Methyl-laudanosine: A new pharmacological tool to investigate the function of small-conductance Ca2+-activated K+ channels

Article abstract of DOI:10.1124/jpet.302.3.1176

Small-conductance Ca²⁺-activated K⁺ channels (SK channels) underlie the prolonged postspike afterhyperpolarization (AHP) observed in many central neurons and play an important role in modulating neuronal activity. However, a lack of

Full text of DOI:10.1124/jpet.302.3.1176

0022-3565/02/3023-1176–1183$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 302:1176–1183, 2002
Vol. 302, No. 3
4758/1005386
Printed in U.S.A.
Methyl-Laudanosine: A New Pharmacological Tool to
Investigate the Function of Small-Conductance Ca2ϩ-Activated
Kϩ Channels
JACQUELINE SCUVEE-MOREAU, JEAN-FRANC¸ OIS LIEGEOIS, LAURENT MASSOTTE, and VINCENT SEUTIN
Research Center for Cellular and Molecular Neurobiology, Laboratory of Pharmacology (J.S.-M., L.M., V.S.), and Natural and Synthetic Drugs
Research Center, Laboratory of Medicinal Chemistry (J.-F.L.), University of Lie` ge, Lie` ge, Belgium
Received November 13, 2001; accepted May 15, 2002
This article is available online at http://jpet.aspetjournals.org
ABSTRACT
Small-conductance Ca2ϩ-activated Kϩ channels (SK channels) effect on the Ih current, action potential parameters, or mem-
underlie the prolonged postspike afterhyperpolarization (AHP) brane resistance of dopaminergic cells, or on the decrease in
observed in many central neurons and play an important role in input resistance induced by muscimol, indicating a lack of
modulating neuronal activity. However, a lack of specific and antagonism at GABAA receptors. Interestingly, 100 ␮M methyl-
reversible blockers of these channels hampers their study in laudanosine induced a significant increase in spiking frequency
various experimental conditions. Because previous work has of dopaminergic neurons but not of CA1 pyramidal cells, sug-
shown that bicuculline salts block these channels, we exam- gesting the possibility of regional selectivity. Binding experi-
ined whether related alkaloids, namely laudanosine quaternary ments on laudanosine derivatives were in good agreement with
derivatives, would produce similar effects. Intracellular record- electrophysiological data. Moreover, methyl-laudanosine has
ings were performed on rat midbrain dopaminergic neurons no affinity for voltage-gated potassium channels, and its affinity
and hippocampus CA1 pyramidal cells. Binding experiments for SK channels (IC50 4 ␮M) is superior to its affinity for mus-
were performed on rat cerebral cortex membranes. Lau- carinic (IC50 114 ␮M) and neuronal nicotinic (IC50 Ն367 ␮M)
danosine, methyl-laudanosine, and ethyl-laudanosine blocked receptors . Methyl-laudanosine may be a valuable pharmaco-
the apamin-sensitive AHP of dopaminergic neurons with mean logical tool to investigate the role of SK channels in various
IC50 values of 152, 15, and 47 ␮M, respectively. The benzyl and experimental models.
butyl derivatives were less potent. Methyl-laudanosine had no
Other than neurotransmitter receptors and transporters, served in many central neurons. Three closely related SK-
ion channels constitute an attractive target to develop new
drugs that will be active on the central nervous system.
Currently, the only ion channel that is well established as a
central nervous system target is the voltage-gated Naϩ chan-
nel, which is blocked by antiepileptic drugs such as phenyt-
oin, carbamazepine, and lamotrigine (McNamara, 1996).
Ca2ϩ-activated Kϩ channels play a fundamental role in the
control of the firing frequency and pattern of neurons (for
reviews, see Sah, 1996; Vergara et al., 1998). Within this
class of Kϩ channels, small-conductance voltage-insensitive
Ca2ϩ-activated Kϩ channels (SK channels) underlie the pro-
longed postspike afterhyperpolarization (AHP) that is ob-
channel subtypes, SK1, SK2, and SK3, have been cloned and
are characterized by different sensitivities to apamin (Ko¨hler
et al., 1996; Strøbæk et al., 2000). These subtypes are differ-
entially expressed in the brain (Stocker and Pedarzani,
2000). Moreover, the currents underlying AHP have been
classified into two groups on the basis of their kinetic and
pharmacological properties. IAHP, the current underlying the
medium AHP, is present in most excitable cells; it is sensitive
to the bee venom toxin apamin, and its activation has been
reported to control firing frequency in tonically spiking neu-
rons. sIAHP, the current underlying the slow AHP, is present
in a few cell types only; it is apamin-insensitive but can be
modulated by various neurotransmitters, and its activation
is responsible for late-spike frequency adaptation (Sah, 1996;
Vergara et al., 1998).
This work was supported in part by Grant 3.4525.98 from the National
Fund for Scientific Research (Brussels, Belgium). This work has been pre-
sented in meeting abstract form: Scuve´e-Moreau J, Lie´geois JF, and Seutin V
(2002) Effect of laudanosine derivatives on the apamin-sensitive afterhyper-
polarization of rat dopaminergic neurons—identification of methyl-lau-
danosine as a new specific blocker (Abstract). Fundam Clin Pharmacol 16:67.
Evidence suggests that SK-channel modulation may be
interesting in a range of central nervous system disorders,
ABBREVIATIONS: SK channels, small-conductance voltage-insensitive Ca2ϩ-activated Kϩ channels; AHP, afterhyperpolarization; ZD7288,
4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium chloride; SR95531, 2-[carboxy-3Ј-propyl]-3-amino-6-paramethoxy-phe-
nyl-pyridazinium bromide.
1176

Methyl-Laudanosine and SK Channels
1177
including Alzheimer’s disease and schizophrenia. Indeed, short pieces of platinum wire. The slice was completely immersed in a
continuously flowing (ϳ2 ml/min), heated solution (35°C) of the same
composition as indicated above. Most recordings of dopaminergic neu-
rons were made from neurons located in the substantia nigra pars
compacta. Identification of dopaminergic and CA1 pyramidal cells was
performed as described previously (Scuve´e-Moreau et al., 1997; Seutin
et al., 1997).
Intracellular recordings were made using glass microelectrodes
filled with 2 M KCl (resistance 70 to 150 M⍀). All recordings were
made in the bridge balance mode, using an NPI SEC1L amplifier
(NPI Electronic GmbH, Tamm, Germany). The accuracy of the bridge
was checked throughout the experiment by examining the voltage
deflection induced by a small (Ϫ50 pA) current injection. The poten-
tial of the extracellular medium was measured at the end of each
experiment, and its absolute value was within 5 mV of that set to 0
at the start. Membrane potentials and injected currents were re-
corded on a Gould TA240 chart recorder (Gould Instrument Systems,
Valley View, OH) and on a Fluke Combiscope oscilloscope (Fluke
Corp., Everett, WA). The Flukeview software was used for off-line
analysis in most cases. Some experimental data were recorded with
pClamp (Axon Instruments, Inc., Foster City, CA).
Drug effects on the prominent apamin-sensitive AHP in dopami-
nergic neurons were quantified as the percentage of reduction of the
surface area of the AHP (in millivolts per second), which was blocked
by a maximally active concentration of apamin (300 nM; Seutin et
al., 1997). Averages of four sweeps were considered in all cases. In
these experiments, the spontaneous firing of the neurons was usu-
ally reduced by constant current injection (Ϫ20 to Ϫ100 pA) to
increase the amplitude of the AHP. Because the amplitude of the
AHP is very sensitive to the firing rate, care was taken to compare all
AHPs of one cell at the same firing rate; this usually necessitated
only very small adjustments of the injected currents (i.e., less than
20 pA). Excitability of dopaminergic neurons and CA1 pyramidal
cells was assessed by applying long (Ն1 s) depolarizing pulses of
increasing intensities, at about 4- to 5-s intervals, and counting the
number of action potentials elicited by the different pulses; averages
of four pulses of similar intensity were considered in the analysis of
the results. In these experiments, the baseline membrane potential
was set at Ϫ60 to Ϫ65 mV by negative current injection.
The antagonism at GABAA receptors was quantified in dopami-
nergic cells as the ability to antagonize the reduction in input resis-
tance induced by 3 ␮M muscimol. Input resistance was measured by
the amplitude of the steady-state voltage deflection elicited by pass-
ing a small hyperpolarizing current (Ϫ20 to Ϫ60 pA). These experi-
ments were performed in the presence of tetrodotoxin (0.5 ␮M) to
minimize indirect effects. Cesium (3 mM) or ZD7288 [4-(N-ethyl-N-
phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium chlo-
ride] (30 ␮M) were used in these experiments to block the activation
of the Ih current (Harris and Constanti, 1995; Mercuri et al., 1995).
All drugs were applied by superfusion. Complete exchange of the
bath solution occurred within 2 to 3 min.
apamin has been shown to improve some aspects of memory
processing in animal models (Ikonen and Riekkinen, 1999;
Fournier et al., 2001), and longer CAG repeats within the
gene of the SK3 channel have been associated with schizo-
phrenia in various ethnic groups (Chandy et al., 1998; Dror et
al., 1999), although the latter observation is controversial
(Antonarakis et al., 1999).
To validate SK channels (or one of their subtypes) as an
interesting drug target, it is critical to develop adequate tools
for studying their function. Apamin is not ideal because it
induces a long-lasting block of SK channels; moreover, its
peptidic nature precludes iontophoretic application during in
vivo experiments. Several currently available nonpeptidic
blockers of SK channels (e.g., (ϩ)-tubocurarine, bicuculline
salts, and gallamine) lack specificity, acting on other targets,
including GABAA, nicotinic, or muscarinic receptors (Lee and
el-Fakahany, 1991; Dunn et al., 1996; Seutin and Johnson,
1999; Strøbæk et al., 2000). Recently, novel nonpeptidic
blockers of SK channels have been synthetised and evalu-
ated. Among these bis-quinolinium cyclophanes, UCL1684
has been found to be the most potent compound, having an
IC50 value of 3 nM on AHP of rat superior cervical ganglion
neurons (Campos Rosa et al., 2000). However, to our knowl-
edge, a comprehensive screening of various receptors and
channels is not available for these compounds, making it
difficult to assess their degree of selectivity.
Given the structural analogy between bicuculline and lau-
danosine, a metabolite of the neuromuscular relaxant atra-
curium, we decided to evaluate the ability of laudanosine and
its methyl, ethyl, butyl, and benzyl derivatives to selectively
block SK channels. Both electrophysiological and binding
experiments were performed. The electrophysiological study
was done on rat midbrain dopaminergic neurons and hip-
pocampus CA1 pyramidal cells. Midbrain dopaminergic neu-
rons display a prominent apamin-sensitive AHP of medium
duration, which appears to be mediated by SK3 channels
(Shepard and Bunney, 1991; Wolfart et al., 2001). Hippocam-
pus CA1 pyramidal cells present both a medium, apamin-
sensitive and a slow, apamin-insensitive AHP (Stocker et al.,
1999). SK1 and SK2 subunits are highly expressed in this
region (Stocker and Pedarzani, 2000). Binding experiments
on SK channels, voltage-gated potassium channels, and mus-
carinic and nicotinic receptors were performed on rat cere-
bral cortex membranes.
Materials and Methods
Curve fitting was carried out using Kaleidagraph (Synergy Soft-
ware, Reading, PA) or GraphPad Prism (GraphPad Software, Inc.,
San Diego, CA) and the standard equation, E ϭ Emax/[1 ϩ (IC50/x)h],
where x is the concentration of the drug and h is the Hill coefficient.
Numerical values are expressed as means Ϯ S.E.M. Statistical anal-
ysis was performed using Student’s t test. The level of significance
was set at p Ͻ 0.05.
Binding Studies. Radioligand binding studies were performed to
assess the affinity of laudanosine and its quaternary derivatives for SK
channels and the affinity of methyl-laudanosine for voltage-gated po-
tassium channels and muscarinic and nicotinic receptors. Assays were
performed by (Cerep/Paris, France) on rat cerebral cortex membranes
using general procedures described by Hugues et al. (1982) for SK
channels, by Sorensen and Blaustein (1989) for voltage-gated potas-
sium channels, by Richards (1990) for nonselective muscarinic recep-
tors, by Pabreza et al. (1991) for neuronal ␣-bungarotoxin-insensitive
Electrophysiological Experiments. The methods used were sim-
ilar to those described previously (Seutin et al., 1997). Male Wistar rats
(150–200 g) were used. They were housed and handled in accordance
with guidelines of the National Institute of Health (National Institutes
of Health Publication 85-23, 1985). Animals were anesthetized with
chloral hydrate (400 mg/kg i.p.) and decapitated. The brain was excised
quickly and placed in cold (ϳ4°C) artificial cerebrospinal fluid of the
following composition 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4,
1.2 mM MgCl2, 2.4 mM CaCl2, 11 mM glucose, and 18 mM NaHCO3,
saturated with 95% O2 and 5% CO2 (pH 7.4). A block of tissue contain-
ing the midbrain or the hippocampus was cut into horizontal or trans-
verse slices, respectively (thickness 350 ␮m), in a Vibratome (Lancer,
St. Louis, MO). The slice containing the region of interest was placed on
a nylon mesh in a recording chamber (volume 500 ␮l). The tissue was
held in position with two electron microscopy grids weighed down by

1178
Scuvee-Moreau et al.
nicotinic receptors, and by Sharples et al. (2000) for neuronal ␣-bunga- Formula 989, Packard) or a solid scintillant (Meltilex B/HS, Wallac).
rotoxin-sensitive nicotinic receptors. Laudanosine derivatives were tested in triplicate on SK channels;
Ligands used and incubation conditions are as follows: 0.004 nM methyl-laudanosine was tested in triplicate on voltage-gated potas-
125I-apamin, 30 min/0°C (SK channels); 0.01 nM 125I-␣-dendrotoxin, sium channels and in duplicate on muscarinic and nicotinic recep-
30 min/22°C (voltage-gated potassium channels), 0.05 nM [3H]3- tors. In each experiment, the respective reference compound was
quinuclidinylbenzilate, 120 min/22°C (muscarinic receptors); 1.5 nM tested at a minimum of eight concentrations in duplicate to obtain a
[3H]cytisine, 75 min/4°C (␣-bungarotoxin-insensitive nicotinic recep- competition curve to validate the experiment. The specific radioli-
tors); and 1 nM 125I-␣-bungarotoxin, 150 min/37°C (␣-bungarotoxin- gand binding to the receptors is defined as the difference between
sensitive nicotinic receptors). Nonspecific binding was assessed us- total binding and nonspecific binding determined in the presence of
ing 0.1 ␮M apamin, 50 nM ␣-dendrotoxin, 1 ␮M atropine, 10 ␮M an excess of unlabeled ligand. IC50 values and Hill coefficients were
nicotine, and 10 ␮M ␣-bungarotoxin, respectively. Following incuba- determined fitting the data to the Hill equation using nonlinear
tion, membranes were rapidly filtered under vacuum through glass regression analysis. The inhibition constants (Ki) were calculated
fiber filters (GF/B, PerkinElmer Life Sciences, Boston, MA; GF/C, from the Cheng-Prusoff equation (Ki ϭ IC50/(1 ϩ L/KD), where L is
Whatman International, Maidstone, UK; or Filtermat A or B, the concentration of radioligand in the assay and KD is the affinity of
PerkinElmer Wallac, Gaithersburg, MD). Filters were then washed the radioligand for the receptor).
several times with an ice-cold buffer using a cell harvester (Packard;
Brandel Inc., Gaithersburg, MD; or Tomtec, Orange, CT). Bound
Compounds. Methyl-laudanosine iodide, ethyl-laudanosine iodide,
butyl-laudanosine iodide, and benzyl-laudanosine chloride (Fig. 1) were
radioactivity was measured with a scintillation counter (Topcount, synthesized in our laboratory according to conventional methods.
Packard; LS series, Beckman Coulter, Inc., Fullerton, CA; or Beta- Briefly, a mixture of laudanosine with an excess of alkyl or aralkyl
plate, Wallac) using a liquid scintillation cocktail (Microscint 0 or halide in acetonitrile was refluxed overnight. Excess of reagent and
Fig. 1. Chemical structure
of bicuculline, laudanosine,
and their quaternary salts,
bicuculline methyl iodide,
and laudanosine methyl io-
dide (R ϭ ϪCH3), ethyl io-
dide (R ϭ ϪC2H5), butyl io-
dide (R
benzyl chloride (R
ϪCH2ϪC6H5).
ϭ ϪC4H9), and
ϭ

Methyl-Laudanosine and SK Channels
1179
Fig. 2. Effect of increasing concentrations of methyl-laudanosine (CH3-L) on the AHP of dopaminergic neurons. A maximal block similar to the one
obtained with 300 nM apamin is obtained with 100 ␮M CH3-L (A). The effect of CH3-L is fully reversible after 10 min in wash (B). Each trace is the
mean of four sweeps. Action potentials are truncated.
solvent was removed under reduced pressure. For each compound, the
crude quaternary salt was isolated in an acetone/diethyl ether mixture.
Results
Midbrain Dopaminergic Neurons. All recorded neu-
rons (n ϭ 32) displayed characteristic features of identified
dopamine neurons (Grace and Onn, 1989), including broad-
action potentials (Ն1 ms at 50% maximum height) with a
threshold close to Ϫ40mV, a prominent Ih current, and a slow
AHP, which reached its peak Ϯ 50 ms after the action poten-
tial; their mean input resistance was 183 Ϯ 11 M⍀.
Methyl-laudanosine induced a marked blockade of the slow
AHP; this effect was concentration-dependent, reaching a
maximum at 100 ␮M. The IC50 and the Hill coefficient were
15 Ϯ 1 ␮M and 1.1 Ϯ 0.03 (n ϭ 7), respectively. The maximal
effect, complete block of the AHP, was similar to that seen
with 300 nM apamin (Fig. 2A). The effect of methyl-lau-
danosine was fully and quickly reversible (about 10 min for
complete recovery) (Fig. 2B), contrary to what is observed
with apamin (Seutin et al., 1993). Ethyl-laudanosine also
blocked the apamin-sensitive AHP in a concentration-depen-
Finally, the products were recrystallized from ethanol, methyl ethyl
ketone, diethyl ether/acetone, or dichloromethane/diethyl ether for
methyl, ethyl, butyl, and benzyl derivatives, respectively. Purity was
checked with classical methods, such as elemental analysis, mass spec-
trometry, and determination of the melting point.
Other compounds used and their sources were as follows: apamin,
cesium chloride, atropine sulfate, (Ϫ)nicotine bitartrate, ␣-dendro-
toxin, ␣-bungarotoxin (obtained from Sigma-Aldrich, St. Louis, MO),
laudanosine (purchased from Aldrich Chemical Co., Milwaukee, WI),
muscimol, tetrodotoxin (obtained from Tocris Cookson Inc., Ballwin,
MO), 125I-apamin, [3H]quinuclidinyl benzilate, [3H]cytisine, 125I-␣-
bungarotoxin (purchased from PerkinElmer Life Sciences, Boston,
MA), 125I-␣-dendrotoxin (purchased from Amersham Biosciences
Inc., Piscataway, NJ), ZD7288 (gift from AstraZeneca Pharmaceuti-
cals LP, Wilmington, DE), and SR95531 [2-[carboxy-3Ј-propyl]-3-
amino-6-paramethoxy-phenyl-pyridazinium bromide] (gift from
Sanofi-Synthelabo, Paris, France). Laudanosine was dissolved in
ethanol or dimethyl sulfoxide; all other drugs were dissolved in
water.
Fig. 3. A, concentration-response curves for the blockade of the slow AHP in midbrain dopaminergic neurons by laudanosine derivatives. Each data
point is presented as the mean Ϯ S.E.M. of three experiments except for methyl- and ethyl-laudanosine (n ϭ 7 and n ϭ 6, respectively). Values for
concentrations higher than 300 ␮M were extrapolated. B, displacement of 125I-apamin binding at rat neocortical SK channels by laudanosine
derivatives. Each data point is presented as the mean Ϯ S.E.M. of three experiments. Values for concentrations higher than 1 mM were extrapolated.
f, Methyl-laudanosine; F, ethyl-laudanosine; Ⅺ, benzyl-laudanosine; ࡗ, butyl-laudanosine; Œ, laudanosine.

1180
Scuvee-Moreau et al.
dent manner, but it was less potent than methyl-lau- tions), or on the voltage deflection induced by activation of Ih
danosine. Its IC50 and Hill coefficient were 47 Ϯ 6 ␮M and (10 Ϯ 0.6 mV) (Fig. 4, A and B). A putative effect of methyl-
1.0 Ϯ 0.02 (n ϭ 6), respectively. The effect of ethyl-lau- laudanosine on GABAA receptors was also investigated in
danosine was also fully and quickly reversible. Laudanosine, four cells; methyl-laudanosine (100 ␮M) did not modify the
benzyl-laudanosine, and butyl-laudanosine had a weak in- decrease in input resistance induced by muscimol (3 ␮M),
hibitory effect on the slow AHP, with respective IC50 values whereas the GABAA antagonist SR95531 (10 ␮M) completely
of 152 Ϯ 8 ␮M, 249 Ϯ 39 ␮M, and Ͼ300 ␮M (n ϭ 3 for each reversed the effect of muscimol (Fig. 4C).
compound). Results are summarized in Fig. 3A.
The consequences of AHP blockade by methyl-laudanosine
Because methyl-laudanosine was the most potent blocker on cell excitability were investigated by applying long (1–2 s)
of the AHP among the various laudanosine derivatives depolarizing current pulses of increasing intensities from a
tested, its influence on different electrophysiological param- resting membrane potential set at about Ϫ60mV. The effect
eters was further investigated. Methyl-laudanosine (3–100 of methyl-laudanosine was compared with that of apamin.
␮M) had no effect on the amplitude (50 Ϯ 2.5 mV from Under control conditions, tonic firing (1 to 4 spikes) was
threshold) and the duration of the action potential (1.3 Ϯ 0.2 induced by depolarizing pulses of increasing intensities (50 to
ms at mid-height in both control and treatment conditions) 200 pA). Both 100 ␮M methyl-laudanosine (n ϭ 8) and 300
(n ϭ 4). Its effect on membrane potential, input resistance, nM apamin (n ϭ 6) induced a significant increase in the
and Ih current was examined in four cells (three in the number of spikes evoked in the recorded cells; the effect of
presence of 1 ␮M tetrodotoxin). For this purpose, neurons methyl-laudanosine was reversible after 10 to 20 min in
were hyperpolarized to Ϫ60 mV by continuous current injec- wash (Fig. 5A). The effect of both compounds was indepen-
tion (Ϫ200 to Ϫ300 pA), their resistance was estimated by dent of the amplitude of the injected current (Fig. 5, B and C).
measuring the voltage deflection induced by a small injection
Hippocampus CA1 Pyramidal Cells. In view of the
of current (Ϫ20 to Ϫ40 pA), and a robust Ih current was different regional distribution of SK subunits, it was inter-
elicited by current injections of Ϫ80 to Ϫ120 pA. Methyl- esting to investigate the influence of methyl-laudanosine on
laudanosine had no effect on membrane potential, input re- the excitability of CA1 pyramidal cells, as these cells have a
sistance (231 Ϯ 22 M⍀ in both control and treatment condi- high level of expression for SK1 and SK2 subunits, contrary
Fig. 4. Effect of 100 ␮M methyl-laudanosine (CH3-L) on cell parameters and GABAA receptors in dopaminergic neurons recorded intracellularly in rat
brain slices. A, CH3-L has no effect on spike amplitude or duration. B, CH3-L has no effect on membrane potential, on input resistance measured by
the amplitude of the voltage deflection elicited by a hyperpolarizing pulse of Ϫ30 pA, or on the Ih current activated by a hyperpolarizing pulse of Ϫ120
pA. The experiment was performed in the presence of 0.5 ␮M tetrodotoxin (TTX); chart speed was reduced during 7 min at the beginning of drug
application. C, CH3-L does not modify the decrease in input resistance induced by muscimol, whereas this decrease is fully antagonized by the GABAA
antagonist SR95531. Traces are means of voltage deflections induced by a current injection of Ϫ60 pA from a potential of Ϫ60 mV in control and
different treatment conditions. ZD7288 (30 ␮M) and tetrodotoxin (0.5 ␮M) were present throughout the experiment.

Methyl-Laudanosine and SK Channels
1181
Fig. 5. Effect of methyl-laudanosine and apamin on the excitability of midbrain dopaminergic neurons. A, action potentials (truncated in the figure)
were elicited by 200 pA depolarizing current injections before, during, and after application of 100 ␮M methyl-laudanosine; the membrane potential
was set at Ϫ60 mV by a continuous injection of Ϫ100 pA. B and C, the number of action potentials elicited by 1- to 1.5-s current injections are plotted
against the level of intensity of the injected current. Methyl-laudanosine (100 ␮M) (B) and apamin (300 nM) (C) induced a significant increase in the
number of action potentials evoked (ૺ, p Ͻ 0.05; ૺૺ, p Ͻ 0.005). The effect of methyl-laudanosine was reversible (see A). Values are means of eight
(methyl-laudanosine) and six (apamin) experiments. Absolute values of current injection were different among experiments; the intensities were
chosen to produce increasing numbers of action potentials from 0 to 4 per injection in control conditions.
Fig. 6. Effect of methyl-laudanosine and apamin on the excitability of hippocampus CA1 pyramidal cells. A, trains of action potentials (truncated in
the figure) characterized by early- and late-frequency adaptation were elicited by 150 pA depolarizing current injections; resting membrane potential
was Ϫ65 mV. Methyl-laudanosine (100 ␮M) did not modify cell excitability. B and C, the number of action potentials elicited by ϳ1-s current injections
are plotted against the level of intensity of the injected current. Methyl-laudanosine (100 ␮M) (B) failed to modify the number of action potentials
evoked, whereas apamin (300 nM) (C) induced a significant increase in this number (ૺ, p Ͻ 0.05). Values are means of eight experiments. Absolute
values of current injection were different among experiments; the intensities were chosen to produce increasing numbers of action potentials (from 0
to 12) in control conditions.

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Scuvee-Moreau et al.
TABLE 1
Discussion
Binding affinities of laudanosine, its quaternary derivatives, and the
reference compound, apamin, at SK channels
The Ki values were calculated by the method of Cheng and Prusoff.
This study was initiated to identify a specific and revers-
ible blocker of SK channels. Drawbacks of blockers such as
apamin, bicuculline, (ϩ)-tubocurarine, and gallamine have
been stressed earlier (see the Introduction).
SK Channel
Compound
IC50
Ki
h
Our results show that quaternary laudanosine derivatives
differentially block the slow AHP in rat dopaminergic neurons,
with the N-methyl derivative being the most potent blocker.
This property, which was previously described for quaternary
salts of bicuculline, namely, bicuculline methyl iodide and bicu-
culline methochloride (Seutin et al., 1997), was suspected given
the similarity of structure between these compounds. Methyl-
laudanosine is slightly more potent as a blocker of the AHP
than bicuculline salts, whose IC50 was found to be 26 ␮M in the
same conditions (Seutin et al., 1997). The difference of potency
(ϳ10 times) between the methyl derivative of laudanosine and
the base is similar to that reported previously between the
quaternary salts of bicuculline and the base (Seutin et al.,
1997). Increasing the length of the N-substituent does not in-
crease the activity on SK channels.
Experiments on cell excitability show that apamin and
methyl-laudanosine significantly increase the number of
spikes evoked in midbrain dopaminergic neurons by positive
current injection. These results are consistent with other
studies indicating a role for SK channels in the control of the
firing frequency of these cells (Shepard and Bunney, 1991;
Wolfart et al., 2001). Interestingly, an increase in the firing
frequency of hippocampus CA1 pyramidal cells was observed
with apamin but not with methyl-laudanosine. Our results
with apamin are in agreement with previous studies indicat-
ing a contribution of apamin-sensitive SK channels to the
firing properties of hippocampal pyramidal neurons (Stocker
et al., 1999). The fact that methyl-laudanosine does not mod-
ify cell excitability at a concentration inducing a maximal
block of the AHP in dopaminergic neurons suggests the pos-
sibility that this compound discriminates between SK sub-
types. In fact, recent studies show a high level of expression
of SK3 subunits in midbrain dopaminergic neurons (Tacconi
et al., 2001; Wolfart et al., 2001) and a high level of expres-
sion of SK1 and SK2 subunits in CA1 to CA3 pyramidal
neurons (Stocker et al., 1999). Electrophysiological results
thus point to a preferential effect of methyl-laudanosine on
the SK3 subtype of SK channels. However, further studies in
slices and in cell lines expressing the various subunits are
needed to address this point thoroughly.
nM
Test compound
Laudanosine
Methyl-
laudanosine
Ethyl-laudanosine
Butyl-laudanosine
Benzyl-
laudanosine
Reference compound
Apamin
40,200
3,850
30,400
2,450
0.9
0.8
47,800
Ϸ294,000*
56,400
30,400
N.C.
35,900
0.9
N.C.
1.0
0.015
0.0095
1.0
IC50, concentration causing a half-maximal inhibition of control-specific binding;
Ki, inhibition constant; h, Hill coefficient; N.C., not calculable.
* This value is approximative; a precise IC50 value could not be obtained.
to midbrain dopaminergic neurons, which express mostly the
SK3 subunit (Stocker and Pedarzani, 2000). Experiments
were performed on eight cells; their mean input resistance
was 55 Ϯ 7.5 M⍀. Under control conditions, long (ϳ1 s)
depolarizing pulses elicited trains of action potentials char-
acterized by early- and late-spike frequency adaptation (Fig.
6A, left panel). Methyl-laudanosine (100 ␮M) failed to signif-
icantly modify spiking frequency and pattern (Fig. 6, A and
B), whereas apamin (300 nM) induced a significant increase
of the number of action potentials evoked without modifying
the late-spike frequency adaptation (not shown). The effect of
apamin was independent of the amplitude of the injected
current (Fig. 6C).
Binding Studies. The IC50 and Ki values determined for
laudanosine, its quaternary derivatives, and the reference com-
pound apamin at neocortical SK channels are presented in
Table 1 and Fig. 3B. The order of affinity was methyl-lau-
danosine Ͼ laudanosine Ն ethyl-laudanosine Ն benzyl-lau-
danosine Ͼ butyl-laudanosine. The IC50 and Ki values deter-
mined for methyl-laudanosine and the reference compounds at
voltage-gated potassium channels and muscarinic and nicotinic
receptors are shown in Table 2. Depending on the parameter
being considered, the affinity of methyl-laudanosine for SK
channels was 8 to 30 times superior to its affinity for muscarinic
receptors. The affinity for nicotinic receptors was weak or neg-
ligible depending upon the subtype examined (␣-bungarotoxin-
insensitive or -sensitive). No affinity for voltage-gated potas-
sium channels could be detected.
Binding data obtained from rat cerebral cortex membranes
are in good agreement with the electrophysiological study on
TABLE 2
Binding affinities of methyl-laudanosine and reference compounds at voltage-gated potassium channels, and muscarinic and nicotinic receptors
The Ki values were calculated by the method of Cheng and Prusoff.
Methyl-Laudanosine
Reference Compounds
Receptor
IC50
Ki
h
IC50
Ki
h
␮M
nM
Voltage-gated potassium channel
Ͼ1,000
114
Ͼ1,000
367
N.C.
19.1
N.C.
84.7
N.C.
0.8
N.C.
1.0
␣-Dendrotoxin
Atropine
0.53
0.37
16
3.8
0.42
0.062
8.9
1.2
0.9
1.0
0.6
Muscarinic (nonselective)
Nicotonic (neuronal) (␣-bungarotoxin-insensitive)
Nicotinic (neuronal) (␣-bungarotoxin-sensitive)
Nicotine
␣-Bungarotoxin
0.89
IC50, concentration causing a half-maximal inhibition of control-specific binding; Ki, inhibition constant; h, Hill coefficient; N.C. value not calculable because of insufficient
inhibition at the highest test concentration (1 mM).

Methyl-Laudanosine and SK Channels
1183
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the two kinds of studies are quite similar (ϳ15 ␮M in elec-
trophysiological studies and ϳ4 ␮M in binding experiments).
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Methyl-laudanosine has a relatively low potency when
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a high off-rate of the compound from the channel that makes
it a rapidly reversible blocker. This property may be inter-
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Our study demonstrates the pharmacological differences
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performed to further study the structural elements required
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In conclusion, the lack of influence of methyl-laudanosine
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Acknowledgments
We are grateful to AstraZeneca and Sanofi-Synthelabo for the gift
of ZD7288 and SR95531, respectively.
Vergara C, Latorre R, Marrion NV, and Adelman JP (1998) Calcium-activated
potassium channels. Curr Opin Neurobiol 8:321–329.
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Address correspondence to: Dr. Jacqueline Scuve´e-Moreau, Laboratory of
Pharmacology, Research Center for Cellular and Molecular Neurobiology,
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