From the 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
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ABSTRACT |
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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 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 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 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 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 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 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 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 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 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
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 (
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
The EBIO-induced increase of
These results suggested that EBIO modulates the interaction between the
SK channel
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.
The second set of experiments probed the SK C terminus, the domain
where CaM interacts with the channel
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 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).
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
(
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 (
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).
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.
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.
-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
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).
-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
-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).
subunits and CaM and that modulating SK
channel activity is an effective mechanism for tuning excitability in central neurons.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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.
80
mV and
120 mV were used, respectively. All data are presented as
mean ± S.D. of n experiments.
1 h.
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.
5000 cells (on
an area of
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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).
View larger version (14K):
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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 (
) 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).
on) and
deactivation (
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
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;
off = 40.2 ± 5.8 ms; n = 4).
View larger version (13K):
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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 on
and
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.
on and
off;
off increased by about 6.8-fold,
whereas
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.
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
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;
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).
-subunit and Ca2+-bound CaM
(Ca2+-CaM). This was further tested in experiments probing
the influence of the SK
-subunit and CaM on the EBIO effect.
View larger version (11K):
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Fig. 3.
EBIO effect on Ca2+ gating of SK
channels requires Ca2+-CaM and the C terminus of the
SK -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.
-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).
-subunit.
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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 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.
decay) was increased by the benzimidazolinone. The values for
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).
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.
View larger version (30K):
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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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
![]() |
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.
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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.
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.
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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.
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REFERENCES |
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