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

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-EBIO or the more potent compound NS309, do not distinguish between the SK subtypes and they also activate the related intermediate conductance Ca(2+)-activated K(+) 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 (EC(50) = 5.6 +/- 1.6 microM, efficacy 90 +/- 1.8 %) and hSK2 (EC(50) = 14 +/- 4 microM, efficacy 71 +/- 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 Ca(2+)-sensitivity of channel activation, changing the EC(50)(Ca(2+)) 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.     

Voltage-dependent Ca2+-channel block by openers of intermediate and small conductance Ca2+-activated K+ channels in urinary bladder smooth muscle cells

We examined effects of small and intermediate conductance Ca(2+)-activated K(+) (SK and IK) channel openers, DCEBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) and NS309 (3-oxime-6,7-dichloro-1H-indole-2,3-dione), on L-type Ca(2+) channel current (I(Ca)) that was measured in smooth muscle cells isolated from mouse urinary bladder under whole cell voltage-clamp. The I(Ca) was concentration-dependently inhibited by DCEBIO and NS309; half inhibition was obtained at 71.6 and 10.6 muM, respectively. The specificity of NS309 to the IK channel over the Ca(2+) channel appears to be high and higher than that of DCEBIO. DCEBIO and even NS309 may, however, substantially block Ca(2+) channels when used as SK channel openers.     

Negative gating modulation by (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) depends on residues in the inner pore vestibule: pharmacological evidence of deep-pore gating of K(Ca)2 channels

Acting as a negative gating modulator, (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) shifts the apparent Ca(2+)-dependence of the small-conductance Ca(2+)-activated K(+) channels K(Ca)2.1-2.3 to higher Ca(2+) concentrations. Similar to the positive K(Ca) channel-gating modulators 1-ethyl-2-benzimidazolinone (1-EBIO) and cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methylpyrimidin-4-yl]-amine (CyPPA), the binding site for NS8593 has been assumed to be located in the C-terminal region, in which these channels interact with their Ca(2+) sensor calmodulin. However, by using a progressive chimeric approach, we were able to localize the site-of-action of NS8593 to the K(Ca)2 pore. For example, when we transferred the C terminus from the NS8593-insensitive intermediate-conductance K(Ca)3.1 channel to K(Ca)2.3, the chimeric channel remained as sensitive to NS8593 as wild-type K(Ca)2.3. In contrast, when we transferred the K(Ca)2.3 pore to K(Ca)3.1, the channel became sensitive to NS8593. Using site-directed mutagenesis, we subsequently identified two specific residues in the inner vestibule of K(Ca)2.3 (Ser507 and Ala532) that determined the effect of NS8593. Mutation of these residues to the corresponding residues in K(Ca)3.1 (Thr250 and Val275) made K(Ca)2.3 insensitive to NS8593, whereas introduction of serine and alanine into K(Ca)3.1 was sufficient to render this channel highly sensitive to NS8593. It is noteworthy that the same two residue positions have been found previously to mediate sensitivity of K(Ca)3.1 to clotrimazole and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34). The location of Ser507 in the pore-loop near the selectivity filter and Ala532 in an adjacent position in S6 are within the region predicted to contain the K(Ca)2 channel gate. Hence, we propose that NS8593-mediated gating modulation occurs via interaction with gating structures at a position deep within the inner pore vestibule.     

Synthesis and structure-activity relationship studies of 2-(N-substituted)-aminobenzimidazoles as potent negative gating modulators ofsmall conductance Ca2+-activated K+ channels

Small conductance Ca2+-activated K+ channels (SK channels) participate in the control of neuronal excitability, in the shaping of action potential firing patterns, and in the regulation of synaptic transmission.SK channel inhibitors have the potential of becoming new drugs for treatment of various psychiatric and neurological diseases such as depression, cognition impairment, and Parkinson's disease. In the present study we describe the structure-activity relationship (SAR) of a class of 2-(N-substituted)-2-aminobenzimidazoles that constitute a novel class of selective SK channel inhibitors that, in contrast to classical SK inhibitors, do not block the pore of the channel. The pore blocker apamin is not displaced by these compounds in binding studies, and they still inhibit SK channels in which the apamin binding site has been abolished by point mutations. These novel SK inhibitors shift the concentration-response curve for Ca2+ toward higher values and represent the first example of negative gating modulation as a mode-of-action for inhibition of SK channels. The first described compound in this class is NS8593 (14), and the most potent analogue identified in this study is the racemic compound 39 (NS11757), which reversibly inhibits SK3-mediated currents with a K(d) value of 9 nM.     

Potent activation of large-conductance Ca2+-activated K+ channels by the diphenylurea 1,3-bis-[2-hydroxy-5-(trifluoromethyl)phenyl]urea (NS1643) in pituitary tumor (GH3) cells

1,3-Bis-[2-hydroxy-5-(trifluoromethyl)phenyl]urea (NS1643) is reported to be an activator of human ether-à-go-go-related gene current. However, it remains unknown whether it has any effects on other types of ion channels. The effects of NS1643 on ion currents and membrane potential were investigated in this study. NS1643 stimulated Ca(2+)-activated K(+) current [I(K(Ca))] in a concentration-dependent manner with an EC(50) value of 1.8 microM in pituitary tumor (GH(3)) cells. In inside-out recordings, this compound applied to the intracellular side of the detached channels stimulated large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels with no change in single-channel conductance. It shifted the activation curve of BK(Ca) channels to less depolarized voltages without altering the gating charge of the channels. NS1643-stimulated channel activity depended on intracellular Ca(2+), and mean closed time during exposure to NS1643 was reduced. NS1643 (3 microM) had little or no effect on peak amplitude of ether-à-go-go-related gene-mediated K(+) current evoked by membrane hyperpolarization, although it increased the amplitude of late-sustained components of K(+) inward current, which was suppressed by paxilline but not by azimilide. NS1643 (3 microM) had no effect on L-type Ca(2+) current. This compound reduced repetitive firing of action potentials, and further application of paxilline attenuated its decrease in firing rate. In addition, NS1643 enhanced BK(Ca)-channel activity in human embryonic kidney 293T cells expressing alpha-hSlo. In summary, we clearly show that NS1643 interacts directly with the BK(Ca) channel to increase the amplitude of I(K(Ca)) in pituitary tumor (GH(3)) cells. The alpha-subunit of the channel may be a target for the action of this small compound.     

Inhibition of the human intermediate conductance Ca(2+)-activated K(+) channel, hIK1, by volatile anesthetics

Ca(2+)-activated K(+) channels (K(Ca)) regulate a wide variety of cellular functions by coupling intracellular Ca(2+) concentration to membrane potential. There are three major groups of K(Ca) classified by their unit conductances: large (BK), intermediate (IK), and small (SK) conductance of channels. BK channel is gated by combined influences of Ca(2+) and voltage, while IK and SK channels are gated solely by Ca(2+). Volatile anesthetics inhibit BK channel activity by interfering with the Ca(2+) gating mechanism. However, the effects of anesthetics on IK and SK channels are unknown. Using cloned IK and SK channels, hIK1 and hSK1-3, respectively, we found that the currents of hIK1 were inhibited rapidly and reversibly by volatile anesthetics, whereas those of SK channels were not affected. The IC(50) values of the volatile anesthetics, halothane, sevoflurane, enflurane, and isoflurane for hIK1 inhibition were 0.69, 0.42, 1.01 and 1.03 mM, respectively, and were in the clinically used concentration range. In contrast to BK channel, halothane inhibition of hIK1 currents was independent of Ca(2+) concentration, suggesting that Ca(2+) gating mechanism is not involved. These results demonstrate that volatile anesthetics, such as halothane, enflurane, isoflurane, and sevoflurane, affect BK, IK, and SK channels in distinct ways.     

The duration of pacing-induced atrial fibrillation is reduced in vivo by inhibition of small conductance Ca(2+)-activated K(+) channels

Atrial fibrillation (AF) is associated with increased morbidity and is in addition the most prevalent cardiac arrhythmia. Compounds used in pharmacological treatment has traditionally been divided into Na(+) channel inhibitors, β-blockers, K(+) channel inhibitors, and Ca(2+) channel inhibitors, whereas newer multichannel blockers such as amiodarone and ranolazine have been introduced later. This study was devoted to the evaluation of an acute pacing-induced in vivo model of AF in rats. Antiarrhythmic effects of well-known compounds such as lidocaine, dofetilide, and ranolazine were confirmed in this model. In addition, antiarrhythmic effects of different inhibitors of Ca(2+)-activated small conductance K(+) (SK) channels were demonstrated. Intravenous application of 5 mg/kg of the negative SK channel modulator NS8593 reduced AF duration by 64.5%, and the lowest significantly effective dose was 1.5 mg/kg. A dose-effect relationship was established based on 6 different dose groups. Furthermore, it was demonstrated that the antiarrhythmic effect of NS8593 and other tested drugs was associated with an increase in atrial effective refractory period. The functional role of SK channels was confirmed by 2 other SK channel inhibitors, UCL1684 and apamin, thereby confirming the hypothesis that these channels might constitute a new promising target for antiarrhythmic treatment.     

The mechanism of actions of 3-(5'-(hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) on Ca(2+)-activated K(+) currents in GH(3) lactotrophs

The effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), an activator of soluble guanylyl cyclase, on ionic currents have been assessed in rat pituitary GH(3) lactotrophs. In GH(3) cells bathed in normal Tyrode's solution, YC-1 (1 microM) reversibly suppressed the amplitude of the Ca(2+)-activated K(+) current (I(K(Ca))). YC-1 at a concentration above 10 microM produced a biphasic response in the amplitude of I(K(Ca)), i.e., an initial decrease followed by a sustained increase. When the pipette solutions were filled with high EGTA (10 mM), the YC-1-induced stimulatory effect on I(K(Ca)) was abolished. Over a similar concentration range, YC-1 also effectively inhibited the voltage-dependent K(+) current (I(K(V))) in GH(3) cells. The IC(50) value required for the inhibition of I(K(V)) by YC-1 was 1 microM. Unlike YC-1, 8-bromo cGMP did not inhibit I(K(Ca)). However, YC-1 (10 microM) did not affect the amplitude of L-type Ca(2+) current. In the cell-attached configuration, application of YC-1 (10 microM) to the bath did not change the single-channel conductance of the large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels; however, it did increase the opening probability of BK(Ca) channels. In contrast, in the outside-out configuration, YC-1 (10 microM) significantly suppressed the opening probability of BK(Ca) channels. The present study shows dual effects of YC-1 on I(K(Ca)) in GH(3) cells. The YC-1-mediated stimulation of I(K(Ca)) may result from elevated cytosolic Ca(2+), whereas the inhibition of I(K(Ca)) and I(K(V)) by YC-1 appears to be direct and independent of the activation of soluble guanylyl cyclase. Caution thus needs to be used in attributing the YC-1-mediated response to the activation of soluble guanylyl cyclase.     

Compounds that block both intermediate-conductance (IK(Ca)) and small-conductance (SK(Ca)) calcium-activated potassium channels

1. Nine bis-quinolinyl and bis-quinolinium compounds related to dequalinium, and previously shown to block apamin-sensitive small conductance Ca(2+)-activated K(+) channels (SK(Ca)), have been tested for their inhibitory effects on actions mediated by intermediate conductance Ca(2+)-activated K(+) channels (IK(Ca)) in rabbit blood cells. 2. In most experiments, a K(+)-sensitive electrode was employed to monitor the IK(Ca)-mediated net loss of cell K(+) that followed the addition of the Ca(2+) ionophore A23187 (2 microM) to red cells suspended at an haematocrit of 1% in a low K(+) (0.12 - 0.17 mM) solution. The remainder used an optical method based on measuring the reduction in light transmission that occurred on applying A23187 (0.4 or 2 microM) to a very dilute suspension of red cells (haematocrit 0.02%). 3. Of the compounds tested, the most potent IK(Ca) blocker was 1,12 bis[(2-methylquinolin-4-yl)amino]dodecane (UCL 1407) which had an IC(50) of 0.85+/-0.06 microM (mean+/-s.d. mean). 4. The inhibitory action of UCL 1407 and its three most active congeners was characterized by (i) a Hill slope greater than unity, (ii) sensitivity to an increase in external [K(+)], and (iii) a time course of onset that suggested use-dependence. Also, the potency of the nonquaternary compounds tested increased with their predicted lipophilicity. These findings suggested that the IK(Ca) blocking action resembles that of cetiedil rather than of clotrimazole. 5. Some quaternized members of the series were also active. The most potent was the monoquaternary UCL 1440 ((1-[N-[1-(3, 5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino]-10-[N'-(2-me thylqu inolinium-4yl)amino] decane (trifluoroacetate) which had an IC(50) of 1.8+/-0.1 microM. The corresponding bisquaternary UCL 1438 (1, 10-bis[N-[1-(3,5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino] decane bis(trifluoroacetate) was almost as active (IC(50) 2.7+/-0.3 microM). 6. A bis-aminoquinolium cyclophane (UCL 1684) had little IK(Ca) blocking action despite its great potency at SK(Ca) channels (IC(50) 4.1+/-0.2 nM). 7. The main outcome is the identification of new intermediate-conductance Ca(2+)-activated K(+) channel blockers with a wide range of IK(Ca)/SK(Ca) selectivities.     

Stimulatory effects of delta-hexachlorocyclohexane on Ca(2+)-activated K(+) currents in GH(3) lactotrophs

delta-Hexachlorocyclohexane (delta-HCH), a lipophilic neurodepressant agent, has been shown to inhibit neurotransmitter release and stimulate ryanodine-sensitive Ca(2+) channels. However, the effect of delta-HCH on neuronal activity remains unclear, although it may enhance the gamma-aminobutyric acid-induced current. Its effects on ionic currents were investigated in rat pituitary GH(3) cells and human neuroblastoma IMR-32 cells. In GH(3) cells, delta-HCH increased the amplitude of Ca(2+)-activated K(+) current (I(K(Ca))). delta-HCH (100 microM) slightly inhibited the amplitude of voltage-dependent K(+) current. delta-HCH (30 microM) suppressed voltage-dependent L-type Ca(2+) current (I(Ca, L)), whereas gamma-HCH (30 microM) had no effect on I(Ca, L). In the inside-out configuration, delta-HCH applied intracellularly did not change the single channel conductance of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels; however, it did increase the channel activity. The delta-HCH-mediated increase in the channel activity is mainly mediated by its increase in the number of long-lived openings. delta-HCH reversibly increased the activity of BK(Ca) channels in a concentration-dependent manner with an EC(50) value of 20 microM. delta-HCH also caused a left shift in the midpoint for the voltage-dependent opening. In contrast, gamma-HCH (30 microM) suppressed the activity of BK(Ca) channels. Under the current-clamp mode, delta-HCH (30 microM) reduced the firing rate of spontaneous action potentials; however, gamma-HCH (30 microM) increased it. In neuroblastoma IMR-32 cells, delta-HCH also increased the amplitude of I(K(Ca)) and stimulated the activity of intermediate-conductance K(Ca) channels. This study provides evidence that delta-HCH is an opener of K(Ca) channels. The effects of delta-HCH on these channels may partially, if not entirely, be responsible for the underlying cellular mechanisms by which delta-HCH affects neuronal or neuroendocrine function.     

Stimulatory actions of thymol, a natural product, on Ca(2+)-activated K(+) current in pituitary GH(3) cells

Drugs that influence the opening of potassium (K(+)) channels and as a consequence cause hyperpolarization of cell membrane possess clinical potential. The large conductance Ca(2+)-activated K(+) (BK) channel is highly selective for K(+). Activation of this channel is Ca(2+)- and voltage-dependent. We have investigated the effects of thymol, a natural product, on ion currents in pituitary GH(3) cells. The patch-clamp technique was used to investigate the effect of thymol (100 microM) in these cells. Thymol reversibly stimulated the Ca(2+)-activated K(+) current with an EC (50) value of 75 microM. In a cell-attached configuration, application of thymol to the bath increased the activity of BK channels. BAPTA (1 mM) attenuated thymol-stimulated channel activity. In an experiment using the inside-out configuration, thymol exposed to the intracellular face of excised patches did not modify the single-channel conductance of these channels whereas it enhanced the channel activity. Neither menthol (100 microM) nor zingerone (100 microM) had an effect on BK-channel activity while AAPH (100 microM) suppressed it significantly. The stimulatory actions of thymol on Ca(2+)-activated K(+) currents may be associated with the underlying cellular mechanisms through which it affects neuronal or neuroendocrine functions.     

The small molecule NS11021 is a potent and specific activator of Ca2+-activated big-conductance K+ channels

Large-conductance Ca(2+)- and voltage-activated K(+) channels (Kca1.1/BK/MaxiK) are widely expressed ion channels. They provide a Ca(2+)-dependent feedback mechanism for the regulation of various body functions such as blood flow, neurotransmitter release, uresis, and immunity. In addition, a mitochondrial K(+) channel with KCa1.1-resembling properties has been found in the heart, where it may be involved in regulation of energy consumption. In the present study, the effect of a novel NeuroSearch compound, 1-(3,5-bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea (NS11021), was investigated on cloned KCa1.1 expressed in Xenopus laevis oocytes and mammalian cells using electrophysiological methods. NS11021 at concentrations above 0.3 microM activated KCa1.1 in a concentration-dependent manner by parallel-shifting the channel activation curves to more negative potentials. Single-channel analysis revealed that NS11021 increased the open probability of the channel by altering gating kinetics without affecting the single-channel conductance. NS11021 (10 microM) influenced neither a number of cloned Kv channels nor endogenous Na(+) and Ca(2+) channels (L- and T-type) in guinea pig cardiac myocytes. In conclusion, NS11021 is a novel KCa1.1 channel activator with better specificity and a 10 times higher potency compared with the most broadly applied KCa1.1 opener, NS1619. Thus, NS11021 might be a valuable tool compound when addressing the physiological and pathophysiological roles of KCa1.1 channels.     

Partial apamin sensitivity of human small conductance Ca2+-activated K+ channels stably expressed in Chinese hamster ovary cells

The bee venom toxin apamin is an important drug tool for characterising small conductance Ca(2+)-activated K(+) channels (SK channels). In recombinant expression systems both rSK2 and rSK3 channels are potently blocked by apamin, whilst the sensitivity of SK1 channels is somewhat less clear. In the present study we have conducted a detailed analysis by patch clamp electrophysiology of the effects of apamin on human SK channels (SK1, SK2 and SK3) stably expressed in Chinese hamster ovary (CHO-K1) cells. CHO-K1 cell lines expressing either hSK1, 2 or 3 channels were first validated using specific antibodies and Western blotting. Specific protein bands of a size corresponding to the predicted channel tetramer (approximately 250-290 kDa) were detected. In each cell line, but not wild-type untransfected cells, large, time-independent inwardly rectifying Ca(2+)-dependent K(+) currents were observed under voltage-clamp. In CHO-hSK1, this current was markedly reduced by apamin (IC(50) value 8 nM), however, a significant fraction of the current remained unblocked (39+/-5%), even at saturating concentrations (1 microM apamin). The apamin-sensitive and -insensitive currents possess very similar biophysical and pharmacological properties. Each are Ca(2+)-dependent, inwardly rectify and have relative ionic permeabilities of K(+)>Cs(+)>Li(+)=Na(+). Both components were resistant to block by charybdotoxin and iberiotoxin, known IK and BK channel blockers, but were attenuated by the tricyclic antidepressant cyproheptadine (>95% block at 1 mM). The SK channel opener 1-EBIO could still produce channel activation in the presence of apamin. Importantly, hSK2 and hSK3 channels also exhibit partial apamin sensitivity in our experimental paradigm (IC(50) values of 0.14 nM and 1.1 nM, respectively, and maximal percentage inhibition values of 47+/-7% and 58+/-9%, respectively). Our data indicate that, at least in a recombinant expression system, all three SK channels can be partially apamin-sensitive. The explanation for this finding is presently unclear but may be due to regulatory subunits, phosphorylation or other types of post translational modification. Ascribing particular SK channels to physiological roles using apamin as a drug tool needs to be done cautiously in light of these findings.     

Large conductance Ca2+-activated K+ (BKCa) channels are involved in the vascular relaxations elicited by piceatannol isolated from Rheum undulatum rhizome

We previously reported that piceatannol isolated from the rhizome extract of RHEUM UNDULATUM has a potent vasorelaxant activity. In the present study, the mechanisms underlying the direct vascular relaxant effect of piceatannol were investigated in isolated rat aorta. Piceatannol induced a concentration-dependent relaxation in aortic preparations precontracted with phenylephrine (EC (50) : 2.4 +/- 0.4 microM), which was completely inhibited by endothelial removal, N(omega)-nitro- L-arginine (nitric oxide synthase inhibitor), methylene blue and 1 H- oxadiazolo [4,3- A]quinoxalin-1-one (guanylyl cyclase inhibitor). The piceatannol-induced relaxation was also blocked by raising the extracellular K (+) (45 mM), 4-aminopyridine (voltage-sensitive K (+) channel blocker) and tetraethylammonium [the non-selective Ca (2+)-activated K (+) (K (Ca)) channel blocker] but not by indomethacin (cyclooxygenase inhibitor), atropine (muscarinic receptor antagonist), propranolol (beta-adrenoceptor antagonist), verapamil and nifedipine (L-type voltage-gated Ca (2+) channel blocker), barium chloride (inward rectifier K (+) channel inhibitor) and glibenclamide (ATP-sensitive K (+) channel blocker). In further studies investigating the role of Ca (2+)-activated K (+) (K (Ca)) channels, piceatannol-induced relaxant responses were decreased by charybdotoxin [large (BK (Ca))- and intermediate (IK (Ca))-conductance K (Ca) channel blocker], iberiotoxin (selective BK (Ca) channels blocker), but not by apamin [small-conductance K (Ca) (SK (Ca)) channel blocker], TRAM-34 [intermediate-conductance K (Ca) (IK (Ca)) channel blocker]. The present results demonstrate that piceatannol-induced vascular relaxation in rat aorta may be mediated by an endothelium-dependent nitric oxide signaling pathway, at least partially, through the activation of BK (Ca).     

Enhancement of hippocampal pyramidal cell excitability by the novel selective slow-afterhyperpolarization channel blocker 3-(triphenylmethylaminomethyl)pyridine (UCL2077)

The slow afterhyperpolarization (sAHP) in hippocampal neurons has been implicated in learning and memory. However, its precise role in cell excitability and central nervous system function has not been explicitly tested for 2 reasons: 1) there are, at present, no selective inhibitors that effectively reduce the underlying current in vivo or in intact in vitro tissue preparations, and 2) although it is known that a small conductance K(+) channel that activates after a rise in [Ca(2+)](i) underlies the sAHP, the exact molecular identity remains unknown. We show that 3-(triphenylmethylaminomethyl)pyridine (UCL2077), a novel compound, suppressed the sAHP present in hippocampal neurons in culture (IC(50) = 0.5 microM) and in the slice preparation (IC(50) approximately 10 microM). UCL2077 was selective, having minimal effects on Ca(2+) channels, action potentials, input resistance and the medium afterhyperpolarization. UCL2077 also had little effect on heterologously expressed small conductance Ca(2+)-activated K(+) (SK) channels. Moreover, UCL2077 and apamin, a selective SK channel blocker, affected spike firing in hippocampal neurons in different ways. These results provide further evidence that SK channels are unlikely to underlie the sAHP. This study also demonstrates that UCL2077, the most potent, selective sAHP blocker described so far, is a useful pharmacological tool for exploring the role of sAHP channels in the regulation of cell excitability in intact tissue preparations and, potentially, in vivo.     

Characterization of an apamin-sensitive small-conductance Ca(2+)-activated K(+) channel in porcine coronary artery endothelium: relevance to EDHF

1. The apamin-sensitive small-conductance Ca(2+)-activated K(+) channel (SK(Ca)) was characterized in porcine coronary arteries. 2. In intact arteries, 100 nM substance P and 600 microM 1-ethyl-2-benzimidazolinone (1-EBIO) produced endothelial cell hyperpolarizations (27.8 +/- 0.8 mV and 24.1 +/- 1.0 mV, respectively). Charybdotoxin (100 nM) abolished the 1-EBIO response but substance P continued to induce a hyperpolarization (25.8 +/- 0.3 mV). 3. In freshly-isolated endothelial cells, outside-out patch recordings revealed a unitary K(+) conductance of 6.8 +/- 0.04 pS. The open-probability was increased by Ca(2+) and reduced by apamin (100 nM). Substance P activated an outward current under whole-cell perforated-patch conditions and a component of this current (38%) was inhibited by apamin. A second conductance of 2.7 +/- 0.03 pS inhibited by d-tubocurarine was observed infrequently. 4. Messenger RNA encoding the SK2 and SK3, but not the SK1, subunits of SK(Ca) was detected by RT - PCR in samples of endothelium. Western blotting indicated that SK3 protein was abundant in samples of endothelium compared to whole arteries. SK2 protein was present in whole artery nuclear fractions. 5. Immunofluorescent labelling confirmed that SK3 was highly expressed at the plasmalemma of endothelial cells and was not expressed in smooth muscle. SK2 was restricted to the peri-nuclear regions of both endothelial and smooth muscle cells. 6. In conclusion, the porcine coronary artery endothelium expresses an apamin-sensitive SK(Ca) containing the SK3 subunit. These channels are likely to confer all or part of the apamin-sensitive component of the endothelium-derived hyperpolarizing factor (EDHF) response.     

Inhibition of Ca2+-release-activated Ca2+ channel (CRAC) and K+ channels by curcumin in Jurkat-T cells

The increase in cytoplasmic Ca(2+) concentration (Δ[Ca(2+)](c)) mediated by the Ca(2+)-release-activated Ca(2+) channel (CRAC) is a critical signal for the activation of lymphocytes. Also, the voltage-gated K(+) channel (K(v)) and intermediate-conductance Ca(2+)-activated K(+) channel (IKCa1/SK4) have drawn attention as pharmacological targets for regulating immune responses. Since polyphenolic agents have various immunomodulatory effects, here we compared the effects of curcumin, rosmarinic acid, resveratrol, and epigallocatechin gallate on the ionic currents through CRAC (I(CRAC)), K(v) (I(Kv)), SK4 (I(SK4)) and on the Δ[Ca(2+)](c) of Jurkat-T cells using the patch clamp technique and fura-2 spectrofluorimetry. Curcumin (10 μM) inhibited store-operated Ca(2+) entry (SOCE). Consistently, dose-dependent inhibition of I(CRAC) by curcumin was confirmed in Jurkat-T (IC(50), 5.9 μM) and the HEK293 cells overexpressing Orai1 and STIM1 (IC(50), 0.6 μM). Also, curcumin inhibited both I(Kv) (IC(50), 11.9 μM) and I(SK4) (IC(50), 4.2 μM). The other polyphenols (rosmarinic acid, resveratrol, and epigallocatechin gallate at 10 - 30 μM) had no effect on SOCE and showed only a partial inhibition of the K(+) currents. In summary, among the tested polyphenolic agents, curcumin showed prominent inhibition of major ion channels in lymphocytes, which might contribute to the anti-inflammatory effects of curcumin. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10209FP].     

Small conductance Ca2+-activated K+ channels as targets of CNS drug development

In most central neurons, small conductance Ca(2+)-activated K(+) channels (SK channels) contribute to afterhyperpolarizations (AHPs), which control neuronal excitability. The medium AHP has pharmacological properties similar to recombinant SK channels, consistent with the hypothesis that SK channels generate this afterhyperpolarization component. It is still unclear how recombinant SK channels are functionally related to the slow AHP component. Cloned SK channels are heteromeric complexes of SK channel subunits and calmodulin. The channels are activated by Ca(2+) binding to calmodulin that induces conformational changes resulting in channel opening. Channel deactivation is the reverse process brought about by dissociation of Ca(2+) from calmodulin. In the mammalian brain, the three SK channel subunits (SK1-3) display partially overlapping distributions. Most of the higher brain regions such as the neocortex and hippocampus show expression of both genes encoding SK1 and SK2 channels, whereas phylogenetically older brain regions such as the thalamus, basal ganglia, cerebellum, and brainstem show high levels of SK3 gene expression. At present, it is still unclear whether native SK channels are generated as heteromeric or homomeric channels. Peptide toxins such as apamin and scyllatoxin, as well as organic compounds such as quaternary salts of bicuculline, dequalinium, UCL 1684 and UCL 1848 serve as non-specific SK channel blockers. The only known exceptions so far are the scorpion toxin tamapin and the peptide inhibitor Lei-Dab(7), which bind preferentially to SK2. Electrophysiological and behavioral studies indicate that blockade of SK channels by apamin increases excitability, lowers the threshold for the induction of synaptic plasticity, and facilitates hippocampus-dependent memory. The potential value of pharmacological SK channel modulation in various pathological states such as increased epileptiform activity, cognitive impairment, pain, mood disorders and schizophrenia will be discussed.     

Maurotoxin: a potent inhibitor of intermediate conductance Ca2+-activated potassium channels

Maurotoxin, a 34-amino acid toxin from Scorpio maurus scorpion venom, was examined for its ability to inhibit cloned human SK (SK1, SK2, and SK3), IK1, and Slo1 calcium-activated potassium (K(Ca)) channels. Maurotoxin was found to produce a potent inhibition of Ca(2+)-activated (86)Rb efflux (IC(50), 1.4 nM) and inwardly rectifying potassium currents (IC(50), 1 nM) in CHO cells stably expressing IK1. In contrast, maurotoxin produced no inhibition of SK1, SK2, and SK3 small-conductance or Slo1 large-conductance K(Ca) channels at up to 1 microM in physiologically relevant ionic strength buffers. Maurotoxin did inhibit (86)Rb efflux (IC(50), 45 nM) through, and (125)I-apamin binding (K(i), 10 nM) to SK channels in low ionic strength buffers (i.e., 18 mM sodium, 250 mM sucrose), which is consistent with previous reports of inhibition of apamin binding to brain synaptosomes. Under similar low ionic strength conditions, the potency for maurotoxin inhibition of IK1 increased by approximately 100-fold (IC(50), 14 pM). In agreement with its ability to inhibit recombinant IK1 potassium channels, maurotoxin was found to potently inhibit the Gardos channel in human red blood cells and to inhibit the K(Ca) in activated human T lymphocytes without affecting the voltage-gated potassium current encoded by Kv1.3. Maurotoxin also did not inhibit Kv1.1 potassium channels but potently blocked Kv1.2 (IC(50), 0.1 nM). Mutation analysis indicates that similar amino acid residues contribute to the blocking activity of both IK1 and Kv1.2. The results from this study show that maurotoxin is a potent inhibitor of the IK1 subclass of K(Ca) potassium channels and may serve as a useful tool for further defining the physiological role of this channel subtype.     

Insulin-induced relaxation of rat mesenteric artery is mediated by Ca(2+)-activated K(+) channels

We tested the hypothesis that relaxation of the rat mesenteric artery in response to insulin is mediated by K(+) channels. Two concentrations of insulin (10 and 100 mU/ml) induced relaxation of the artery by 6+/-1%, 24+/-3% (mean+/-S.E.M.). Denudation of the endothelium or precontraction by KCl (30 mM), clotrimazole (10 microM), a cytochrome P450 inhibitor, charybdotoxin (30 nM) an inhibitor of large-conductance Ca(2+)-activated K(+) channels, abolished the relaxation of the artery in response to insulin. However, N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 microM), an inhibitor of nitric oxide synthase, apamin (1 microM), an inhibitor of small-conductance Ca(2+)-activated K(+) channels, or glibenclamide (10 microM), an ATP-sensitive K(+) channels blocker, did not attenuate the relaxation of the artery caused by insulin. These results suggest that the relaxation of rat mesenteric artery in response to insulin is mediated mostly by large-conductance Ca(2+)-activated K(+) channels, perhaps an endothelium-derived hyperpolarizing factor (EDHF).