Dorte Strøbæk

Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel

The human intermediate-conductance, Ca2+-activated K+ channel (hIK) was identified by searching the expressed sequence tag database. hIK was found to be identical to two recently cloned K+ channels, hSK4 and hIK1. RNA dot blot analysis showed a widespread tissue expression, with the highest levels in salivary gland, placenta, trachea, and lung. With use of fluorescent in situ hybridization and radiation hybrid mapping, hIK mapped to chromosome 19q13.2 in the same region as the disease Diamond-Blackfan anemia. Stable expression of hIK in HEK-293 cells revealed single Ca2+-activated K+ channels exhibiting weak inward rectification (30 and 11 pS at -100 and +100 mV, respectively). Whole cell recordings showed a noninactivating, inwardly rectifying K+ conductance. Ionic selectivity estimated from bi-ionic reversal potentials gave the permeability ( P K/ P X) sequence K+ = Rb+ (1.0) > Cs+ (10.4) ≫ Na+, Li+, N-methyl-d-glucamine (>51).[Formula: see text] blocked the channel completely. hIK was blocked by the classical inhibitors of the Gardos channel charybdotoxin (IC50 28 nM) and clotrimazole (IC50 153 nM) as well as by nitrendipine (IC50 27 nM), Stichodactyla toxin (IC50 291 nM), margatoxin (IC50 459 nM), miconazole (IC50 785 nM), econazole (IC50 2.4 μM), and cetiedil (IC50 79 μM). Finally, 1-ethyl-2-benzimidazolinone, an opener of the T84 cell IK channel, activated hIK with an EC50 of 74 μM.

Pharmacological characterization of small‐conductance Ca2+‐activated K+ channels stably expressed in HEK 293 cells

Three genes encode the small‐conductance Ca2+‐activated K+ channels (SK channels). We have stably expressed hSK1 and rSK2 in HEK 293 cells and addressed the pharmacology of these subtypes using whole‐cell patch clamp recordings. The bee venom peptide apamin blocked hSK1 as well as rSK2 with IC50 values of 3.3 nM and 83 pM, respectively. The pharmacological separation between the subtypes was even more prominent when applying the scorpion peptide blocker scyllatoxin, which blocked hSK1 with an IC50 value of 80 nM and rSK2 at 287 pM. The potent small molecule blockers showed little differentiation between the channel subtypes. The bis‐quinolinium cyclophane UCL 1684 blocked hSK1 with an IC50 value of 762 pM and rSK2 at 364 pM. The antiseptic compound dequalinium chloride blocked hSK1 and rSK2 with IC50 values of 444 nM and 162 nM, respectively. The nicotinic acetylcholine receptor antagonist d‐tubocurarine was found to block hSK1 and rSK2 with IC50 values of 27 μM and 17 μM when measured at +80 mV. The inhibition by d‐tubocurarine was voltage‐dependent with increasing affinities at more hyperpolarized potentials. The GABAA receptor antagonist bicuculline methiodide also blocked hSK1 and rSK2 in a voltage‐dependent manner with IC50 values of 15 and 25 μM when measured at +80 mV. In conclusion, the pharmacological separation between SK channel subtypes expressed in mammalian cells is too small to support the notion that the apamin‐insensitive afterhyperpolarization of neurones is mediated by hSK1.

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.

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

Positive modulators of small conductance Ca2+‐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 Ca2+‐activated K+ channel (IK). This paper demonstrates, for the first time, subtype‐selective positive modulation of SK channels.

KCNQ4 channel activation by BMS-204352 and retigabine

Activation of potassium channels generally reduces cellular excitability, making potassium channel openers potential drug candidates for the treatment of diseases related to hyperexcitabilty such as epilepsy, neuropathic pain, and neurodegeneration. Two compounds, BMS-204352 and retigabine, presently in clinical trials for the treatment of stroke and epilepsy, respectively, have been proposed to exert their protective action via an activation of potassium channels. Here we show that KCNQ4 channels, stably expressed in HEK293 cells, were activated by retigabine and BMS-204352 in a reversible and concentration-dependent manner in the concentration range 0.1-10 microM. Both compounds shifted the KCNQ4 channel activation curves towards more negative potentials by about 10 mV. Further, the maximal current obtainable at large positive voltages was also increased concentration-dependently by both compounds. Finally, a pronounced slowing of the deactivation kinetics was induced in particular by BMS-204352. The M-current blocker linopirdine inhibited the baseline current, as well as the BMS-204352-induced activation of the KCNQ4 channels. KCNQ2, KCNQ2/Q3, and KCNQ3/Q4 channels were activated to a similar degree as KCNQ4 channels by 10 microM of BMS-204352 and retigabine, respectively. The compounds are, thus, likely to be general activators of M-like currents.

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 Ca2+-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 Ca2+ sensitivity of SK channels. In the present study, we describe inhibitory gating modulation as a novel principle for selective inhibition of SK channels. In wholecell 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 125I-apamin binding. Using excised patches, it was demonstrated that NS8593 decreased the Ca2+ sensitivity by shifting the activation curve for Ca2+ to the right, only slightly affecting the maximal Ca2+-activated SK current. NS8593 inhibited all the SK1-3 subtypes Ca2+-dependently (Kd = 0.42, 0.60, and 0.73 μM, respectively, at 0.5 μM Ca2+), whereas the compound did not affect the Ca2+-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 μM.

Modulation of the Ca2+-dependent K+ Channel, hslo, by the Substituted Diphenylurea NS 1608, Paxilline and Internal Ca2+

The high-conductance Ca(2+)-activated K channel (BK channel) is not only regulated by a number of physiological stimuli, but it is also sensitive to pharmacological modulation. We have stably expressed the alpha-subunit of the human BK channel, hslo, in HEK 293 cells and studied by patch-clamp technique how its gating is modulated by the channel activator NS 1608, by the selective channel blocker paxilline, as well as by changes in [Ca2+]i and Vm. The cells expressed 200-800 hslo channels per patch. The channel activity was determined by tail current analysis, and the activation curves were fitted to single Boltzmann functions, from which a gating charge for the hslo channel of 1.2 elementary charges was deduced. The hslo channel was very sensitive to changes in [Ca2+]i within the physiological range, whereas Ca(2+)-independent openings were seen at Ca2+ concentrations of 15 nM or below. NS 1608 shifted the hslo channel activation curve towards negative membrane potentials with an EC50 of 2.1 microM and a maximal shift of -74 mV. The channels activated by NS 1608 were sensitive to block by paxilline, but the two molecules apparently did not interact within the same site, since paxilline reduced the size of the tail current at all voltages, whereas NS 1608 shifted the activation curve along the voltage axis. Further, the effect of paxilline was Ca(2+)-sensitive, whereas NS 1608 elicited identical effects in the presence of either < 0.5 nM or 500 nM [Ca2+]i. NS 1608 hyperpolarized the cells by -50 to -70 mV, and paxilline depolarized them towards 0 mV. In addition to the effects on the steady state current NS 1608 also significantly influenced the non-stationary channel kinetics. In the presence of NS 1608 the time constants for deactivation of tail currents were more than tripled at all potentials. We have shown, that NS 1608 modulates steady-state BK currents and channel gating kinetics through a Ca(2+)-independent interaction with the alpha-subunit of the channel.

Activation of the human intermediate-conductance Ca2+-activated K+ channel by 1-ethyl-2-benzimidazolinone is strongly Ca2+-dependent

Modulation of the cloned human intermediate-conductance Ca(2+)-activated K(+) channel (hIK) by the compound 1-ethyl-2-benzimidazolinone (EBIO) was studied by patch-clamp technique using human embryonic kidney cells (HEK 293) stably expressing the hIK channels. In whole-cell studies, intracellular concentrations of free Ca(2+) were systematically varied, by buffering the pipette solutions. In voltage-clamp, the hIK specific currents increased gradually from 0 to approximately 300 pA/pF without reaching saturation even at the highest Ca(2+) concentration tested (300 nM). In the presence of EBIO (100 microM), the Ca(2+)-activation curve was shifted leftwards, and maximal currents were attained at 100 nM Ca(2+). In current-clamp, steeply Ca(2+)-dependent membrane potentials were recorded and the cells gradually hyperpolarised from -20 to -85 mV when Ca(2+) was augmented from 0 to 300 nM. EBIO strongly hyperpolarised cells buffered at intermediate Ca(2+) concentrations. In contrast, no effects were detected either below 10 nM (no basic channel activation) or at 300 nM Ca(2+) (V(m) close to E(K)). Without Ca(2+), EBIO-induced hyperpolarisations were not obtainable, indicating an obligatory Ca(2+)-dependent mechanism of action. When applied to inside-out patches, EBIO exerted a Ca(2+)-dependent increase in the single-channel open-state probability, showing that the compound modulates hIK channels by a direct action on the alpha-subunit or on a closely associated protein. In conclusion, EBIO activates hIK channels in whole-cell and inside-out patches by a direct mechanism, which requires the presence of internal Ca(2+).

Selective positive modulator of calcium-activated potassium channels exerts beneficial effects in a mouse model of spinocerebellar ataxia type 2.

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder caused by a polyglutamine expansion within the Ataxin-2 (Atxn2) protein. Purkinje cells (PC) of the cerebellum fire irregularly and eventually die in SCA2. We show here that the type 2 small conductance calcium-activated potassium channel (SK2) play a key role in control of normal PC activity. Using cerebellar slices from transgenic SCA2 mice we demonstrate that SK channel modulators restore regular pacemaker activity of SCA2 PCs. Furthermore, we also show that oral delivery of a more selective positive modulator of SK2/3 channels (NS13001) alleviates behavioral and neuropathological phenotypes of aging SCA2 transgenic mice. We conclude that SK2 channels constitute a therapeutic target for SCA2 treatment and that the developed selective SK2/3 modulator NS13001 holds promise as a potential therapeutic agent for treatment of SCA2 and possibly other cerebellar ataxias.

Stable expression of the human large-conductance Ca2+-activated K+ channel α- and β-subunits in HEK293 cells

We have generated HEK293 cell lines stably expressing high levels of either the human BK channel α‐subunit alone or the BK channel α‐subunit and β‐subunit together. For co‐expression a plasmid with three expression cassettes was constructed. Patch‐clamp recordings on inside‐out patches from the transfected cells resulted in macroscopic currents reflecting the expression of 200–800 BK channels per patch. No decrease in channel expression could be detected in cells grown for more than 50 passages. The α‐subunit when expressed alone conducted currents which were sensitive to intracellular Ca2+ in the physiological range. In the presence of the β‐subunit the steady‐state activation curves were shifted by −20 to −30 mV and channel deactivation kinetics were slowed. The BK channel opener NS1608 (10 μM) shifted the steady‐state activation curves for the α‐subunit as well as for the αβ‐subunits by −40 to −50 mV.