Selective positive modulation of the SK3 and SK2 subtypes of small conductance Ca2+-activated K+ channels

Article abstract of DOI:10.1038/sj.bjp.0707281

Background and purpose: Positive modulators of small conductance Ca ²⁺-activated K⁺ channels (SK1, SK2, and SK3) exert hyperpolarizing effects that influence the activity of excitable and non-excitable cells. The prototype compound 1

Full text of DOI:10.1038/sj.bjp.0707281

British Journal of Pharmacology (2007) 151, 655–665
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RESEARCH PAPER
Selective positive modulation of the SK3 and SK2
subtypes of small conductance Ca2 þ -activated
Kþ channels
C Hougaard, BL Eriksen, S Jørgensen, TH Johansen, T Dyhring, LS Madsen, D Strøbæk
and P Christophersen
NeuroSearch A/S, Pederstrupvej 93, Ballerup, Denmark
Background and purpose: Positive modulators of small conductance Ca2 þ -activated Kþ channels (SK1, SK2, and SK3) exert
hyperpolarizing effects that influence the activity of excitable and non-excitable cells. The prototype compound 1-EBIO or the
more potent compound NS309, do not distinguish between the SK subtypes and they also activate the related intermediate
conductance Ca2 þ -activated Kþ channel (IK). This paper demonstrates, for the first time, subtype-selective positive
modulation of SK channels.
Experimental approach: Using patch clamp and fluorescence techniques we studied the effect of the compound cyclohexyl-
[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) on recombinant hSK1-3 and hIK channels expressed in
HEK293 cells. CyPPA was also tested on SK3 and IK channels endogenously expressed in TE671 and HeLa cells.
Key results: CyPPA was found to be a positive modulator of hSK3 (EC50 ¼ 5.6 7 1.6 mM, efficacy 90 7 1.8 %) and
hSK2 (EC50 ¼ 14 7 4 mM, efficacy 71 7 1.8 %) when measured in inside-out patch clamp experiments. CyPPA was inactive
on both hSK1 and hIK channels. At hSK3 channels, CyPPA induced a concentration-dependent increase in the apparent
Ca2 þ -sensitivity of channel activation, changing the EC50(Ca2 þ ) from 429 nM to 59 nM.
Conclusions and implications: As a pharmacological tool, CyPPA may be used in parallel with the IK/SK openers 1-EBIO and
NS309 to distinguish SK3/SK2- from SK1/IK-mediated pharmacological responses. This is important for the SK2 and SK1
subtypes, since they have overlapping expression patterns in the neocortical and hippocampal regions, and for SK3 and IK
channels, since they co-express in certain peripheral tissues.
British Journal of Pharmacology (2007) 151, 655–665; doi:10.1038/sj.bjp.0707281; published online 8 May 2007
Keywords: SK channel; IK channel; gating; positive modulator; 1-EBIO; NS309; patch clamp; subtype-selective
Abbreviations: AHP, afterhyperpolarization; BK channel, large conductance Ca2 þ -activated Kþ channel; BMB, bicuculline
methobromide; BSA, bovine serum albumin; BTC-AM, benzothiazole coumarin acetoxymethyl ester; CAM,
calmodulin; CAMBD, calmodulin-binding domain; ChTX, charybdotoxin; CyPPA,
cyclohexyl-[2-(3,5-
dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine; DC-EBIO, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzi-
midazol-2-one; DMSO, dimethylsulphoxide; DRG, dorsal root ganglion; 1-EBIO, 1-ethyl-2-benzimidazolinone;
FLIPR, fluorometric imaging plate reader; HEK, human embryonic kidney; hERG, human ether-a-go-go-related
gene; h, human; IAHP, afterhyperpolarizing current; IK channel, intermediate conductance Ca2 þ -activated Kþ
channel; I-V, current–voltage; NS309, 6,7-dichloro-1H-indole-2,3-dione 3-oxime; PBS, phosphate-buffered
saline; SK channel, small conductance Ca2 þ -activated Kþ channel.
Introduction
Small conductance Ca2 þ -activated Kþ channels (SK chan-
nels) are important regulators of excitability, endogenous
firing pattern and synaptic integration in many neurons
(Bond et al., 2005). Three SK channel subtypes exist (SK1-3;
Kohler et al., 1996) and are widely distributed in the central
nervous system (CNS) (Stocker and Pedarzani, 2000) as well
as in the periphery. SK channels and the peripherally
expressed intermediate conductance Ca2 þ -activated Kþ
channel (IK; Ishii et al., 1997), constitute a molecular family
of voltage-independent channels, that are gated by Ca2 þ
binding to calmodulin (CAM) tightly associated with a CAM-
binding domain (CAMBD) in the C-terminal region (Xia
et al., 1998; Khanna et al., 1999). Crystallographic data from
C-terminal peptides of the SK2 channel indicate that dimers
Correspondence: Dr P Christophersen, NeuroSearch A/S, Pederstrupvej 93,
DK 2750, Ballerup, Denmark.
E-mail: pc@neurosearch.dk
Received 28 December 2006; revised 20 March 2007; accepted 21 March
2007; published online 8 May 2007

Subtype-selective activation of SK channels
656
C Hougaard et al
of CAMBD associate with two CAM molecules, each binding
1 or 2 Ca2 þ at the EF hand motifs 1 and 2 (Schumacher et al.,
2001). The binding of Ca2 þ is potent (effector concentration
for half-maximum response (EC50) E400 nM), highly co-
operative (nH ¼ 4–5) and thought to involve binding of 4–8
Ca2 þ in all the SK channel subtypes (Xia et al., 1998). In
contrast, the intermediate conductance Ca2 þ -activated Kþ
channel (IK channel) exhibits a significantly lower Hill
coefficient for Ca2 þ activation (but identical EC50 value;
Ishii et al., 1997), indicating that molecular differences exist
between the gating mechanisms of SK and IK channels
despite their overall similarity in Ca2 þ binding.
Ca2 þ -dependent activation of IK/SK channels is facilitated
by certain small organic compounds. 1-Ethyl-2-benzimida-
zolinone (1-EBIO) was the first described positive modulator
of IK channels (Devor et al., 1996; Jensen et al., 1998) and SK
channels (Syme et al., 2000). 1-EBIO causes a leftward shift in
the Ca2 þ -activation curves for IK/SK channels (Pedersen
et al., 1999; Pedarzani et al., 2001) and strongly reduces the
rate of deactivation upon Ca2 þ removal. The 1-EBIO
interaction site has been localized close to the CAMBD in
the C-terminal region (Pedarzani et al., 2001). In addition,
the more potent 1-EBIO analogue, 5,6-dichloro-1-ethyl-1,3-
dihydro-2H-benzimidazol-2-one (DC-EBIO) (Singh et al.,
2001; Pedarzani et al., 2005), the centrally acting muscle
relaxant drugs zoxazolamine and chlorzoxazone (Cao et al.,
2001), as well as the neuroprotectant riluzole (Cao et al.,
2002), all have IK/SK activating properties. 6,7-Dichloro-1H-
indole-2,3-dione 3-oxime (NS309) is a recently described
compound in this functional class and is orders of magni-
tude more potent than 1-EBIO (Strøbæk et al., 2004;
Pedarzani et al., 2005). However, NS309 still exhibits the
characteristic 1-EBIO-like selectivity for the IK/SK channels,
being most potent on IK and 10–20 times less active on the
SK subtypes.
channel Ca2 þ sensitivity can be decreased by phosphoryla-
tion of CAM threonine-80 via protein kinase CK2, and
conversely increased by dephosphorylation via protein
phosphatase 2A (Bildl et al., 2004). Therefore, SK channels
may under some regulatory conditions, be too insensitive to
intracellular Ca2 þ to contribute significantly to spike
frequency adaptation unless their Ca2 þ sensitivity is
increased by dephosphorylation or by the presence of
a positive modulator of SK channels (Faber and Sah, 2002).
Subtype-selective SK channel modulators are clearly
needed to assign specific SK channel subtypes to observed
pharmacological responses. However, owing to the con-
served gating mechanism within the IK/SK channel family
and the identical activation curves for Ca2 þ obtained with
the SK1-3 channels (Xia et al., 1998), the identification of
subtype-selective positive modulators has only been
a
hypothetic possibility. In this study we demonstrate that
the compound, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-
methyl-pyrimidin-4-yl]-amine (CyPPA) is a selective positive
modulator of SK3 and SK2 channels exhibiting the following
selectivity profile: hSK3 4hSK2 44hSK1 (inactive) ¼ hIK
(inactive). The EC50 value of CyPPA on SK3 channels was
5.6 mM, which although higher than that of NS309 (0.3 mM),
was lower than those of DC-EBIO (16 mM), 1-EBIO (1040 mM),
and other available non selective SK activators.
Methods
Cell cultures
Establishment of human embryonic kidney (HEK) 293 cell
lines expressing hSK1, hSK2, hSK3 and hIK channels has
been described previously (Strøbæk et al., 2004). HeLa and
TE671 cells were obtained from ATCC (Rockville, MD, USA).
Cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM; Cambrex BioScience, Verviers, Belgium) supple-
mented with 10% fetal calf serum (FCS; Sigma-Aldrich,
Vallensbæk Strand, Denmark) at 371C and 5% CO2. For
TE671 cells, the DMEM also contained 5% horse serum
(Sigma-Aldrich). At 60–80% confluence, cells were washed
with phosphate-buffered saline (PBS), harvested by trypsin/
Despite their lack of SK subtype selectivity, 1-EBIO and
other positive modulators of IK/SK channels have been used
to elucidate the role of SK channels in the regulation of
neuronal excitability. For example debate continues as to the
contribution of SK channels to hippocampal CA1 neuron
spike frequency adaptation (Bond et al., 2004; Gu et al.,
2005). The involvement of SK channels is strongly supported
by the effects of 1-EBIO and NS309, which both increase the
size and duration of the Ca2 þ -activated afterhyperpolarizing
current (IAHP), increase the intermediate duration after-
hyperpolarization (mAHP), augment spike frequency adapta-
tion and reduce excitability following constant current
stimuli (Pedarzani et al., 2001, 2005). Furthermore, the
negative modulator (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetra-
hydro-1-naphtylamine (NS8593), which decreases the appar-
ent SK channel Ca2 þ sensitivity, also reduces IAHP (Strøbæk
et al., 2006). Because the SK channel modulators exclusively
affect Ca2 þ sensitivity and have no effect on maximally
activated SK channels, these results show that SK channels
are not saturated with Ca2 þ following action potentials or
even long depolarizing stimuli. Therefore, these pharmaco-
logical agents indirectly demonstrate the possibility that
modulation of SK channel Ca2 þ sensitivity could be a
sensitive endogenous mechanism for fine-tuning neuronal
excitability. Importantly, it has also been shown that SK2
ethylenediaminetetraacetic
acid
(EDTA;
Cambrex
BioScience, Verviers, Belgium) treatment.
Electrophysiology
HEK293 cells expressing hSK1, hSK2, hSK3 or hIK
were transferred to Petri dishes containing coverslips
(+ 3.5 mm, custom made at VWR International APS,
Albertslund, Denmark). Membrane currents were recorded
using the whole-cell or inside-out configuration of the patch
clamp technique. Patch pipettes (1.8–2.3 MO) were pulled
from borosilicate tubes with an outside diameter of 1.32 mm
(Modulohm; Copenhagen, Denmark) using a horizontal
electrode puller (Zeitz Instruments; Augsburg, Germany).
An electronically controlled micromanipulator (Eppendorf,
Radiometer, Copenhagen, Denmark) was used for the
positioning of pipettes. Cells seeded on coverslips were
transferred to
a 15 ml recording chamber continuously
superfused at 1 ml minÀ1 at room temperature. An Ag/AgCl
British Journal of Pharmacology (2007) 151 655–665

Subtype-selective activation of SK channels
C Hougaard et al
657
pellet was fixed in the chamber. Experiments were performed
using an EPC-9 amplifier (HEKA; Lambrecht, Germany)
connected to a Macintosh computer by an ITC-16 interface.
Data were filtered at 3 kHz. Ca2 þ -activated Kþ currents were
recorded upon application of 200 ms linear voltage ramps
(from À80 to þ 80 mV) every 5 s from a holding potential of
0 mV. For analyses, currents were measured at À75 mV. In
whole-cell experiments, the cell capacitance and series
resistance (Rs below 8 MO, 80% compensation) were updated
before each application of the voltage ramp.
wells. The data were subsequently analysed by fitting the
fluorescence peak values for each concentration to the Hill
equation (see below).
Calculations and statistics
EC50 values (EC50(Ca2 þ ), EC50(NS309) and EC50(CyPPA))
were estimated from equilibrium concentration–response
experiments by fitting data from individual experiments to
the Hill equation (Equation 1):
For voltage-clamp experiments, an extracellular solution
containing a high [Kþ ] was used (in mM): 150 KCl, 2 CaCl2,
1 MgCl2 and 10 HEPES with pH adjusted to 7.4 with KOH.
For current-clamp experiments, the extracellular solution
contained (in mM): 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2 and
10 HEPES; pH adjusted to 7.4 with NaOH.
Rmax ꢀ Cn
BðCÞ ¼
ð1Þ
ECn50 þ Cn
where B is the current or peak fluorescence response, Rmax is
the maximal current or maximal peak fluorescent response,
C is the test concentration and n is the Hill coefficient.
All results are given as the mean7standard error of the
mean (s.e.m.). Tests for statistical differences were performed by
two-way analysis of variance (ANOVA) (and Bonferroni post hoc
comparisons between individual groups) or Student’s t-test.
The intracellular solutions contained 154 mM KCl and
10 mM HEPES as well as 10 mM EGTA or a combination of
EGTA and NTA (10 mM in total). Concentrations of CaCl2
and MgCl2 needed to obtain the desired free concentrations
of Ca2 þ (0.01–10 mM, Mg2 þ always 1 mM) were calculated by
EqCal software (Cambridge, UK) and added. The free Ca2 þ
concentrations were controlled by a Ca2 þ -sensitive electrode
(WPI; Sarasota, FL, USA). All intracellular solutions were
adjusted to pH 7.2 with concentrated HCl or KOH.
Chemicals
CyPPA (see International Patent Publication WO 2006/
100212) was either purchased from Specs (Delft, Nether-
lands) or synthesized according to the following procedure.
2,4-Dichloro-6-methylpyrimidine (7.0 g, 42.9 mmol) was
dissolved in acetonitrile (20 ml). Cyclohexylamine (5.1 g,
51.5 mmol) and triethylamine (6.83 ml, 67.3 mmol) were
added. The mixture was shaken in a sealed vial on a sand
bath at 601C for 12 h. Evaporation of the solvent followed by
column chromatography on silica gel (60–120 mesh) with
ethyl acetate/hexane as eluent, gave (2-chloro-6-methyl-
pyrimidine-4-yl)-cyclohexylamine (6 g, 62%) and (4-chloro-
Fluorescence-based Tlþ assay
HEK293 cells expressing hSK1, hSK2, hSK3 or hIK channels
were seeded in 384-well, clear-bottomed, black-walled plates
(Corning Inc., NY, USA). The plates were coated with poly-D-
lysine (10 mg mlÀ1), and the cells were seeded at a density of
B3 Â 106 cells mlÀ1 in 20 ml DMEM containing 10% FCS per
well and left overnight at 371C in a 5% CO2 incubator.
Before the experiment, the cells were loaded with the
fluorescent dye benzothiazole coumarin acetoxymethyl ester
(BTC-AM), essentially as described by Weaver et al. (2004).
In brief, the cells were washed thrice in ClÀ-free assay buffer
(in mM: 140 Naþ -gluconate, 2.5 Kþ -gluconate, 6 Ca2 þ
-gluconate, 1 Mg2 þ gluconate, 5 glucose, 10 HEPES, pH
7.3), the buffer was aspirated, and 25 ml ClÀ-free loading
buffer (assay buffer containing an additional 2 mM BTC-AM,
2 mM amaranth and 1 mM tartrazine) was added to each well.
The cells were incubated at room temperature for 1 h and,
subsequently, transferred to a fluorometric imaging plate
reader (FLIPR; Molecular Devices, Sunnyvale, CA, USA). For
excitation, the 488 nm line of an argon laser was used,
whereas on the emission side, a 540730 nm bandpass filter
was inserted. Dimethylsulphoxide (DMSO) stock solutions of
the compounds were diluted in ClÀ-free assay buffer
containing Tl2SO4, giving a final [Tlþ ] of 2 mM and final
compound concentrations of 47 nM to 100 mM (final DMSO
concentration o0.1%). An initial baseline signal was
collected before compound addition. The cells were stimu-
lated in quadruplicate, that is, four individual wells were
stimulated per concentration. In addition, on each plate, 16
wells served as negative controls (buffer addition) and
another 16 wells as positive controls (1 mM ionomycin). The
data from the individual wells were background corrected by
subtraction of the averaged value of the negative control
6-methyl-pyrimidine-2-yl)-cyclohexylamine
(2 g,
21%).
(2-Chloro-6-methyl-pyrimidine-4-yl)-cyclohexylamine (6 g,
26.6 mmol) was dissolved in acetonitrile (20 ml) and
3,5-dimethylpyrazole (3.8 mg, 39.9 mmol) was added. The
mixture was heated in a microwave oven at 1301C for
30 min. The mixture was concentrated and the residue
basified with sodium hydrogen carbonate, extracted with
chloroform, dried over anhydrous sodium sulphate, filtrated
and evaporated. The crude product was purified by column
chromatography (as described above) to give cyclohexyl-
[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine
(2.9 g, 38%). The melting point was 133.2–136.91C. 1H-NMR
analysis in CDCl3 gave the following chemical shifts: d 6.03
(1H, s), 5.97 (1H, s), 5.14 (s, 1H), 3.53–3.65 (1H, m), 2.63 (3H, s),
2.40 (3H, s), 2.32 (3H, s), 2.00–2.03 (2H, m), 1.76–1.80 (3H,
m) and 1.23–1.40 (5H, m). Analysis by mass spectro-
metry (LC-ESI-HRMS) of [MH]þ was found to be
286.2037 Da. The calculated value was 286.20317. Elemen-
tary analysis (Department of Chemistry, University
of Copenhagen, Copenhagen, Denmark): Found: C, 67.35;
H, 8.21; N, 24.45; calculated for C16H23N5: C, 67.34;
H, 8.12; N, 24.54.
NS309 (Strøbæk et al., 2004) was also synthesized at
NeuroSearch A/S (Ballerup, Denmark). Clotrimazole (Clot)
and charybdotoxin (ChTX) were purchased from Sigma-
British Journal of Pharmacology (2007) 151 655–665

Subtype-selective activation of SK channels
658
C Hougaard et al
Aldrich and bicuculline methobromide (BMB) from MP
Biomedicals Inc. (Illkirch, France). All other chemicals were
purchased from regular commercial sources and were of the
purest grade available. Clotrimazole, BMB, NS309 and CyPPA
were dissolved in DMSO and diluted at least 1000 times in
the experimental solutions.
tively. For both figures, the panels to the left show the
currents recorded upon application of voltage ramps and the
panels to the right show the time courses of the experiments
(analysed as the current at À75mV). Application of 40nM
NS309 activated both hSK3 (Figure 1b) and hIK (Figure 1c)
channels, with the largest effect on hIK channels (8.570.4
(n ¼ 3)-fold increase in current compared to 1.970.1 (n ¼ 5)-
fold increase in current for hSK3 channels; Po0.0001). In
contrast to this, CyPPA prominently activated the hSK3
current when applied at 300 nM (3.070.4-fold increase,
n ¼ 5), whereas it was inactive at hIK even at a concentration
of 10mM (1.170.2-fold increase, n ¼ 3). The figures also
illustrate that the hSK3 and hIK channels are inhibited by
their respective blockers to the same extent in the absence
and presence of CyPPA. BMB applied at 30mM blocked hSK3
channels 7073.4 and 7874.1% (n ¼ 5) in the absence or
presence of CyPPA, whereas ChTX applied at 30nM blocked
hIK channels 5073.7 and 4675% (n ¼ 3), respectively, in the
absence or presence of CyPPA, in good agreement with
previous reports on SK and IK channel block by these
compounds (Jensen et al., 1998; Strøbæk et al., 2000).
Results
Effects of CyPPA on recombinant hSK3 and hIK channels
in the whole-cell configuration
Figure 1a shows the chemical structures of CyPPA as well as of
the reference compound NS309. CyPPA is a small (MW: 285),
trisubstituted pyrimidine that belongs to
a completely
different compound class from that of the prototype IK/SK
channel activators 1-EBIO and NS309, which are rigid, fused
heteroaromatic compounds. In Figure 1b and c, the effect of
CyPPA is compared to that of NS309. Whole-cell patch clamp
recordings were performed in symmetrical high Kþ solutions
using HEK293 cells stably expressing hSK3 and hIK, respec-
Figure 1 CyPPA is a positive modulator of hSK3 but not hIK channels. (a) Chemical structures of the subtype-selective SK channel activator
CyPPA and the pan-selective compound NS309. (b) The left panel shows whole-cell current–voltage (I-V) relationships from a HEK293 cell
expressing hSK3. The currents were recorded upon application of 200 ms long voltage ramps (À80 to þ 80 mV) elicited every 5 s from a
holding potential of 0 mV. The free [Ca2 þ ] in the pipette solution was buffered at 0.3 mM and the [Kþ ] was 154 mM in the intra- as well as
extracellular solutions. The traces were obtained before (Ctrl) and during application of 40 nM NS309 or 300 nM CyPPA as indicated. The right
panel shows the current at À75 mV as a function of time. The first 7 min after obtaining the whole-cell configuration are not shown. During this
period the hSK3 current increased as a result of Ca2 þ equilibration between the pipette solution and the cell; also 0.1% DMSO was shown to
be without effect on the currents. NS309 (40 nM), BMB (30 mM) and CyPPA (300 nM) were present in the bath solution as indicated by the bars.
BMB inhibited the current by 75 and 77% in the absence and presence of CyPPA, respectively. (c) Same experimental conditions as in (b) but
using a hIK-expressing HEK293 cell. The hIK current was activated by 40 nM NS309 and inhibited by 30 nM ChTX. The hIK current was inhibited
by 54 and 51% before and during application of 10 mM CyPPA.
British Journal of Pharmacology (2007) 151 655–665

Subtype-selective activation of SK channels
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659
CyPPA affinities for the individual SK channel subtypes studied
by inside-out patches
Inside-out patches enable measurements under controlled
intracellular [Ca2 þ ] and furthermore it is feasible to obtain
full activation curves for Ca2 þ and test compounds. Multi-
channel inside-out patches were pulled from hSK3, hSK2 and
hSK1 expressing HEK293 cells. Figure 2a shows typical SK3
current traces measured upon application of voltage ramps
in symmetrical Kþ concentrations with [Ca2 þ ]i buffered at
0.2 mM and 10 mM (solid lines) yielding 2.5 and 100%
activation, respectively (data from full Ca2 þ -activation
curves, see Figure 3). At 0.2 mM Ca2 þ
, application of
increasing concentrations of CyPPA (0.1 –100 mM, dotted
curves) to the inside of the patch, caused a concentration-
dependent increase of the hSK3 current, reaching nearly
100% activation (compared to 10 mM Ca2 þ ) at the highest
concentration. Note that the characteristic inward rectifica-
tion of the current was maintained when activated by a
combination of low Ca2 þ and CyPPA. Figure 2b shows the
time course of this experiment. A number of identical
experiments were performed and the data were normalized
to the maximal current attained at 10 mM Ca2 þ in the
individual experiments. Figure 2c shows the CyPPA concen-
tration–response plot for hSK3, hSK2 and hSK1 based on
these averaged and normalized data (n ¼ 3–5). Fitting the
data to Equation 1 resulted in an EC50 value of 5.671.6 mM
for activation of hSK3. The corresponding Hill coefficient
was 1.770.2 and the efficacy relative to 10 mM Ca2 þ was
9071.8%. CyPPA activated hSK2 with an EC50 value of
1474 mM, a Hill coefficient of 2.070.1 and an efficacy
of 7171.8% (middle curve), whereas no activation was
observed with hSK1 at concentrations of CyPPA up to 100 mM
(lower curve). CyPPA was not tested on hIK in inside-out
Figure 2 CyPPA is a SK subtype-selective potentiator. (a) I-V
relationships measured on an inside-out patch obtained from a
HEK293 cell stably expressing hSK3. The voltage protocol was as
described in the legend to Figure 1. The solid lines are I-V
relationships measured at a bath/intracellular [Ca2 þ ] of 0.2 or
10 mM in the absence of CyPPA. The dotted lines are I-V relationships
obtained in the presence of CyPPA (0.2 mM Ca2 þ , [CyPPA] from 0.1
to 100 mM as indicated). (b) hSK3 current at À75 mV as a function of
time. The patch was exposed to a bath/intracellular [Ca2 þ ] of 0.01,
0.2 or 10 mM as indicated. In the presence of 0.2 mM Ca2 þ , the patch
was exposed to increasing CyPPA concentrations (0.1–100 mM).
(c) Concentration–response relationship for CyPPA obtained from
experiments conducted as in (b). Currents were normalized with
respect to the effect of 10 mM Ca2 þ , which induces maximal SK
channel activity, and data points represent mean7s.e.m. of 3–5
experiments. The solid lines are the fit of the Hill equation to mean
data. EC50 values and Hill coefficients, estimated from separate
experiments were as follows: hSK3, 5.671.6 mM and 1.770.2
(n ¼ 3); hSK2, 1474 mM and 2.070.1 (n ¼ 3); hSK1, not detectable.
Efficacies with respect to 10 mM Ca2 þ were as follows: hSK3,
9071.8%; and hSK2, 7171.8%.
experiments due to
a prominent rundown of channel
activity in excised patches. For comparisons, the non
selective SK channel activators, NS309, DC-EBIO and 1-EBIO
activated hSK3 with EC50 values of 0.370.03 mM (n ¼ 3),
1672 mM (n ¼ 3) and 10407150 mM (n ¼ 5), respectively (not
shown).
Similar effects of CyPPA were obtained in experiments
with the corresponding rat SK3 and SK2 channels (results not
shown). As the rat isoform of the recombinant SK1 channel
does not express as a homomer, we have not been able to
investigate selectivity towards the rat SK1 channel.
The effect of CyPPA on the Ca2 þ -activation curves for
SK channels elucidated using inside-out patches
Figure 3a (left panel) shows the time course of an experiment
where the effect of 10 mM CyPPA was tested at 0.01, 0.2 and
10 mM intracellular Ca2 þ . No effect of CyPPA was observed at
a very low (0.01 mM Ca2 þ ) or a very high (10 mM Ca2 þ ) degree
of hSK3 channel activation, whereas a prominent stimula-
tion (from 6 to 80% of full activation) occurred at the
intermediate Ca2 þ concentration. Figure 3a (right panel)
shows the Ca2 þ -activation curves for hSK3 in the absence
and presence of 10 mM CyPPA. The Ca2 þ activation curve was
significantly affected by CyPPA (Po0.001, two-way ANOVA)
and the main effect is clearly to left-shift the activation curve
for Ca2 þ , but we also consistently found a reduced Hill
coefficient (from 6.370.4 to 3.670.2). The influence
of CyPPA on the Ca2 þ activation curve for hSK2 and hSK1
was investigated under identical experimental conditions
(Figure 3b and c). CyPPA also significantly (Po0.001, two-
way ANOVA) affected the Ca2 þ activation curve at hSK2
channels, although less prominently than at hSK3. In
contrast to the effect on hSK3, however, CyPPA induced a
small reduction of the current at high Ca2 þ concentrations
(Figure 3b, left panel). Consequently, the effect of CyPPA on
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Subtype-selective activation of SK channels
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C Hougaard et al
Figure 3 SK subtype-dependent effects of CyPPA on the Ca2 þ activation curves. In the left panels, currents at À75 mV are depicted as a
function of time. Currents were measured from inside-out patches obtained from HEK293 cells stably expressing hSK3 (a), hSK2 (b) or hSK1
(c). The patches were exposed to a bath/intracellular [Ca2 þ ] of 0.01, 0.2 or 10 mM as indicated, and CyPPA (10 mM) was applied at the different
[Ca2 þ ] as shown by the bars. In the right panels, the Ca2 þ concentration–response relationships for hSK3, hSK2 and hSK1 are depicted in the
absence or presence of 10 mM CyPPA. Currents from individual patches were normalized with respect to the effect of 10 mM Ca2 þ (in the
absence of CyPPA). The solid lines are the fit of the Hill equation to mean data. The following EC50(Ca2 þ ) and Hill coefficients were estimated
from separate experiments: hSK3, 0.4370.01 mM and 6.370.4 (control, n ¼ 14), 0.1270.02 mM and 3.470.2 ( þ 10 mM CyPPA, n ¼ 3); hSK2,
0.4270.02 mM and 5.270.1 (control, n ¼ 8), 0.2070.01 and 3.970.3 ( þ 10 mM CyPPA, efficacy: 9174%, n ¼ 3); hSK1, 0.4270.01 mM and
4.570.2 (control, n ¼ 8), 0.3870.03 mM and 3.570.2 ( þ 10 mM CyPPA, efficacy: 8477%, n ¼ 3 ); ***Po0.001 compared to corresponding
control values (Bonferroni post hoc comparisons).
the Ca2 þ activation curve for hSK2 is more complex than for
hSK3, consisting of a leftward shift and a slight reduction in
the maximal activation (right panel). No overall influence
on the activation of hSK1 was observed at any Ca2 þ
concentration (Figure 3c, left panel, P40.05, two-way
ANOVA), but the slight reduction in current at high Ca2 þ
concentrations, as was observed for hSK2, was also seen with
hSK1 (right panel).
(EC50(Ca2 þ )) vs the concentration of NS309 and CyPPA,
respectively. Both plots reveal saturating curves with a 350–
400 nM maximal change in the Ca2 þ -sensitivity of the drug-
modified hSK3 channel, reducing the EC50(Ca2 þ ) to below
75 nM. This suggests that, in the presence of high concentra-
tions of any of these positive modulators, the SK3 channel
can be activated at the resting intracellular Ca2 þ concentra-
tion. The EC50 values for the leftward shift in the hSK3
activation curve for Ca2 þ were 0.15 mM for NS309 and
1.01 mM for CyPPA.
The mechanism of action of CyPPA was compared to that
of the IK/SK channel activator, NS309, by performing
detailed concentration–response evaluations on the hSK3
activation curves for Ca2 þ . Figure 4a shows the results for
NS309 (0.03–30 mM) and Figure 4b shows the corresponding
results obtained with CyPPA (0.1–100 mM). The effects of the
two compounds are qualitatively very similar: both produce
a concentration-dependent leftward shift of the activation
curve for Ca2 þ , without increasing the maximal degree of
SK3 channel activation. This is quantified for both com-
pounds in Figure 4c, by plotting the EC50 values for Ca2 þ
Interaction studies with CyPPA and NS309 at the hSK1 channel
Given the very similar functional effects of CyPPA and
NS309 on hSK3, these compounds might be anticipated to
interact with this channel in a similar fashion, perhaps even
interacting with the same binding site. The question is then:
how does selectivity against hSK1 arise? The straightforward
possibility is that CyPPA does not bind at all to hSK1. An
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661
Figure 4 NS309 and CyPPA modulate the Ca2 þ sensitivity of SK3 channels in a similarly concentration-dependent manner. Ca2 þ
concentration–response relationships for hSK3, depicted in the absence or presence of increasing concentrations of NS309 (a) or CyPPA
(b) (n ¼ 3–14). Currents from individual patches were normalized with respect to the effect of 10 mM Ca2 þ (in the absence of compound). The
solid lines are the fit of data to the Hill equation, yielding the following EC50(Ca2 þ ) and Hill coefficients: (a) control: 429 nM and 5.6; 0.03 mM
NS309: 322 nM and 4.6; 0.3 mM NS309: 177 nM and 4.0; 3 mM NS309: 77 nM and 3.6; 30 mM NS309: 24 nM and 2.1; (b) Control: 429 nM and
5.6; 0.1 mM CyPPA: 332 nM and 3.7; 1 mM CyPPA: 239 nM and 3.6; 10 mM CyPPA: 119 nM and 3.1; 100 mM CyPPA: 59 nM and 2.8. At 100 mM
CyPPA, the fitted maximal value was 95%, for all other experiments the fitted maximal value was 499% with respect to the effect of 10 mM
Ca2 þ . (c) EC50(Ca2 þ ) as a function of the NS309 or CyPPA concentration. EC50(Ca2 þ ) values were obtained from the experiments
summarized in (a) þ (b). The solid lines are the fit of the Hill equation to mean data yielding the following minimal values for EC50(Ca2 þ ), EC50
and Hill coefficients: NS309: 24 nM, 0.15 mM and 0.6; CyPPA: 59 nM, 1.01 mM and 0.5.
alternative possibility is that CyPPA binds, but that it
selectively lacks the capability of transmitting the stimula-
tory action to the gating machinery of hSK1. To see if CyPPA
possibly interacts with or even occludes the activating effect
of NS309 an interaction study with these two compounds on
hSK1 was performed. Figure 5a shows the time course of an
inside-out experiment addressing this question: After max-
imal activation/deactivation of the hSK1 channel by high
and low Ca2 þ , respectively, the activity was adjusted to a low
level by 0.2 mM intracellular Ca2 þ . As expected, addition of
10 mM NS309 fully activated the hSK1 channel. After washout
of NS309, 10 mM CyPPA was added for 1.5 min (no activation)
before 10 mM NS309 was co-applied with CyPPA for another
1.5 min. Clearly, NS309 activated/deactivated the hSK1
channel as fully and as fast as in the absence of CyPPA. In
the bar diagrams of Figure 5b, the increase in hSK1 current
induced by 0.1 and 10 mM NS309 in the presence and absence
of 10 mM CyPPA is shown. Clearly, the presence of CyPPA is
not a significant hindrance for the NS309-induced activation
of hSK1 even at low concentrations of NS309.
Figure 5 CyPPA does not inhibit NS309-induced activation of
hSK1. (a) hSK1 current at À75 mV as a function of time. The current
was measured from an inside-out patch obtained from a HEK293 cell
stably expressing hSK1. The patch was exposed to
a bath/
intracellular [Ca2 þ ] of 0.01, 0.2 or 10 mM as indicated. NS309
(10 mM) was added at a [Ca2 þ ] of 0.2 mM either in the absence or
presence of 10 mM CyPPA. (b) Increase in hSK1 current induced by
NS309 (0.1 or 10 mM) in the absence or presence of 10 mM CyPPA;
n ¼ 6–13.
Effect of CyPPA on intact, unclamped cells in population using
a fluorescent Tlþ flux assay
As with all Kþ -selective ion channels (for data on KcsA, see
LeMasurier et al., 2001), SK channels are permeable for Tlþ
and we exploited this feature to carry out an influx assay
British Journal of Pharmacology (2007) 151 655–665

Subtype-selective activation of SK channels
662
C Hougaard et al
with cells loaded with a Tlþ -sensitive fluorescent dye (BTC-
AM). Figure 6a shows the Ca2 þ -induced activation of hSK1-3
and hIK expressed in HEK293 cells as measured by the
fluorescence increase over time. The effect of 1 mM ionomy-
cin (maximal response) was compared to the responses
obtained with 10 and 100 mM CyPPA, respectively. In the
hSK3 and hSK2 cell lines, CyPPA gave a clear increase in
fluorescence, whereas no response was detected using the
hSK1 and hIK cell lines. The distinct Tlþ fluorescence
increase seen upon CyPPA application to hSK3 and hSK2
cell lines emphasizes that CyPPA is capable of activating SK
channels at the resting concentration of intracellular Ca2 þ
prevailing in these cells. In SK-expressing cells both the
ionomycin- and CyPPA-induced signals were completely
blocked by apamin, whereas the ionomycin-induced fluor-
escence from the hIK cell line was blocked by clotrimazole
(data not shown). In contrast to CyPPA, NS309 concentra-
tion dependently increased the fluorescence from all four
cell lines (data not shown). Figure 6b shows the compiled
concentration–response curves (normalized to the maximal
ionomycin response) from the experiments with CyPPA. The
following selectivity sequence was found from the Tlþ
experiments: SK3 (EC50 ¼ 4.371.0 mM; efficacy ¼ 10572%);
SK2 (EC50 ¼ 1372.0 mM; efficacy ¼ 9174%); SK1 (undetect-
able); hIK (undetectable).
Effects of CyPPA on endogeneously expressed SK3 and IK channels
in the whole-cell configuration
The selectivity pattern for CyPPA described here is based on
the effect on recombinant channels overexpressed in a
mammalian cell line. To verify that the same selectivity
properties of CyPPA apply to endogenous SK/IK channels, we
also tested it on the SK3 expressing TE671 cells as well on the
IK expressing HeLa cells. Figure 7a shows whole-cell current-
voltage ramps (symmetrical Kþ ) from a TE671 cell. Applica-
tion of 10 mM CyPPA stimulated the current by a factor of 2,
which was essentially the same effect as obtained with a
subsequent 1 mM NS309 application. The experiment was
ended by application of 100 mM of the selective SK channel
blocker BMB. A very similar experimental protocol was
applied to the HeLa cells (Figure 7b; note the much less
pronounced inward rectification compared to the SK3-
mediated current from TE671 cells, compare also with
Figure 1). CyPPA at 10 mM had no effect on the IK current,
whereas NS309 at 1 mM exerted a significant activation. Again
the experiment was concluded with the application of the
established IK blocker, clotrimazole, at a concentration of
1 mM. Figure 7c and d show corresponding experiments with
TE671 and HeLa cells recorded under current-clamp condi-
tions with cells superfused with near-physiological saline.
Both cells exhibited a rather depolarized resting membrane
potential when equilibrated with a pipette solution buffered
at 100 nM Ca2 þ . In accordance with the voltage-clamp
experiments, CyPPA (10 mM) strongly hyperpolarized the
TE671 cell, whereas no effect was observed on the IK channel
expressing HeLa cell. Both cell lines responded with
substantial hyperpolarization upon application of NS309.
Finally, to investigate selectivity towards other classes of
ion channels, CyPPA was tested on a number of cloned ion
channels expressed in HEK293 cells (hERG, BK, KV7.2 þ 7.3,
NaV1.2) as well as voltage-dependent Naþ channels, delayed
rectifier Kþ channels, and high threshold voltage-dependent
Ca2 þ channels from rat dorsal root ganglia neurons (see
Table 1). The compound did not activate any ion channel
tested except for SK3 and SK2, indicating that this effect is
specific for SK channels. However, CyPPA caused an inhibi-
tion of BK (half-maximal inhibitory concentration
(IC50) ¼ 5.5 mM) and of NaV (IC50 E10 mM) channels.
Figure 6 Subtype selectivity of CyPPA in a Tlþ -based FLIPR assay.
(a) Relative fluorescence units (RFUs) measured over time in HEK293
cells expressing hSK1, hSK2, hSK3 or hIK channels loaded with the
fluorescent dye BTC-AM (see Materials and methods). At the time
indicated by the arrow, ionomycin (1 mM) or CyPPA (10 or 100 mM)
dissolved in Tlþ -containing ClÀ-free assay buffer was added to the
wells. Opening of either channel subtype permits an influx of Tlþ
,
resulting in an increase in RFUs due to interaction of Tlþ with BTC.
(b) Concentration–response relationship for CyPPA on the four cell
types expressing hSK1, hSK2, hSK3 or hIK, based on experiments
similar to those shown in (a). The increase in RFUs was normalized to
the effect of 1 mM ionomycin. The data points represent the
mean7s.e.m. of four independent experiments. The solid lines are
the fit of mean data to the Hill equation. The following efficacies,
EC50 values and Hill coefficients were estimated from separate
experiments (n ¼ 4): hSK3, 10572%, 4.371.0 mM and 1.170.1;
hSK2, 9174%, 13.072.0 mM and 1.670.2; hSK1, n.d. hIK, n.d.
Discussion and conclusions
In this study we have identified and characterized CyPPA, a
subtype-selective positive modulator of small conductance
British Journal of Pharmacology (2007) 151 655–665

Subtype-selective activation of SK channels
C Hougaard et al
663
Figure 7 CyPPA activates the endogenous SK channels in TE671 cells but not the endogenous IK channels in HeLa cells. Whole cell I-V
relationships measured in a TE671 (a) or HeLa (b) cell upon application of 200 ms long voltage ramps (À80 to þ 80 mV) elicited every 5 s from
a holding potential of 0 mV; [Ca2 þ ] in the pipette solution was 0.4 mM. Whole-cell I-V relationships were measured in the absence of compound
(Ctrl) and in the presence of NS309 (1 mM), or CyPPA (10 mM). BMB (100 mM) was applied as an inhibitor of SK channel activity in TE671 cells
(a) and clotrimazole (clot, 1 mM) as an inhibitor of IK channels in HeLa cells (b). Membrane potential measured under current-clamp conditions
in a TE671 cells (c) or a HeLa cell (d). The cells were exposed to a near-physiological Kþ gradient at an intracellular [Ca2 þ ] of 0.1 mM. NS309
(1 mM) and CyPPA (10 mM) were applied to the extracellular solution as indicated and experiments terminated by addition of BMB (100 mM, C)
or clot (1 mM, D); n ¼ 3–5.
Table 1 Effect of CyPPA on selected ion channels
voltage-clamp studies with symmetric Kþ distributions,
and in current-clamp studies using near-physiological ion
gradients. In contrast, using identical experimental condi-
tions, the compound had no activity on IK channels from
Ion channel
Cell type
Result (mM)
KCa1.1 (BK)
Kv7.2 þ 7.3 (KCNQ2 þ 3)
HEK293
HEK293
HEK293
HEK293
DRG
IC50 ¼ 5.571.3 (n ¼ 6)
No effect at 30 (n ¼ 3)
No effect at 10 (n ¼ 4)
IC50 ¼ 1071.9 (n ¼ 3)
IC50 ¼ 6878.8 (n ¼ 5)
IC50 ¼ 1170.48 (n ¼ 3)
No effect at 10 (n ¼ 3)
´
HeLa cells (Sauve et al., 1986). Hence, the selectivity profile
Kv11.3 (hERG)
of CyPPA is not a cloning artifact, is not related to the level of
expression or the expression system used, and is not linked
to a specific experimental technique.
Nav1.2
Voltage-dependent Kþ
Voltage-dependent Naþ
Voltage-dependent Ca2 þ
DRG
DRG
Using inside-out patches, the Ca2 þ concentration giving
50% channel activation, the EC50(Ca2 þ ) value, was found to
be 420–430 nM for all the human SK channel subtypes.
CyPPA was shown to induce a leftward shift in the activation
curves for Ca2 þ on hSK3 and, to a lesser degree, on hSK2
channels, whereas no shift was recorded for the hSK1
channel. A detailed analysis of the concentration depen-
Abbreviations: BK, large conductance Ca2 þ -activated Kþ channel; CyPPA,
cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine;
DRG, dorsal root ganglion; HEK, human embryonic kidney; hERG, human
ether-a-go-go-related gene; IC50, half-maximal inhibitory concentration.
CyPPA was tested in whole-cell voltage-clamp experiments using standard
experimental protocols using recombinant ion channels stably expressed in
HEK293 cells or endogeneously expressed channels in isolated DRG neurons.
IC50 values were calculated from the blocker kinetics, assuming that
dence of the CyPPA-mediated effect on EC50(Ca2 þ
)
)
À1, as described in Strøbæk et al. (2006).
was performed for the hSK3 channel: CyPPA left-shifts the
Ca2 þ -activation curve with an EC50 value of 1.01 mM to a
limiting minimal EC50(Ca2 þ ) value of 59 nM. This implies
that in presence of high concentrations of CyPPA, the hSK3
channel can be activated by the resting intracellular Ca2 þ
concentration of most cells. For comparison, a similar
analysis of hSK3 activation was performed for the potent
prototype IK/SK activator NS309. Qualitatively the two
compounds acted similarly, although NS309 was signifi-
cantly more potent (EC50(NS309) ¼ 150 nM) and caused a
slightly larger maximal shift in the EC50(Ca2 þ ) value (to
24 nM). Their similar modes of action on hSK3 might suggest
that NS309 and CyPPA bind to the same site and modulate
IC50 ¼ Kd ¼ (koff) (kon
Ca2 þ -activated Kþ channels. Using the whole-cell as well as
the inside-out patch-clamp technique, CyPPA was shown to
stimulate recombinant hSK3 and hSK2 channel activity,
whereas it was inactive on hSK1 and hIK channels. Using the
same cell lines, the selectivity sequence was confirmed with
a fluorescence-based Tlþ flux assay with unclamped popula-
tions of intact cells. The described selectivity of CyPPA holds
true for endogeneously expressed channels as well: CyPPA
activated SK channels in the TE671 human medulloblastoma
cell line (predominantly SK3, Carignani et al., 2002) both in
British Journal of Pharmacology (2007) 151 655–665

Subtype-selective activation of SK channels
664
C Hougaard et al
channel activity by
a
similar mechanism. There was,
function mutation in P/Q type Ca2 þ channels as human
patients), the precision of cerebellar Purkinje cell pacemaker
firing was shown to be significantly impaired owing to
inefficient periodic activation of SK channels. By increasing
the Ca2 þ sensitivity of the SK channels via local chronic
administration of 1-EBIO to the cerebellum, the precision of
Purkinje cell firing was restored and partial correction of the
ataxic behaviour was attained.
however, no apparent interaction between NS309 and CyPPA
on the hSK1 channels. The selectivity of CyPPA could
therefore result from lack of binding to
a common
modulator site of hSK1-3 and hIK or indicate that additional
sites are present in hSK2 and hSK3 subtypes. Mutagenesis
work has localized the 1-EBIO interaction site of SK2 to the
C-terminal region close to the CAMBD (Pedarzani et al.,
2001). A similar approach could be used to elucidate regions
important for CyPPA action and whether NS309 and CyPPA
possibly act through a common site.
The identification of a subtype-selective SK channel acti-
vator, such as CyPPA, has a number of consequences. Firstly,
this finding strongly indicates that positive modulation of SK
channel gating involves amino-acid residues on the a-subunit
that are nonconserved among the SK subtypes and also
compared to the IK channel. In this respect, the selectivity
profile of CyPPA is narrower than the recently described
negative gating modulator, NS8593 (Strøbæk et al., 2006),
which is inactive on IK, although it does not discriminate
between the SK members. Secondly, as a pharmacological tool,
CyPPA may be used in parallel with the IK/SK openers such as
1-EBIO and NS309 to distinguish SK3/SK2 from SK1-mediated
pharmacological responses; this is particularly important for
the SK2 and SK1 subtypes, which show considerable overlap
in expression in the neocortical and hippocampal regions
(Sailer et al., 2004). This also applies to peripheral cells where
both SK and IK channels are present, for instance in vascular
endothelial cells (SK3/IK, Eichler et al., 2003). However, it
must be stressed that CyPPA, owing to its inhibitory action on
Naþ channels, should be used cautiously in complex
biological systems such as epilepsy models, where the
causality of responses may be misinterpreted. Thirdly, CyPPA
may be used to address an important selectivity question –
which this study does not consider – namely whether CyPPA
can activate heteromeric SK channels (Benton et al., 2003;
Strassmaier et al., 2005). The answer to this question is
obviously of pharmacological relevance, in particular for SK1
and SK2, but it may also help to understand if the physical
binding site for SK channel openers possibly involves one or
more SK a-subunits.
The activation of neuronal SK channels by intracellular
Ca2 þ occurs by a fast association/dissociation reaction (Xia
et al., 1998), allowing SK channel opening and generation of
the mAHP following single or a few action potentials. In
presence of 1-EBIO, the deactivation time constant is greatly
prolonged (Pedarzani et al., 2001) and the use of 1-EBIO and
other positive modulators of IK/SK have revealed pharmaco-
logical sensitization of SK channels as
a principle for
intervention with CNS hyperactivity disorders. However,
owing to the lack of selectivity of these channel modulators,
the specific SK subtypes responsible are often ill –defined,
particularly with in vivo studies. 1-EBIO eliminates low Mg2 þ
-
induced network oscillations in cultures of dissociated cortical
neurons, suggesting a role for positive modulators of SK
channels in epilepsy (Pedarzani et al., 2001). Accordingly,
using hippocampal slices, 1-EBIO was shown to be an effective
general inhibitor of in vitro epileptiform activity in CA3
neurons, both when induced by increasing glutamatergic
drive (low extracellular Mg2 þ ), general excitability (slightly
increased extracellular Kþ ), or via GABA-ergic disinhibition
using antagonists of GABA-A (pentylenetetrazole, gabazine)
and GABA-B (CGP55845)-mediated neurotransmission, re-
spectively (Lappin et al., 2005). Recently, the anticonvulsive
effect of 1-EBIO has been confirmed in vivo using the mouse
maximal electroshock and the pentylenetetrazole models of
epilepsy; notably, however, CNS-related adverse effects were
observed at the effective doses (Anderson et al., 2006).
SK channels contribute to the precision of autonomous
activity in several pacemaking CNS neurons. Dopaminergic
neurons in the substantia nigra compacta exhibit a slow
(1–3 Hz) regular electrical activity when recorded in slices of
the midbrain (Shepard and Bunney, 1988). 1-EBIO reduces
the frequency of this spontaneous activity (Wolfart et al.,
2001), an effect that is blocked by apamin. Pharmacological
modulation of SK channels also affects firing of dopaminer-
gic neurons in vivo: the highly irregular firing mode recorded
by extracellular single-unit recording is regularized by
systemic administration of 1-EBIO, whereas local apamin
(Ji and Shepard, 2006) or N-methyl-laudanosine (Waroux
et al., 2005) application induce burst firing. These observa-
tions may help explain the action of the centrally acting
muscle relaxant drugs, zoxazolamine and chlorzoxazone
(Matthews et al., 1984). SK channels are also essential for
firing control in cerebellar Purkinje neurons, where 1-EBIO
and apamin exert opposite effects and induce pace-making
and bursting, respectively (Cingolani et al., 2002). Recently,
elegant experiments have been performed with a disease
model of Human Ataxia Type 2 (Walter et al., 2006). Using an
ataxic tottering mouse (which carries a similar loss-of-
Acknowledgements
Aino Munch is acknowledged for help with the thallium flux
characterization. Jette Sonne, Vibeke Meyland-Smith and
Anne Stryhn Meincke are acknowledged for help with the
initial patch-clamp characterization of CyPPA. Dr Gordon
John Blackburn-Munro is greatly acknowledged for reading
this paper and correcting the language.
Conflict of interest
The authors state no conflict of interest.
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