping the electrical response patterns of central neurons and suggest that EBIO-mediated modulation of SK channel gating may be a potent mechanism for controlling excitability in the central nervous system.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results presented here demonstrate that EBIO modifies Ca2+ gating of SK-type K+ channels so that low nanomolar concentrations of intracellular Ca2+ are sufficient to initiate robust channel activity. In neurons, the increased channel activity results in a substantial enhancement of AHP currents, which, in turn, has profound effects on excitability and firing patterns.

The most prominent effect of EBIO on SK channels is the increase in the apparent Ca2+ sensitivity by almost 1 order of magnitude that allows for channel activation at [Ca2+]i of as low as 50 nM (Fig. 1). This observation is consistent with the reported effects of EBIO on the structurally and functionally related IK1 channel (21) but differs from a previous report describing an increased open probability for SK2 channels without any obvious change in their Ca2+ concentration-response relation (34). The results show that EBIO does not work as a channel opener on its own because EBIO did not induce channel activity in the absence of [Ca2+]i (Fig. 1). Rather, EBIO requires [Ca2+]i in the lower nanomolar range to be effective.

Mechanistically, the EBIO-induced increase in Ca2+ sensitivity is suggested to result from the interaction of the benzimidazolinone with the Ca2+ gating apparatus of SK/IK channels. As indicated by the fast Ca2+ application experiments (Fig. 2), EBIO predominantly affects channel deactivation, whereas channel activation is only slightly changed by the compound. Given that channel deactivation occurs upon dissociation of Ca2+ from CaM and alteration of the Ca2+-CaM-SK interaction (13-15), the slowed deactivation may indicate that EBIO acts by stabilizing the Ca2+-CaM-SK interaction that drives channel opening (13, 15). The roughly 7-fold decreased deactivation rate in turn increases the apparent Ca2+ sensitivity of SK channels by about the same amount (Figs. 1 and 2). Indeed, for CaM, the properties of substrate binding and Ca2+ affinity are linked; the EF hand affinities for Ca2+ may be increased by interactions with target proteins (35-38).

A consequence of this model is that channel activation should be less affected by EBIO and rather depend on [Ca2+]i and Ca2+ binding to CaM that is prerequiste for the Ca2+-CaM-SK interaction. This is consistent with the results from fast Ca2+ application experiments (Fig. 2).

When interpreted with respect to the multiple-state model derived from single-channel analysis (14, 18), the suggested EBIO-induced stabilization of the Ca2+-CaM-SK interaction is equivalent to stabilization of the (closed) state(s) from which channel opening occurs. Entering this state(s) strongly depends on [Ca2+]i, whereas the opening transition is rapid and independent of [Ca2+]i (14, 18). Consequently, at nonsaturating [Ca2+]i, EBIO should increase channel open probability without significant changes in the mean open time, as was hypothesized for IK1 channels (21).

EBIO likely interacts with the intracellular C terminus of the channels, perhaps the CaM binding domain (CaMBD). The concentration-response relations of SK and IK channels to EBIO are different; IK channels are more sensitive, and the sensitivity of IK1 was endowed upon SK2 by replacing its intracellular C terminus with that of IK1 (Fig. 3B). CaM binds to the CaMBD of SK or IK channels (13-15). The CaMBD contains many conserved residues among the two channel subtypes. However, there are also residues conserved among SK channels that are not shared in the IK channel. C-terminal to the CaMBD there is almost no recognizable homology between IK1 and SK2. The different efficacies seen for EBIO with SK and IK channels may be due to differences in the CaMBD sequences and reflect subtle conformational differences between the two channel types. Structural rearrangements in the CaMBD upon interactions with CaM and Ca2+-CaM have been monitored by changes in the fluorescence emission profile of the single tryptophan residue in recombinant CaMBD (14). We examined whether EBIO had additional effects; however, none were detected. Whereas a positive biochemical result would have been further supportive of our model, it seems likely that the isolated CaMBD does not reflect the disposition of the much more complex, complete channel incorporated into the plasma membrane.

Prominent effects of EBIO on native systems have thus far been reported exclusively for epithelial and endothelial cells (20, 39-43). Although previous studies have inferred physiological roles for native SK channels in the central nervous system using specific inhibitors such as apamin, the results presented here using CA1 pyramidal neurons in freshly prepared brain slices and cultured cortical neurons demonstrate that native SK channel function may also be investigated using a selective enhancer of SK channel activity. The selectivity of EBIO for SK channels was inferred from experiments as described in Fig. 4, where, under physiological conditions, EBIO induced an increase in outward current that was fully inhibited by the combined action of apamin and 8-CPT cAMP (Fig. 4B). Both agents are known to specifically affect IAHP and sIAHP, respectively, currents that are thought to be mediated by SK-type K+ channels (1-3).

We also tested the specificity of EBIO (at 2 mM concentration) on a panel of cloned K+ channels, including BK channels, Kv channels (Kv1.1, Kv1.4, Kv3.4, and Kv4.1), KCNQ channels (KCNQ1-4), Kir channels (Kir1.1, Kir2.1, Kir4.1, and Kir6.2/SURs), and ligand-gated ion channels, including NMDA- and AMPA-type glutamate receptors as well as neuronal nicotinic acetylcholine-receptors (25). We also examined EBIO on native Ca2+ channels or hyperpolarization-activated pacemaker channels when applied to sensory hair cells or dopaminergic midbrain neurons (25, 44).2 In no case did EBIO exhibit any discernable effect (data not shown).

Recordings from CA1 pyramidal cells show that EBIO substantially increased the amplitude of the AHP currents (Fig. 4), most likely as a result of the shift in Ca2+ sensitivity observed for cloned SK channels in the presence of the benzimidazolinone. By increasing the Ca2+ sensitivity of the channels (Fig. 1), EBIO is able to activate SK channels that are silent in the absence of the compound. The EBIO-mediated enhancement of SK channel activity is limited to a range in [Ca2+]i between ~0.1 and 0.5 µM. Whereas these Ca2+ concentrations are saturating for SK channel activation in the presence of EBIO, there is only modest channel activity in the absence of EBIO (Fig. 1). Thus, application of EBIO will recruit an otherwise silent population of SK channels.

Alternatively, the EBIO-induced increase in AHP currents might be due to other effects. Thus, EBIO might prolong Ca2+ entry through L-type Ca2+ channels whose gating is modulated by Ca2+-CaM (45, 46); L-type Ca2+ channels are located close to SK channels in CA1 neurons and can provide the source of Ca2+ for SK channel gating (47). However, measuring the unclamped Ca2 current during the depolarizing pulse provided no indication for increased Ca2+ influx induced by EBIO. Instead, a decrease in Ca2+ inward current with prolonged application of EBIO was observed.

Other channels that influence Ca2+ homeostasis, ryanodine receptors and IP3 receptors, are also modulated by Ca2+-CaM (48-54). Potential involvement of these molecules in the EBIO effect will be addressed in further investigations. However, in light of the observations on cloned SK channels, it seems most likely that the EBIO-mediated increase in AHP currents is predominantly brought about by EBIO acting directly on SK channels.

The EBIO-induced increase in AHP currents had profound effects on the excitability and firing patterns of hippocampal neurons (Fig. 5) as well as those of hyperexcitable networks of cultured cortical neurons (Fig. 6). In both cases, the compound strongly reduced firing. The action of EBIO as a SK channel enhancer may make it a useful tool to study the functional role of SK channels in various cell types. Moreover, EBIO and SK channels may be used to estimate the Ca2+ concentration underneath the cell membrane as it occurs at rest or during excitation (25). In this study, for example, the resting [Ca2+]i around the SK channels in CA1 pyramidal cells is estimated to be <50 nM because an increase in the Ca2+ sensitivity of SK channels by EBIO did not activate K+ currents before pulse-triggered Ca2+ entry into the cells. Additionally, the results obtained on cortical networks suggest that EBIO may serve as a prototype compound for the development of agents that reduce states of neuronal hyperexcitability.

    ACKNOWLEDGEMENTS

We thank Drs. S. Alberi, T. Baukrowitz, U. Schulte, J. P. Ruppersberg, and R. Warth for stimulating discussion; Dr. A. Schwab for hardware advice; and Prof. W. Stühmer for generous support.

    FOOTNOTES

* This work was supported by grants from the Deutsche Forschungsgemeinschaft (Grant Fa 332/3-1 to B. F. and Grant SFB 406/C8 to P. P. and M. S.) and the Human Frontier Science Program (Grant RG0233).

** Present address: Wellcome Laboratory J. Molecular Pharmacology, UCL, Gower St., London WC1E 6BT, UK.

Dagger Dagger To whom correspondence should be addressed: Dept. of Physiology II, Ob dem Himmelreich 7, 72074 Tübingen, Germany. Tel.: 49-7071-297-7173; Fax: 49-7071-87815; E-mail: bernd.fakler@uni-tuebingen.de.

Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M010001200

2 J. Roeper, personal communication.

    ABBREVIATIONS

The abbreviations used are: AP, action potential; AHP, afterhyperpolarization; SK, small conductance Ca2+-activated potassium; EBIO, 1-ethyl-2-benzimidazolinone; mAHP, medium AHP; CaM, calmodulin; 8CPT-cAMP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate; CaMBD, CaM binding domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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