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See detailThe interactions of apamin and tetraethylammonium are differentially affected by single mutations in the pore mouth of small conductance calcium-activated potassium (SK) channels
Dilly, Sébastien ULg; Philippart, Fabian ULg; Lamy, Cédric et al

in Biochemical Pharmacology (2013), 85

Valine residues in the pore region of SK2 (V366) and SK3 (V520) were replaced by either an alanine or a phenylalanine to evaluate the impact on the interactions with the allosteric blocker apamin. Unlike ... [more ▼]

Valine residues in the pore region of SK2 (V366) and SK3 (V520) were replaced by either an alanine or a phenylalanine to evaluate the impact on the interactions with the allosteric blocker apamin. Unlike TEA which showed high sensitivity to phenylalanine mutated channels, the binding affinity of apamin to the phenylalanine mutants was strongly reduced. In addition, currents from phenylalanine mutants were largely resistant to block by apamin. On the other hand, when the valine residue was replaced by an alanine residue, an increase of the binding affinity and the amount of block by apamin was observed for alanine mutated SK2 channels, but not for mutated SK3 channels. Interestingly, the VA mutation reduced the sensitivity to TEA. In silico data confirmed these experimental results. Therefore, such mutations in the pore region of SK channels show that the three-dimensional structure of the SK tetramers can be disorganized in the outer pore region leading to reduced interaction of apamin with its target. [less ▲]

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See detailPhysiologie, pharmacologie et modélisation de canaux potassiques : zoom sur les canaux SK
Dilly, Sébastien ULg; Poncin, Sylvie; Lamy, Cédric et al

in Medecine Sciences : M/S (2012), 28

Various types of ion channels are involved in the control of neuronal activity. Among them, SK channels represent an interesting therapeutic target. Indeed, they underlie medium duration ... [more ▼]

Various types of ion channels are involved in the control of neuronal activity. Among them, SK channels represent an interesting therapeutic target. Indeed, they underlie medium duration afterhyperpolarizations in many types of neurons, thus inhibiting cell excitability. A thorough knowledge of the physiology of these channels and the discovery of non-peptidic selective modulators able to pass the blood-brain barrier are essential in view of developing future drugs for brain diseases such as those related to a dysfunction of dopaminergic and serotonergic systems. [less ▲]

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See detailInteractions of apamin with pore mutated SK3 channels
Dilly, Sébastien ULg; Lamy, Cédric; Poncin, Sylvie et al

Poster (2012, March 16)

In the present work, we have tested the impact of the replacement of valine residues in the pore region of SK3 (520) by either an alanine or a phenylalanine residue in terms of the interactions of apamin ... [more ▼]

In the present work, we have tested the impact of the replacement of valine residues in the pore region of SK3 (520) by either an alanine or a phenylalanine residue in terms of the interactions of apamin with these mutants in comparison with the corresponding native channels. Replacing valine residue at position 520 of the SK3 channel by a phenylalanine significantly increased the sensitivity of the channel to be blocked by tetraethylammonium (TEA) as previously reported. Indeed, an aromatic residue, such as a phenylalanine or a tyrosine, is frequently found in the pore region of several potassium channels more sensitive to TEA than SK channels. We measured the affinity (Kd) of apamin in saturation experiments and studied SK currents in transfected cells using patch clamp techniques. In parallel, molecular modelling techniques were used to examine the impact of these local modifications on the interaction of apamin with the corresponding channels. The presence of a phenylalanine in the pore region of potassium channels led to a higher sensitivity for TEA by creating more hydrophobic interactions as found by the docking procedure. In the in vitro binding experiments, the phenylalanine mutant (SK3VF) displayed a very low affinity for apamin. In patch clamp experiments, the SK current was only very partially blocked by apamin in the SK3VF mutant. Furthermore, apamin displayed an affinity and a blocking activity for the alanine mutant close to that for the corresponding native channels. In conclusion, the presence of a bulky and hydrophobic residue at a position near the pore mouth of SK3 channels has a negative impact on their interactions with apamin. [less ▲]

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See detailNew pyridobenzoxazepine derivatives derived from 5-(4-methylpiperazin-1-yl)-8-chloro-pyrido[2,3-b][1,5]benzoxazepine (JL13): chemical synthesis and pharmacological evaluation
Liégeois, Jean-François ULg; Deville, Marine; Dilly, Sébastien ULg et al

in Journal of Medicinal Chemistry (2012), 55(1572), 1582

A series of new pyridobenzoxazepine derivatives with various heterocyclic amine side chains were synthesized in order to explore two main parameters related to the distal basic nitrogen. These compounds ... [more ▼]

A series of new pyridobenzoxazepine derivatives with various heterocyclic amine side chains were synthesized in order to explore two main parameters related to the distal basic nitrogen. These compounds were tested for their affinity for dopamine D2L, D4, serotonin 5-HT1A, 5-HT2A and adrenergic 2A receptors in comparison with 5-(4-methylpiperazin-1-yl)-8-chloro-pyrido[2,3-b][1,5]benzoxazepine, JL13 (1) and other diarylazepine derivatives. In terms of multi-receptor target strategy, 2 and 5 present the most promising in vitro binding profile. Bulky, polar and more flexible side chains are not favourable in this context. 2 and 5 were tested in adult rats to evaluate their long-term effects on dopamine and serotonin receptors density in different brain areas. Similar to 1 and other second-generation antipsychotic drugs, repeated treatment with 2 significantly increased D1 and D4 receptors in nucleus accumbens and caudate putamen, and D2 receptors in medial prefrontal cortex and hippocampus while 5 significantly increased D2 and D4 receptors in nucleus accumbens. In addition, 2 increased 5-HT1A and decreased 5-HT2A receptors in cerebral cortex. In contrast, 5 did not alter levels of any 5-HT receptor subtype in any brain region examined. These results encourage further development of 2 as a novel second-generation antipsychotic agent. [less ▲]

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See detailIon channel modulators: more diversity than previously thought
Dilly, Sébastien ULg; Lamy, Cédric; Marrion, Neil et al

in Chembiochem : A European Journal of Chemical Biology (2011), 12(12), 1808-1812

Ion channel function can be modified in various ways. For example, numerous studies have shown that currents through voltage-gated ion channels are affected by pore block or modification of voltage ... [more ▼]

Ion channel function can be modified in various ways. For example, numerous studies have shown that currents through voltage-gated ion channels are affected by pore block or modification of voltage-dependence of activation/inactivation. Recent experiments performed on various ion channels show that allosteric modulation is an important mechanism to affect channel function. For instance, in KCa2 (formerly SK) channels, the prototypic “blocker” apamin prevents conduction by an allosteric mechanism, while TRPV1 channels are prevented from closing by a tarantula toxin, DkTx, through an interaction with residues located away from the selectivity filter. The recent evidence therefore suggests that, in several ion channels, the region around the outer mouth of the pore is rich in binding sites which may be exploited therapeutically. These discoveries also suggest that the pharmacological vocabulary should be adapted to define these various actions. [less ▲]

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See detailBlock of SK channels by the sigma agonist 1,3-di-o-tolyl-guanidine: evidence for a novel site of action for SK blockers
Dilly, Sébastien ULg; Lamy, Cédric; Snyders, Dirk et al

Poster (2010, October 16)

Among ion channels involved in the control of neuronal activity, small conductance calcium-activated potassium channels (SK) represent an interesting therapeutic target. Indeed, they underlie medium ... [more ▼]

Among ion channels involved in the control of neuronal activity, small conductance calcium-activated potassium channels (SK) represent an interesting therapeutic target. Indeed, they underlie medium duration afterhyperpolarizations (mAHPs) in many types of neurons, thus inhibiting cell excitability. Three subtypes of SK subunits, SK1, SK2 and SK3, have been cloned and are expressed differentially within the central nervous system (CNS). Blocking SK channels might be beneficial in the treatment of several CNS disorders such as depression (SK3), Parkinson’s disease (SK3) and cognitive disorders (SK2). So far, the prototypical blocker of SK channels is apamin, an octadecapeptide from bee venom. We have recently shown that apamin blocks SK channels by binding to a site distinct from that used by classical pore blockers such as tetraethylammonium (TEA) (Lamy et al. J. Biol. Chem. 2010, 285, 27067-77). We have also demonstrated that the nonpeptide blocker N-methyl-laudanosine (NML) (Scuvée-Moreau et al. J. Pharmacol. Exp. Ther. 2002, 302, 1176-83) competes for the binding site of the toxin. Further, our research team has recently shown that the sigma agonist 1,3-di-o-tolyl-guanidine (DTG) directly blocks SK currents in a voltage-independent manner (Lamy et al. Eur. J. Pharmacol. 2010, 641, 23-8). We have combined patch clamp experiments on cell lines with molecular modelling and mutagenesis, to try to identify the site where DTG blocks. DTG was found to be equipotent on wild-type (WT) and apamin-insensitive (e.g. SK2H337N) channels. Moreover, mutated channels with increased sensitivity to TEA (SK3V520F: mean IC50 of TEA: 0.34 mM versus 11 mM for WT channels) were blocked by DTG with the same potency as WT channels. Thus, DTG does not seem to share the site of either apamin or TEA. Modelling data were in agreement with this possibility because of the identification of various potential binding sites. Although preliminary, these results suggest the existence of yet another binding site in the outer pore region of SK channels. [less ▲]

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See detailEtude du mode de liaison des canaux potassiques de type SK et l’apamine par modélisation moléculaire
Dilly, Sébastien ULg; Lamy, Cédric; Seutin, Vincent ULg et al

Poster (2010, May 20)

Parmi les canaux ioniques impliqués dans le contrôle de l'activité neuronale, les canaux potassiques calcium-dépendants de basse conductance, dénommés canaux SK, constituent une cible thérapeutique ... [more ▼]

Parmi les canaux ioniques impliqués dans le contrôle de l'activité neuronale, les canaux potassiques calcium-dépendants de basse conductance, dénommés canaux SK, constituent une cible thérapeutique intéressante. En effet, ils sous-tendent la posthyperpolarisation ("AfterHyperPolarization") de durée moyenne (mAHP) qui limite l'excitabilité de divers types de neurones du système nerveux central (SNC). A ce jour, 3 types de sous-unités, SK1, SK2 et SK3, ont été identifiés dans différentes régions du cerveau. Le blocage sélectif de ces canaux pourrait être bénéfique dans le traitement de divers troubles du SNC comme la maladie de Parkinson (SK3), la dépression (SK3) ou encore les désordres cognitifs (SK2) (Liégeois et al., 2003 ; Pedarzani et al., 2008). Jusqu’à présent, le site d’interaction entre les canaux SK et leurs bloqueurs n’a pas encore été précisément élucidé. Dans ce contexte, nous avons entrepris la modélisation de ces canaux par homologie comparative en se basant sur la structure cristalline du canal potassique KCSA (Doyle et al., 1998). La construction de ces canaux constitue la première étape dans la détermination des requis structuraux essentiels à l’affinité de bloqueurs et à la compréhension des modes de liaison de ces ligands. Le mode de liaison de l’apamine, bloqueur peptidique issu du venin d’abeille, a été ensuite exploré par « docking ». Afin de confirmer ce site de liaison potentiel, des expériences de mutagénèse dirigée ont été réalisées. Les premiers canaux mutants testés dans des expériences électrophysiologiques par la technique de « patch clamp » ont permis de valider certaines données théoriques. Grâce à cette stratégie, nous espérons préciser le mécanisme d'action des bloqueurs des canaux SK et, idéalement, découvrir des pistes pour concevoir des bloqueurs sélectifs. [less ▲]

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See detailCombined experimental and computational approaches to study the action of blockers of small conductance calcium-activated potassium (SK) channels
Dilly, Sébastien ULg; Lamy, Cédric; Liégeois, Jean-François ULg et al

in Acta Physiologica Scandinavica (2010), 199(supplement 678), -10

Small conductance calcium-activated potassium channels (SK) are widely expressed throughout the central nervous system (CNS) and underlie medium duration afterhyperpolarizations in many types of neurons ... [more ▼]

Small conductance calcium-activated potassium channels (SK) are widely expressed throughout the central nervous system (CNS) and underlie medium duration afterhyperpolarizations in many types of neurons. Three subtypes of SK channels, SK1, SK2 and SK3, have been identified so far in different parts of the brain. Blocking SK channels might be beneficial in the treatment of several CNS disorders such as depression, Parkinson’s disease and cognitive disorders. Until now, the precise site of interaction between these channels and their blockers has not yet been elucidated. In this context, molecular modeling is a theoretical approach that can quickly provide ideas on the binding mode of SK blockers. We first performed homology modeling of the S5-H5-S6 portion of the channels on the basis of the crystal structure of the KcsA potassium channel (Zhou et al. Nature. 2001, 414, 43-48). The binding sites of N-methyl-laudanosine (NML) (Scuvée-Moreau et al. J. Pharmacol. Exp. Ther. 2002, 302, 1176-83), a non-selective and non-peptidic ligand, and apamin (Blatz et al. Nature. 1986, 323, 718-20), an octadecapeptide with a preference for the SK2 subtype, were subsequently explored by docking analysis. Different amino-acids were suggested to interact with the two blockers. The docking of NML revealed a binding site in the turret region, far from the pore. The docking of apamin identified a very large binding site that includes a portion of the site of NML. In order to confirm the predicted binding sites, site-directed mutagenesis was used. The first mutant channels tested in electrophysiological experiments by the patch clamp technique validated some of the theoretical data. Using this strategy, we hope to get a better understanding of the mechanism of action of SK blockers and eventually find strategies to obtain subtype-selective blockers. [less ▲]

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See detailDirect block of SK2 and SK3 current by the sigma agonist 1,3-di-(2-tolyl)guanidine
Lamy, Cédric; Moreau, Jacqueline ULg; Dilly, Sébastien ULg et al

Poster (2008, November 17)

Sigma receptors are widely distributed in the central nervous system where they modulate neurotransmitter release, receptor function, ionic channel activity and calcium homeostasis. Two subtypes of sigma ... [more ▼]

Sigma receptors are widely distributed in the central nervous system where they modulate neurotransmitter release, receptor function, ionic channel activity and calcium homeostasis. Two subtypes of sigma receptors have been identified (sigma-1 and sigma-2) with different pharmacological profiles, anatomical distribution and physiological functions. 1,3-Di-(2-tolyl)guanidine (DTG) is a sigma-1 and sigma-2 agonist which is widely used to probe the function of these receptors. It has recently been shown that sigma-1 receptor activation reduces the opening of SK channels in the hippocampus. We have observed that DTG (100 µM) reduces the apamin-sensitive afterhyperpolarization (AHP) of dopaminergic neurons within a slice preparation by ~60%, an effect not observed with other sigma agonists. In addition, neither the selective sigma-1 antagonist BD 1047 (30 µM) nor haloperidol (1 µM) blocked the effect of DTG, which suggested that the inhibition of the AHP might result from a direct block of the underlying SK channels. Whole-cell recordings were made from HEK293 cells transiently transfected with rSK2 or hSK3 cDNA in symmetrical K+ conditions with currents activated by a [Cai] of 1 µM. Expressed SK2 and SK3 channels displayed a classical pharmacology, being blocked by apamin with mean IC50’s of 100 pM and 4 nM, respectively. In contrast, both channel subtypes were blocked with equal sensitivity by N-methyl-laudanosine (NML). DTG inhibited both SK2 and SK3 currents with the same potency (IC50’s were ~30 µM). A mutation that rendered both SK2 and SK3 insensitive to apamin and NML produced a current that was still sensitive to DTG. This direct block of SK channels may be important to consider in relation to the pharmacological effects of this compound. [less ▲]

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See detailSK Channel blockade promotes burst firing in dorsal raphe serotonergic neurons
Rouchet, Nathalie; Waroux, Olivier ULg; Lamy, Cédric et al

in European Journal of Neuroscience (2008), 28(6), 1108-15

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See detailDirect induction of burst firing by SK channel blockade in serotonergic neurons in vivo
Rouchet, Nathalie; Waroux, Olivier ULg; Moreau, Jacqueline ULg et al

Scientific conference (2007, November 04)

Small conductance calcium-activated potassium channels (SK channels) are widely expressed throughout the central nervous system and underlie the medium afterhyperpolarization following a single or a train ... [more ▼]

Small conductance calcium-activated potassium channels (SK channels) are widely expressed throughout the central nervous system and underlie the medium afterhyperpolarization following a single or a train of action potentials. It has been shown that they are involved in the regulation of the excitability and the firing pattern of several types of neurons. In vivo, serotonergic (5-HT) neurons of the dorsal raphe nucleus usually show a tonic pattern of discharge, but they can also display repetitive burst firing activity, usually involving doublets of closely spaced (< 20 ms) action potentials. It has been shown that burst firing is correlated with an increase in transmitter release and postsynaptic effects (Gartside et al., Neuroscience, 98, 295-300, 2000). We hypothesized that SK channels modulate the firing pattern of 5-HT neurons. In a preliminary study, extracellular single-cell recordings combined with iontophoresis showed that UCL1684, a water soluble SK blocker (200 µM), significantly increased the % of spikes produced in bursts in 60% of presumed serotonergic neurons in the anesthetized rat. We confirm here this observation by demonstrating that UCL1684 significantly increased the production of doublets in 17 out of 25 serotonergic neurons. In order to explore whether a GABAergic input was involved in this effect, additional experiments were performed in the presence of the specific GABAA antagonist SR 95531. In these conditions, 50 % (5 out of 10) of serotonergic neurons showed an increase in the production of doublets when UCL 1684 was applied (p = 0.31 vs control), suggesting that a GABAergic input is not implicated in the regulation of the firing pattern of 5-HT neurons by the SK blocker. Finally, the effect of SK channel blockade was explored in vitro in slices. Bath application of the SK blocker apamin (300 nM) did not induce bursting in 15 out of 18 neurons (p < 0.001 vs in vivo control conditions), although it did increase the coefficient of variation of the interspike intervals.Taken together, our results suggest that SK blockade induces burst firing in a majority of dorsal raphe serotonergic neurons. This effect does not involve GABAergic interneurons, but requires an input that is only present in vivo. [less ▲]

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See detailEffect of SK channel blockade on the firing of dorsal raphe neurons in anaesthetized rats
Alleva, Livia ULg; Rouchet, Nathalie; Waroux, Olivier ULg et al

Poster (2006, October 17)

K channels are small conductance calcium-activated potassium channels which trigger an outward current generating an afterhyperpolarization (AHP). This AHP follows a single or a train of action potential ... [more ▼]

K channels are small conductance calcium-activated potassium channels which trigger an outward current generating an afterhyperpolarization (AHP). This AHP follows a single or a train of action potential, and therefore is important in the regulation of the firing frequency and/or pattern of many types of neurons. Serotonergic (5-HT) neurons from the raphe nuclei express SK channels and exhibit a significant AHP which can be efficiently blocked in vitro by apamin and N-methyl laudanosine (NML) (Scuvée-Moreau et al, 2004). In the later study, we found that some but not all neurons (50%) had a significantly increase in their firing rate when positive current was injected after SK channel blockade. In order to determine the physiological relevance of these channels in vivo, single unit extracellular recordings were carried out in anesthetized rats and combined with iontophoresis of the specific non-peptidic SK channel blocker, UCL1684. 5-HT neurons were tested for their inhibitory response to locally applied 5-HT and histological analysis confirmed the localization of the recording site. UCL 1684 was used at a concentration of 200 µM. Out of 11 neurons recorded, 6 showed a significant increase in the production of doublets, with no effect on their mean firing rate as compared to the control condition. The other neurons were completely unaffected. These results suggest that the responsiveness of presumed 5-HT neurons to SK channel block is variable. Although the use of 200 µM UCL allow us to be sure of a sufficient SK blockade at the recording site (Waroux et al, 2005), we can not rule out the possibility that SK channels present at the dendritic level were not completely blocked. In conclusion, SK channels in vivo might play a role in controlling the firing pattern of a subgroup of 5-HT neurons. [less ▲]

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