Positive Gating Modulators Research Articles

Exploring the binding site and mechanism of action of NS309, a superagonistic positive gating modulator for the calcium-activated potassium channel KCa3.1

Exploring the binding site and mechanism of action of NS309, a superagonistic positive gating modulator for the calcium-activated potassium channel KCa3.1

Endothelial KCa channels: Novel targets to reduce atherosclerosis-driven vascular dysfunction.

Elevated levels of cholesterol in the blood can induce endothelial dysfunction, a condition characterized by impaired nitric oxide production and decreased vasodilatory capacity. Endothelial dysfunction can promote vascular disease, such as atherosclerosis, where macrophages accumulate in the vascular intima and fatty plaques form that impair normal blood flow in conduit arteries. Current pharmacological strategies to treat atherosclerosis mostly focus on lipid lowering to prevent high levels of plasma cholesterol that induce endothelial dysfunction and atherosclerosis. While this approach is effective for most patients with atherosclerosis, for some, lipid lowering is not enough to reduce their cardiovascular risk factors associated with atherosclerosis (e.g., hypertension, cardiac dysfunction, stroke, etc.). For such patients, additional strategies targeted at reducing endothelial dysfunction may be beneficial. One novel strategy to restore endothelial function and mitigate atherosclerosis risk is to enhance the activity of Ca2+-activated K+ (KCa) channels in the endothelium with positive gating modulator drugs. Here, we review the mechanism of action of these small molecules and discuss their ability to improve endothelial function. We then explore how this strategy could mitigate endothelial dysfunction in the context of atherosclerosis by examining how KCa modulators can improve cardiovascular function in other settings, such as aging and type 2 diabetes. Finally, we consider questions that will need to be addressed to determine whether KCa channel activation could be used as a long-term add-on to lipid lowering to augment atherosclerosis treatment, particularly in patients where lipid-lowering is not adequate to improve their cardiovascular health.

Mapping the binding site of NS309, a superagonistic positive gating modulator for the calcium-activated potassium channel KCa3.1.

The intermediate-conductance Ca2+-activated K+ channel KCa3.1 is expressed in erythrocytes, vascular endothelial cells, and immune cells. KCa3.1 is involved in a wide variety of disease pathologies, suggesting that both positive and negative gating modulators would be of interest as pharmacological tools and potential drugs. NS309 is the most potent positive gating modulator for KCa3.1, with an EC50 of 75 nM, and interestingly, it is capable of increasing channel activity even at saturating calcium concentrations. However, its binding site is currently unknown. Previous X-ray crystallographic and solution-state NMR studies suggested that NS309 was binding between calmodulin's N-lobe and the C-terminal region of the calmodulin binding domain and interacting with residues M51 and R372. Unfortunately, the crystal dimer used in these studies was later found to be an artifact and disproved when the full-length crystal structure of KCa3.1 was published in 2018. This full-length structure suggested that the “real” binding site for positive gating modulators was located within the interface between calmodulin's N-lobe and the S4-S5 linker, a hypothesis we have now confirmed via site-directed mutagenesis and whole-cell patch clamp. In addition, we have also been investigating a second binding pocket between the distal end of the S4-S5 intracellular loop and the S6 transmembrane segment (a site highlighted by Schroedinger's SiteMap software). And while NS309 does not seem to be binding in this second pocket, several mutations within this pocket cause the channel to become hypersensitive to calcium, indicating that this pocket could serve as another functional binding site for positive gating channel modulators.

Mapping the binding site of NS309, a superagonistic positive gating modulator for the calcium-activated potassium channel KCa3.1

The intermediate-conductance Ca2+-activated K+ channel KCa3.1 is expressed in erythrocytes, vascular endothelial cells, and immune cells. KCa3.1 is involved in a wide variety of disease pathologies, suggesting that both positive and negative gating modulators would be of interest as pharmacological tools and potential drugs. NS309 is the most potent positive gating modulator for KCa3.1, with an EC50 of ∼30 nM, and interestingly, it is capable of increasing channel activity even at saturating calcium concentrations.

Pharmacology of Small- and Intermediate-Conductance Calcium-Activated Potassium Channels.

The three small-conductance calcium-activated potassium (KCa2) channels and the related intermediate-conductance KCa3.1 channel are voltage-independent K+ channels that mediate calcium-induced membrane hyperpolarization. When intracellular calcium increases in the channel vicinity, it calcifies the flexible N lobe of the channel-bound calmodulin, which then swings over to the S4-S5 linker and opens the channel. KCa2 and KCa3.1 channels are highly druggable and offer multiple binding sites for venom peptides and small-molecule blockers as well as for positive- and negative-gating modulators. In this review, we briefly summarize the physiological role of KCa channels and then discuss the pharmacophores and the mechanism of action of the most commonly used peptidic and small-molecule KCa2 and KCa3.1 modulators. Finally, we describe the progress that has been made in advancing KCa3.1 blockers and KCa2.2 negative- and positive-gating modulators toward the clinic for neurological and cardiovascular diseases and discuss the remaining challenges.

Propofol inhibits prokaryotic voltage-gated Na+ channels by promoting activation-coupled inactivation.

Propofol is widely used in the clinic for the induction and maintenance of general anesthesia. As with most general anesthetics, however, our understanding of its mechanism of action remains incomplete. Local and general anesthetics largely inhibit voltage-gated Na+ channels (Navs) by inducing an apparent stabilization of the inactivated state, associated in some instances with pore block. To determine the biophysical and molecular basis of propofol action in Navs, we investigated NaChBac and NavMs, two prokaryotic Navs with distinct voltage dependencies and gating kinetics, by whole-cell patch clamp electrophysiology in the absence and presence of propofol at clinically relevant concentrations (2-10 µM). In both Navs, propofol induced a hyperpolarizing shift of the pre-pulse inactivation curve without any significant effects on recovery from inactivation at strongly hyperpolarized voltages, demonstrating that propofol does not stabilize the inactivated state. Moreover, there was no evidence of fast or slow pore block by propofol in a non-inactivating NaChBac mutant (T220A). Propofol also induced hyperpolarizing shifts of the conductance-voltage relationships with negligible effects on the time constants of deactivation at hyperpolarized voltages, indicating that propofol does not stabilize the open state. Instead, propofol decreases the time constants of macroscopic activation and inactivation. Adopting a kinetic scheme of Nav gating that assumes preferential closed-state recovery from inactivation, a 1.7-fold acceleration of the rate constant of activation and a 1.4-fold acceleration of the rate constant of inactivation were sufficient to reproduce experimental observations with computer simulations. In addition, molecular dynamics simulations and molecular docking suggest that propofol binding involves interactions with gating machinery in the S4-S5 linker and external pore regions. Our findings show that propofol is primarily a positive gating modulator of prokaryotic Navs, which ultimately inhibits the channels by promoting activation-coupled inactivation.

Inhibition of Intermediate-Conductance Calcium-Activated K Channel (KCa3.1) and Fibroblast Mitogenesis by α-Linolenic Acid and Alterations of Channel Expression in the Lysosomal Storage Disorders, Fabry Disease, and Niemann Pick C.

The calcium/calmodulin-gated KCa3.1 channel regulates normal and abnormal mitogenesis by controlling K+-efflux, cell volume, and membrane hyperpolarization-driven calcium-entry. Recent studies suggest modulation of KCa3.1 by omega-3 fatty acids as negative modulators and impaired KCa3.1 functions in the inherited lysosomal storage disorder (LSD), Fabry disease (FD). In the first part of present study, we characterize KCa3.1 in murine and human fibroblasts and test the impact of omega-3 fatty acids on fibroblast proliferation. In the second, we study whether KCa3.1 is altered in the LSDs, FD, and Niemann-Pick disease type C (NPC). Our patch-clamp and mRNA-expression studies on murine and human fibroblasts show functional expression of KCa3.1. KCa currents display the typical pharmacological fingerprint of KCa3.1: Ca2+-activation, potentiation by the positive-gating modulators, SKA-31 and SKA-121, and inhibition by TRAM-34, Senicapoc (ICA-17043), and the negative-gating modulator, 13b. Considering modulation by omega-3 fatty acids we found that α-linolenic acid (α-LA) and docosahexanenoic acid (DHA) inhibit KCa3.1 currents and strongly reduce fibroblast growth. The α-LA-rich linseed oil and γ-LA-rich borage oil at 0.5% produce channel inhibition while α-LA/γ-LA-low oils has no anti-proliferative effect. Concerning KCa3.1 in LSD, mRNA expression studies, and patch-clamp on primary fibroblasts from FD and NPC patients reveal lower KCa3.1-gene expression and membrane expression than in control fibroblasts. In conclusion, the omega-3 fatty acid, α-LA, and α-LA/γ-LA-rich plant oils, inhibit fibroblast KCa3.1 channels and mitogenesis. Reduced fibroblast KCa3.1 functions are a feature and possible biomarker of cell dysfunction in FD and NPC and supports the concept that biased lipid metabolism is capable of negatively modulating KCa3.1 expression.

Vascular Reactivity Profile of Novel KCa 3.1-Selective Positive-Gating Modulators in the Coronary Vascular Bed.

Opening of intermediate-conductance calcium-activated potassium channels (KC a 3.1) produces membrane hyperpolarization in the vascular endothelium. Here, we studied the ability of two new KC a 3.1-selective positive-gating modulators, SKA-111 and SKA-121, to (1) evoke porcine endothelial cell KC a 3.1 membrane hyperpolarization, (2) induce endothelium-dependent and, particularly, endothelium-derived hyperpolarization (EDH)-type relaxation in porcine coronary arteries (PCA) and (3) influence coronary artery tone in isolated rat hearts. In whole-cell patch-clamp experiments on endothelial cells of PCA (PCAEC), KC a currents evoked by bradykinin (BK) were potentiated ≈7-fold by either SKA-111 or SKA-121 (both at 1 μM) and were blocked by a KC a 3.1 blocker, TRAM-34. In membrane potential measurements, SKA-111 and SKA-121 augmented bradykinin-induced hyperpolarization. Isometric tension measurements in large- and small-calibre PCA showed that SKA-111 and SKA-121 potentiated endothelium-dependent relaxation with intact NO synthesis and EDH-type relaxation to BK by ≈2-fold. Potentiation of the BK response was prevented by KC a 3.1 inhibition. In Langendorff-perfused rat hearts, SKA-111 potentiated coronary vasodilation elicited by BK. In conclusion, our data show that positive-gating modulation of KC a 3.1 channels improves BK-induced membrane hyperpolarization and endothelium-dependent relaxation in small and large PCA as well as in the coronary circulation of rats. Positive-gating modulators of KC a 3.1 could be therapeutically useful to improve coronary blood flow and counteract impaired coronary endothelial dysfunction in cardiovascular disease.

Structural Determinants for Selectivity of the Positive KCa Channel Gating Modulator, SKA-121

Small-conductance (KCa2) and intermediate-conductance (KCa3.1) calcium-activated K+ channels are voltage-independent and share a common calcium/calmodulin mediated gating mechanism. Existing positive gating modulators like EBIO, NS309 or SKA-31 activate both KCa2 and KCa3.1 channels with similar potency. We recently performed a structure activity relationship (SAR) study with the aim of optimizing the benzothiazole pharmacophore of SKA-31 towards KCa3.1 selectivity and have identified SKA-121 (5-methylnaphtho[2,1-d]oxazol-2-amine), which displays 41-fold selectivity for KCa3.1 (EC50 109 nM ± 14 nM) over KCa2.3 (EC50 4.4 ± 1.6 μM). SKA-121 is a typical positive-gating modulator, which shift the calcium-concentration response curves of KCa3.1 and KCa2.3 to the left but also increases open probability at saturating Ca2+ concentrations. In order to understand why introduction of a “simple” CH3 group in 5-position of the benzothiazole/oxazol system could achieve such a significant gain in selectivity for KCa3.1 over KCa2.3 we are currently performing site-directed mutagenesis of the calmodulin binding domain (CaMBD) guided by the crystal structure of the KCa2.2 CAMBD and structural modeling. We used the RosettaLigand method to generate models of SKA-121 binding to both the KCa3.1 and KCa2.3 C-terminal CaMBD in complex with calmodulin and then mutated channel residues suggested to be within 4A of SKA-121. Based on the results of the mutagenesis and the model we hypothesize that an extensive hydrogen bond network is responsible for the selectivity of SKA-121 for KCa3.1 over KCa2.3. The NH2 group of SKA-121 forms hydrogen bonds with M51 and E54 on calmodulin. E54 is part of a hydrogen bond network including R362, E295 and N300 of the KCa3.1 channel. Mutation of R362 to S breaks this hydrogen bond network and significantly reduces SKA-121 activity on KCa3.1.Supported by U54NS079202 and T32-HL086350 from NIH.

Positive KCa Channel Gating Modulators with Selectivity for KCa3.1

Positive KCa Channel Gating Modulators with Selectivity for KCa3.1

Small Molecule Modulation of Voltage-Gated Ion Channels

Small Molecule Modulation of Voltage-Gated Ion Channels

A New Class of Positive Gating Modulators of hKv3.2 Channels: Insights into the Mechanism of Action

Neuronal KV3 channels are implicated in neurological (tinnitus, hearing loss) and psychiatric (schizophrenia) disorders, and are potential therapeutic targets. Here, we investigated two new compounds (AUT3 and AUT5) developed by Autifony Therapeutics, which may act as specific modulators of this K+ channel sub-family. Initial screening of heterologously expressed KV channels in Xenopus oocytes demonstrated that AUT5 acts as a relatively selective positive modulator of hKV3.2. AUT5 (2 µM) induces a substantial parallel negative shift in the conductance-voltage relation (ΔV1/2 = −24.1±2.0 mV, n = 9). However, 2 µM AUT5 has little to no effect on Kv1.2, Kv3.4, Δ28-Kv3.4 and Kv4.2. The positive modulatory effect on hKv3.2 is concentration-dependent with an EC50 = 2 – 4 µM. AUT3 also acts as a positive modulator of hKV3.2, albeit the effect is ≥ 10-fold weaker. Toward investigating the mechanism of action, we examined tail current kinetics and found a dramatic slowing of deactivation with 2 µM AUT5 (τa = 24.7±6.2 ms vs 6.5±1.1 ms of control at −50 mV, n = 6). We observed similar positive modulation by AUT3 and AUT5 in mammalian cell lines (HEK-293 and stably transfected cells). We hypothesize that a direct interaction between AUT compounds and the channel's activation machinery favorably affects the energetics of hKV3.2 activation gating. To test this hypothesis, we are currently investigating possible effects on voltage sensor movements, extending the screening to additional Kv channels, and searching for putative binding sites in the Kv3.2 protein.

A novel pan-negative-gating modulator of KCa2/3 channels, fluoro-di-benzoate, RA-2, inhibits endothelium-derived hyperpolarization-type relaxation in coronary artery and produces bradycardia in vivo.

Small/intermediate conductance KCa channels (KCa2/3) are Ca(2+)/calmodulin regulated K(+) channels that produce membrane hyperpolarization and shape neurologic, epithelial, cardiovascular, and immunologic functions. Moreover, they emerged as therapeutic targets to treat cardiovascular disease, chronic inflammation, and some cancers. Here, we aimed to generate a new pharmacophore for negative-gating modulation of KCa2/3 channels. We synthesized a series of mono- and dibenzoates and identified three dibenzoates [1,3-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate) (RA-2), 1,2-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate), and 1,4-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate)] with inhibitory efficacy as determined by patch clamp. Among them, RA-2 was the most drug-like and inhibited human KCa3.1 with an IC50 of 17 nM and all three human KCa2 subtypes with similar potencies. RA-2 at 100 nM right-shifted the KCa3.1 concentration-response curve for Ca(2+) activation. The positive-gating modulator naphtho[1,2-d]thiazol-2-ylamine (SKA-31) reversed channel inhibition at nanomolar RA-2 concentrations. RA-2 had no considerable blocking effects on distantly related large-conductance KCa1.1, Kv1.2/1.3, Kv7.4, hERG, or inwardly rectifying K(+) channels. In isometric myography on porcine coronary arteries, RA-2 inhibited bradykinin-induced endothelium-derived hyperpolarization (EDH)-type relaxation in U46619-precontracted rings. Blood pressure telemetry in mice showed that intraperitoneal application of RA-2 (≤100 mg/kg) did not increase blood pressure or cause gross behavioral deficits. However, RA-2 decreased heart rate by ≈145 beats per minute, which was not seen in KCa3.1(-/-) mice. In conclusion, we identified the KCa2/3-negative-gating modulator, RA-2, as a new pharmacophore with nanomolar potency. RA-2 may be of use to generate structurally new types of negative-gating modulators that could help to define the physiologic and pathomechanistic roles of KCa2/3 in the vasculature, central nervous system, and during inflammation in vivo.

New positive Ca2+-activated K+ channel gating modulators with selectivity for KCa3.1.

Small-conductance (KCa2) and intermediate-conductance (KCa3.1) calcium-activated K(+) channels are voltage-independent and share a common calcium/calmodulin-mediated gating mechanism. Existing positive gating modulators like EBIO, NS309, or SKA-31 activate both KCa2 and KCa3.1 channels with similar potency or, as in the case of CyPPA and NS13001, selectively activate KCa2.2 and KCa2.3 channels. We performed a structure-activity relationship (SAR) study with the aim of optimizing the benzothiazole pharmacophore of SKA-31 toward KCa3.1 selectivity. We identified SKA-111 (5-methylnaphtho[1,2-d]thiazol-2-amine), which displays 123-fold selectivity for KCa3.1 (EC50 111 ± 27 nM) over KCa2.3 (EC50 13.7 ± 6.9 μM), and SKA-121 (5-methylnaphtho[2,1-d]oxazol-2-amine), which displays 41-fold selectivity for KCa3.1 (EC50 109 nM ± 14 nM) over KCa2.3 (EC50 4.4 ± 1.6 μM). Both compounds are 200- to 400-fold selective over representative KV (KV1.3, KV2.1, KV3.1, and KV11.1), NaV (NaV1.2, NaV1.4, NaV1.5, and NaV1.7), as well as CaV1.2 channels. SKA-121 is a typical positive-gating modulator and shifts the calcium-concentration response curve of KCa3.1 to the left. In blood pressure telemetry experiments, SKA-121 (100 mg/kg i.p.) significantly lowered mean arterial blood pressure in normotensive and hypertensive wild-type but not in KCa3.1(-/-) mice. SKA-111, which was found in pharmacokinetic experiments to have a much longer half-life and to be much more brain penetrant than SKA-121, not only lowered blood pressure but also drastically reduced heart rate, presumably through cardiac and neuronal KCa2 activation when dosed at 100 mg/kg. In conclusion, with SKA-121, we generated a KCa3.1-specific positive gating modulator suitable for further exploring the therapeutical potential of KCa3.1 activation.

Development of a Qpatch Automated Electrophysiology Assay for Identifying Kca3.1 Inhibitors and Activators

The intermediate-conductance Ca2+-activated K+ channel KCa3.1 (also known as KCNN4, IK1 or the Gardos channel) plays an important role in the activation of T- and B-cells, mast cells, macrophages and microglia by regulating membrane potential, cellular volume and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization (EDH) responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorder, while KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca2+ sensitivity of KCa3.1 was studied using varying intracellular Ca2+ concentrations. A free Ca2+ concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca2+ concentration (∼200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay.Supported by U54NS079202, R21NS072585 and T32-GM008799 from NIH.

Development of a QPatch automated electrophysiology assay for identifying KCa3.1 inhibitors and activators.

The intermediate-conductance Ca(2+)-activated K(+) channel KCa3.1 (also known as KCNN4, IK1, or the Gárdos channel) plays an important role in the activation of T and B cells, mast cells, macrophages, and microglia by regulating membrane potential, cellular volume, and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders, whereas KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca(2+) sensitivity of KCa3.1 was studied using varying intracellular Ca(2+) concentrations. A free Ca(2+) concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca(2+) concentration (∼200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay.

NS8593-Mediated Negative Gating Modulation Depends on Residues in the Inner Pore Vestibule of Kca2 Channels

The identification of NS8593 has provided a selective and novel means for modulating the activity of the small-conductance calcium-activated potassium channels KCa2.1-2.3. Acting as a negative gating modulator, NS8593 shifts the apparent calcium dependence of channel gating to higher calcium concentrations. It has been assumed that the binding site for NS8593 was located in the C-terminal region, similar to that of some positive gating modulators (e.g. EBIO and CyPPA, but not GW542573X). However, by employing a progressive chimera approach, (where all critical constructs were tested for normal Ca2+-sensitivity in inside-out patches) we were able to localize the site-of-action to the pore. For example, when we transferred the C-terminus from the NS8593-insensitive intermediate-conductance KCa3.1 channel to KCa2.3 the chimeric channel remained as sensitive to NS8593 as WT-KCa2.3. In contrast, when we transferred the KCa2.3 pore, KCa3.1 became sensitive to NS8593. Subsequently, by using site-directed mutagenesis we identified two residues in the inner vestibule of KCa2.3 (Ser-507 and Ala-532) that mediate the activity of NS8593. By mutating these residues to the corresponding residues in KCa3.1 (Thr-250 and Val-275), we were able to make KCa2.3 insensitive. Conversely, replacement of these two residues was sufficient to render KCa3.1 sensitive to NS8593. The positions of these residues, Ser-507 in the pore-loop near the selectivity filter and Ala-532 in an adjacent position in the S6 segment, are within in the region predicted to contain the channel gate. Based on these results, we propose that NS8593 mediated gating modulation of KCa2.3 occurs at a position deep within the inner pore vestibule.

Inhibitory Gating Modulation of Small Conductance Ca2+-Activated K+Channels by the Synthetic Compound (R)-N-(Benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) Reduces Afterhyperpolarizing Current in Hippocampal CA1 Neurons

SK channels are small conductance Ca(2+)-activated K(+) channels important for the control of neuronal excitability, the fine tuning of firing patterns, and the regulation of synaptic mechanisms. The classic SK channel pharmacology has largely focused on the peptide apamin, which acts extracellularly by a pore-blocking mechanism. 1-Ethyl-2-benzimidazolinone (1-EBIO) and 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) have been identified as positive gating modulators that increase the apparent Ca(2+) sensitivity of SK channels. In the present study, we describe inhibitory gating modulation as a novel principle for selective inhibition of SK channels. In whole-cell patch-clamp experiments, the compound (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) reversibly inhibited recombinant SK3-mediated currents (human SK3 and rat SK3) with potencies around 100 nM. However, in contrast to known pore blockers, NS8593 did not inhibit (125)I-apamin binding. Using excised patches, it was demonstrated that NS8593 decreased the Ca(2+) sensitivity by shifting the activation curve for Ca(2+) to the right, only slightly affecting the maximal Ca(2+)-activated SK current. NS8593 inhibited all the SK1-3 subtypes Ca(2+)-dependently (K(d) = 0.42, 0.60, and 0.73 microM, respectively, at 0.5 microM Ca(2+)), whereas the compound did not affect the Ca(2+)-activated K(+) channels of intermediate and large conductance (hIK and hBK channels, respectively). The site of action was accessible from both sides of the membrane, and the NS8593-mediated inhibition was prevented in the presence of a high concentration of the positive modulator NS309. NS8593 was further tested on mouse CA1 neurons in hippocampal slices and shown to inhibit the apaminand tubocurarine-sensitive SK-mediated afterhyperpolarizing current, at a concentration of 3 microM.