Synthesis and biological activity of 3,4-dihydroquinazolines for selective T-type Ca2+ channel blockers

We have synthesized 3,4-dihydroquinazoline derivatives for the potent and selective T-type Ca(2+) channel blockers and evaluated for their inhibitory activities against two subtypes T-type Ca(2+) channels and N-type Ca(2+) channels. Among them, 5b (KYS05044, IC(50)=0.56+/-0.10 microM) was identified as potent T-type Ca(2+) channel blocker with in vitro selectivity profile at meaningful level (T/N-type, SI=>100).     

Stage-dependent inhibition of Plasmodium falciparum by potent Ca2+ and calmodulin modulators

The effects of Ca2+ channel blockers, verapamil, nicardipine and diltiazem, and of potent calmodulin (CaM) inhibitors, trifluoperazine (TFP), calmidazolium, W-7 and W-5, on Plasmodium falciparum in culture were examined. Among Ca2+ blockers, nicardipine was the most potent with the 50% inhibitory concentration (IC50) of 4.3 microM at 72 h after culture. Parasites were more sensitive to calmidazolium and W-7 with IC50 of 3.4 and 4.5 microM, respectively, than to TFP and W-5. All Ca2+ blockers and CaM inhibitors suppressed parasite development at later stages. Nicardipine, diltiazem, calmidazolium and W-5 also retarded parasite development at earlier stages and/or subsequent growth following pretreatment. Verapamil, nicardipine, TFP and calmidazolium reduced erythrocyte invasion by merozoites. Fluorescence microscopy with the cationic fluorescent dye rhodamine 123 revealed that nicardipine, TFP and calmidazolium depolarized both the plasma membrane and mitochondrial membrane potentials of the parasite. It is therefore considered that although all Ca2+ and CaM antagonists tested here influence parasite development at later stages, they are multifunctional, having effects not directly associated with Ca2+ channels or CaM.     

Cloning and functional expression of a liver isoform of the small conductance Ca2+-activated K+ channel SK3

Small conductance Ca2+-activated K+ (SK) channels have been cloned from mammalian brain, but little is known about the molecular characteristics of SK channels in nonexcitable tissues. Here, we report the isolation from rat liver of an isoform of SK3. The sequence of the rat liver isoform differs from rat brain SK3 in five amino acid residues in the NH3 terminus, where it more closely resembles human brain SK3. SK3 immunoreactivity was detectable in hepatocytes in rat liver and in HTC rat hepatoma cells. Human embryonic kidney (HEK-293) cells transfected with liver SK3 expressed 10 pS K+ channels that were Ca2+ dependent (EC(50) 630 nM) and were blocked by the SK channel inhibitor apamin (IC(50) 0.6 nM); whole cell SK3 currents inactivated at membrane potentials more positive than -40 mV. Notably, the Ca2+ dependence, apamin sensitivity, and voltage-dependent inactivation of SK3 are strikingly similar to the properties of hepatocellular and biliary epithelial SK channels evoked by metabolic stress. These observations raise the possibility that SK3 channels influence membrane K+ permeability in hepatobiliary cells during liver injury.     

Stage Depandent Inhibition of Plasmodiun falcipaium falciparum by Potent Ca2+ and Calmodulin Modulators

The effects Ca2+ channel blockers, verapamil, nicardipine and diltiazem, and of potent calmodulin (CaM) inhibitors, trifluoperazine (TFP), calmidazolium, W-7 and W-5, on Plasmodium falciparum in culture were examined. Among Ca2+ blockers, nicardipine was the most potent with the 50% inhibitory concentration (IC50) of 4.3 μM at 72 h after culture. Parasites were more sensitive to calmidazolium and W-7 with IC50 of 3.4 and 4.5 μM, respectively, than to TFP and W-5. All Ca2+ blockers and CaM inhibitors suppressed parasite development at later stages. Nicardipine, ditiazem, calmidazolium and W-5 also retarded parasite development at earlier stages and/or subsequent growth following pretreatment. Verapamil, nicardipine, TFP and calmidazolium reduced erythocyte invasion by merozoites. Fluroscence microscopy with the cationic flurescent dye rhodamine 123 revealed that nicardipine. TFP and calmidazolium depolarized both the plasma membrane and mitochondrial membrane potentials of the parasite. It is therefore considered that although al Ca2+ and CaM antagonists tested here influence parasite development at later stages, they are multifunctional, having effects not directly associated with Ca2+ channels or CaM.     

BmBKTx1, a novel Ca2+-activated K+ channel blocker purified from the Asian scorpion Buthus martensi Karsch

BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch. It is composed of 31 residues and is structurally related to SK toxins. However, when tested on the cloned rat SK2 channel, it only partially inhibited rSK2 currents, even at a concentration of 1 microm. To screen for other possible targets, BmBKTx1 was then tested on isolated metathoracic dorsal unpaired median neurons of Locusta migratoria, in which a wide variety of ion channels are expressed. The results suggested that BmBKTx1 could specifically block voltage-gated Ca(2+)-activated K(+) currents (BK-type). This was confirmed by testing the BmBKTx1 effect on the alpha subunits of BK channels of the cockroach (pSlo), fruit fly (dSlo), and human (hSlo), heterologously expressed in HEK293 cells. The IC(50) for channel blocking by BmBKTx1 was 82 nm for pSlo and 194 nm for dSlo. Interestingly, BmBKTx1 hardly affected hSlo currents, even at concentrations as high as 10 microm, suggesting that the toxin might be insect specific. In contrast to most other scorpion BK blockers that also act on the Kv1.3 channel, BmBKTx1 did not affect this channel as well as other Kv channels. These results show that BmBKTx1 is a novel kind of blocker of BK-type Ca(2+)-activated K(+) channels. As the first reported toxin active on the Drosophila Slo channel dSlo, it will also greatly facilitate studying the physiological role of BK channels in this model organism.     

Tamapin,a venom peptide from the Indian red scorpion (Mesobuthus tamulus) that targets small conductance Ca2+-activated K+ channels and afterhyperpolarization currents in central neurons

The biophysical properties of small conductance Ca(2+)-activated K(+) (SK) channels are well suited to underlie afterhyperpolarizations (AHPs) shaping the firing patterns of a conspicuous number of central and peripheral neurons. We have identified a new scorpion toxin (tamapin) that binds to SK channels with high affinity and inhibits SK channel-mediated currents in pyramidal neurons of the hippocampus as well as in cell lines expressing distinct SK channel subunits. This toxin distinguished between the SK channels underlying the apamin-sensitive I(AHP) and the Ca(2+)-activated K(+) channels mediating the slow I(AHP) (sI(AHP)) in hippocampal neurons. Compared with related scorpion toxins, tamapin displayed a unique, remarkable selectivity for SK2 versus SK1 ( approximately 1750-fold) and SK3 ( approximately 70-fold) channels and is the most potent SK2 channel blocker characterized so far (IC(50) for SK2 channels = 24 pm). Tamapin will facilitate the characterization of the subunit composition of native SK channels and help determine their involvement in electrical and biochemical signaling.     

3,4-Dihydroquinazoline derivatives as novel selective T-type Ca2+ channel blockers

For LVA T-type Ca2+ channel blockers, 3,4-dihydroquinazoline derivatives as new scaffolds were prepared and evaluated for the inhibitory activity against two members of the recombinant T-type Ca2+ channel family. Among them, 8a (KYS05001, IC50=0.9 microM) was nearly equipotent with mibefradil (IC50=0.84 microM) and inhibited LVA T-type Ca2+ channel with greater efficacy than HVA Ca2+ channel.     

Bis-tetrahydroisoquinoline derivatives: AG525E1, a new step in the search for non-quaternary non-peptidic small conductance Ca(2+)-activated K(+) channel blockers

So far, small conductance Ca(2+)-activated K(+) channel (SK) blockers mostly consist of quaternary ammonium derivatives or peptides. Due to their physicochemical properties, these blockers are not suitable to study the physiological roles of SK channels in the central nervous system in vivo. Herein, we report the discovery of a chiral bis-tertiary amine with SK blocking properties from chemical modulation of laudanosine. AG525E1 has an affinity for SK channels (K(i)=293nM) approximately 100-fold higher than the tertiary compound laudanosine (K(i) approximately 30muM) and similar to the charged compound dequalinium (K(i)=221nM). AG525E1 equipotently blocks SK1, SK2 and SK3 currents in transfected cell lines. Because of its basic and lipophilic properties, it can reach central SK targets.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.     

High affinity inhibition of Ca(2+)-dependent K+ channels by cytochrome P-450 inhibitors.

The Ca(2+)-dependent K+ channel of human red cells was inhibited with high affinity by several imidazole antimycotics which are potent inhibitors of cytochrome P-450. IC50 values were (in microM): clotrimazole, 0.05; tioconazole, 0.3; miconazole, 1.5; econazole, 1.8. Inhibition of the channel was also found with other drugs with known cytochrome P-450 inhibitory effect. However, no inhibition was obtained with carbon monoxide (CO). This suggests that, given the high selectivity of the above inhibitors for the heme moiety, a different but closely related to cytochrome P-450 kind of hemoprotein may be involved in the regulation of the red cell Ca(2+)-dependent K+ channel. Clotrimazole also inhibited two other charybdotoxin-sensitive Ca(2+)-dependent K+ channels, those of rat thymocytes (IC50 = 0.1-0.2 microM) and of Ehrlich ascites tumor cells (IC50 = 0.5 microM). Imidazole antimycotics inhibit also receptor-operated Ca2+ channels (Montero, M., Alvarez, J. and García-Sancho, J. (1991) Biochem. J. 277, 73-79). This suggests that both Ca2+ and Ca(2+)-dependent K+ channels might have a similar regulatory mechanism involving a cytochrome.     

Expression and modulation of the intermediate- conductance Ca2+-activated K+ channel in glioblastoma GL-15 cells.

We report here the expression and properties of the intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) channel in the GL-15 human glioblastoma cell line. Macroscopic IK(Ca) currents on GL-15 cells displayed a mean amplitude of 7.2+/-0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC(50)=257 nM), TRAM-34 (IC(50)=55 nM), and charybdotoxin (CTX, IC(50)=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IK(Ca) channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a K(D) for Ca(2+) of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 muM, for 5 days) virtually suppressed the IK(Ca) current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17+/-0.75 pA/pF at 1-2 days, and 3.11+/-1.35 pA/pF at 5-6 days after plating. This time-dependent downregulation of the IK(Ca) current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ~35% reduction of the IK(Ca) channel mRNA resulting from ERK inhibition and a approximately 50% reduction with time in culture.     

Synthesis and SAR studies of a novel series of T-type calcium channel blockers

For the novel, potent, and selective T-type Ca2+ channel blockers, a series of sulfonamido-containing 3,4-dihydroquinazoline derivatives were prepared and evaluated for their blocking actions on T- and N-type Ca2+ channels. Among them, 9c (KYS05064, IC50 = 0.96 +/- 0.22 microM) was found to be as potent as Mibefradil and also showed the highest selectivity for T-type Ca2+ channel with no effect on N-type Ca2+ channel.     

Interactions of N-terminal and C-terminal parts of the small conductance Ca2+ activated K+ channel, hSK3.

Ca(2+) activated K(+) channels modulate the afterhyperpolarization in neurons. Using a variety of different techniques we obtained information about the function of N- and C-terminal parts of the Ca(2+)-activated K(+) channel, SK3. By means of the yeast two hybrid technique we found an interaction between N-C and N-N- terminal parts of SK3. The strong N-C and N-N interaction was specific for SK3 and could not be observed for SK1 and SK2. Possibly a homotetrameric assembly of SK3 is favored in tissues were all SK channels are expressed. In addition, the interaction in SK3 was independent of the length of the polymorphic glutamine repeat in the N-terminus of SK3. Electrophysiological investigations showed that expression of amino acids 1-299 of SK3 (SK3N_299) modified the 1-EBIO pharmacology of endogenous SK3 channels in PC12 cells without affecting the Ca(2+)-sensitvity. The activation by 0.5 mM 1-EBIO in cells expressing amino acids 1-299 of SK3 was reduced by 32% in comparison to control experiments. Considering the N-C interaction in yeast, we conclude that the sensitivity of SK3 channels to 1-EBIO was modified by N-C interactions with SK3N_299. Therefore we conclude that N-C interactions influence SK3 channel function.     

Morpholin-2-one derivatives as novel selective T-type Ca2+ channel blockers

Morpholin-2-one-5-carboxamide derivatives were prepared by using the one-pot Ugi multicomponent reaction and evaluated for blocking effects on T- and N-type Ca(2+) channels. Among them, compound 5i produced the highest potency (IC(50)=0.45+/-0.02 microM), while compounds 5d, 5f, 5k, 5n, 5o, and 6m produced relatively high potency as well as selectivity on T-type Ca(2+) channels. These novel scaffolds showed potent and selective T-type Ca(2+) channel blocking activities.     

Small conductance Ca2+-activated K+ channels and calmodulin: cell surface expression and gating

Small conductance Ca2+-activated K+ channels (SK channels) are heteromeric complexes of pore-forming alpha subunits and constitutively bound calmodulin (CaM). The binding of CaM is mediated in part by the electrostatic interaction between residues Arg-464 and Lys-467 of SK2 and Glu-84 and Glu-87 of CaM. Heterologous expression of the double charge reversal in SK2, SK2 R464E/K467E (SK2:64/67), did not yield detectable surface expression or channel activity in whole cell or inside-out patch recordings. Coexpression of SK2:64/67 with wild type CaM or CaM1,2,3,4, a mutant lacking the ability to bind Ca2+, rescued surface expression. In patches from cells coexpressing SK2:64/67 and wild type CaM, currents were recorded immediately following excision into Ca2+-containing solution but disappeared within minutes after excision or immediately upon exposure to Ca2+-free solution and were not reactivated upon reapplication of Ca2+-containing solution. Channel activity was restored by application of purified recombinant Ca2+-CaM or exposure to Ca2+-free CaM followed by application of Ca2+-containing solution. Coexpression of the double charge reversal E84R/E87K in CaM (CaM:84/87) with SK2:64/67 reconstituted stable Ca2+-dependent channel activity that was not lost with exposure to Ca2+-free solution. Therefore, Ca2+-independent interactions with CaM are required for surface expression of SK channels, whereas the constitutive association between the two channel subunits is not an essential requirement for gating.     

Gene expression, mutation, and structure-function relationship of scorpion toxin BmP05 active on SK(Ca) channels

Four peptide inhibitors of small-conductance Ca(2+)-activated, apamin-sensitive K(+) channels (SK(Ca)) have been isolated from the venom of the Chinese scorpion Buthus martensi, named BmP01, BmP02, BmP03, and BmP05, respectively [Romi-Lebrun, R. (1997) Eur. J. Biochem. 245, 457-464]. Among them BmP05 with 31 amino acid residues has been intensively studied due to its most potent toxicity. To investigate the structure-function relationship of BmP05, its wild type and seven mutants (their C-termini unamidated) were successfully expressed in the yeast secretion system and purified with a high yield over 8 mg/L. Their toxicity to mice and electrophysiological activity on the K(+) currents (SK(Ca) and Kv) in rat adrenal chromaffin cells were measured and compared. The results indicated the following: (1) As a selective antagonist against SK(Ca), 1 microM rBmP05 is equivalent to 0.2 microM apamin, and its IC(50) is 0.92 microM. (2) The basic residues Lys and Arg located at positions 6 and 13 in the N-terminal alpha-helix region are essential and synergetic in the interaction of the toxin with SK(Ca). (3) Disruption of the alpha-helix by mutation of Gln at position 9 with Pro results in almost total loss of toxicity. (4) The C-terminal residue His31 plays an auxiliary role in the interaction of the toxin with SK(Ca). (5) The beta-turn connecting two beta-sheets near the C-terminal part is responsible for the specificity of the toxin to the different subtypes of K(+) channels.     

A helical region in the C terminus of small-conductance Ca2+-activated K+ channels controls assembly with apo-calmodulin.

Small conductance Ca(2+)-activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca(2+) in the submicromolar range. This high affinity for Ca(2+) results from Ca(2+)-independent association of the SK alpha-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396-487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423-437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca(2+)-free CaM, and results in SK channels that are no longer gated by Ca(2+). The results show that the Ca(2+)-independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaM-interacting proteins.     

Control of electrical activity in central neurons by modulating the gating of small conductance Ca2+-activated K+ channels

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

Synthesis and characterization of Pi4, a scorpion toxin from Pandinus imperator that acts on K+ channels.

Pi4 is a 38-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the Chactidae scorpion Pandinus imperator. Together with maurotoxin, Pi1, Pi7 and HsTx1, Pi4 belongs to the alpha KTX6 subfamily of short four-disulfide-bridged scorpion toxins acting on K+ channels. Due to its very low abundance in venom, Pi4 was chemically synthesized in order to better characterize its pharmacology and structural properties. An enzyme-based cleavage of synthetic Pi4 (sPi4) indicated half-cystine pairings between Cys6-Cys27, Cys12-32, Cys16-34 and Cys22-37, which denotes a conventional pattern of scorpion toxin reticulation (Pi1/HsTx1 type). In vivo, sPi4 was lethal after intracerebroventricular injection to mice (LD50 of 0.2 microg per mouse). In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage-gated K+ channel subtypes showed potent inhibition of currents from rat Kv1.2 (IC50 of 8 pm) and Shaker B (IC50 of 3 nm) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels. The sPi4 was also found to compete with 125I-labeled apamin for binding to small-conductance Ca(2+)-activated K+ (SK) channels from rat brain synaptosomes (IC50 value of 0.5 microm). sPi4 is a high affinity blocker of the Kv1.2 channel. The toxin was docked (BIGGER program) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region. The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the interaction and the associated blockage of the Kv1.2 channel.     

Specific enhancement of SK channel activity selectively potentiates the afterhyperpolarizing current I(AHP) and modulates the firing properties of hippocampal pyramidal neurons

SK channels are Ca2+-activated K+ channels that underlie after hyperpolarizing (AHP) currents and contribute to the shaping of the firing patterns and regulation of Ca2+ influx in a variety of neurons. The elucidation of SK channel function has recently benefited from the discovery of SK channel enhancers, the prototype of which is 1-EBIO. 1-EBIO exerts profound effects on neuronal excitability but displays a low potency and limited selectivity. This study reports the effects of DCEBIO, an intermediate conductance Ca2+-activated K+ channel modulator, and the effects of the recently identified potent SK channel enhancer NS309 on recombinant SK2 channels, neuronal apamin-sensitive AHP currents, and the excitability of CA1 neurons. NS309 and DCEBIO increased the amplitude and duration of the apamin-sensitive afterhyperpolarizing current without affecting the slow afterhyperpolarizing current in contrast to 1-EBIO. The potentiation by DCEBIO and NS309 was reversed by SK channel blockers. In current clamp experiments, NS309 enhanced the medium afterhyperpolarization (but not the slow afterhyperpolarization sAHP) and profoundly affected excitability by facilitating spike frequency adaptation in a frequency-independent manner. The potent and specific effect of NS309 on the excitability of CA1 pyramidal neurons makes this compound an ideal tool to assess the role of SK channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability.