Charlotte Hougaard

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.

Deregulation of apoptotic volume decrease and ionic movements in multidrug-resistant tumor cells: role of chloride channels

Changes in cell volume and ion gradients across the plasma membrane play a pivotal role in the initiation of apoptosis. Here we explore the kinetics of apoptotic volume decrease (AVD) and ion content dynamics in wild-type (WT) and multidrug-resistant (MDR) Ehrlich ascites tumor cells (EATC). In WT EATC, induction of apoptosis with cisplatin (5 muM) leads to three distinctive AVD stages: an early AVD(1) (4-12 h), associated with a 30% cell water loss; a transition stage AVD(T) ( approximately 12 to 32 h), where cell volume is partly recovered; and a secondary AVD(2) (past 32 h), where cell volume was further reduced. AVD(1) and AVD(2) were coupled to net loss of Cl(-), K(+), Na(+), and amino acids (ninhydrin-positive substances), whereas during AVD(T), Na(+) and Cl(-) were accumulated. MDR EATC was resistant to cisplatin, showing increased viability and less caspase 3 activation. Compared with WT EATC, MDR EATC underwent a less pronounced AVD(1,) an augmented AVD(T), and a delay in induction of AVD(2). Changes in AVD were associated with inhibition of Cl(-) loss during AVD(1), augmented NaCl uptake during AVD(T), and a delay of Cl(-) loss during AVD(2). Application of the anion channel inhibitor NS3728 inhibited AVD and completely abolished the differences in AVD, ionic movements, and caspase 3 activation between WT and MDR EATC. Finally, the maximal capacity of volume-regulated anion channel was found to be strongly repressed in MDR EATC. Together, these data suggest that impairment of AVD, primarily via modulation of NaCl movements, contribute to protection against apoptosis in MDR EATC.

Rho family GTP binding proteins are involved in the regulatory volume decrease process in NIH3T3 mouse fibroblasts

The role of Rho GTPases in the regulatory volume decrease (RVD) process following osmotic cell swelling is controversial and has so far only been investigated for the swelling‐activated Cl− efflux. We investigated the involvement of RhoA in the RVD process in NIH3T3 mouse fibroblasts, using wild‐type cells and three clones expressing constitutively active RhoA (RhoAV14). RhoAV14 expression resulted in an up to fourfold increase in the rate of RVD, measured by large‐angle light scattering. The increase in RVD rate correlated with RhoAV14 expression. RVD in wild‐type cells was unaffected by the Rho kinase inhibitor Y‐27632 and the phosphatidyl‐inositol 3 kinase (PI3K) inhibitor wortmannin. The maximal rates of swelling‐activated K+ (86Rb+ as tracer) and taurine ([3H]taurine as tracer) efflux after a 30 % reduction in extracellular osmolarity were increased about twofold in cells with maximal RhoAV14 expression compared to wild‐type cells, but were unaffected by Y‐27632. The volume set points for activation of release of both osmolytes appeared to be reduced by RhoAV14 expression. The maximal taurine efflux rate constant was potentiated by the tyrosine phosphatase inhibitor Na3VO4, and inhibited by the tyrosine kinase inhibitor genistein. The magnitude of the swelling‐activated Cl− current (ICl,swell) was higher in RhoAV14 than in wild‐type cells after a 7.5 % reduction in extracellular osmolarity, but, in contrast to 86Rb+ and [3H]taurine efflux, similar in both strains after a 30 % reduction in extracellular osmolarity. ICl,swell was inhibited by Y‐27632 and strongly potentiated by the myosin light chain kinase inhibitors ML‐7 and AV25. It is suggested that RhoA, although not the volume sensor per se, is an important upstream modulator shared by multiple swelling‐activated channels on which RhoA exerts its effects via divergent signalling pathways.

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.

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.

Cell cycle-dependent activity of the volume- and Ca2+-activated anion currents in Ehrlich lettre ascites cells

Recent evidence implicates the volume‐regulated anion current (VRAC) and other anion currents in control or modulation of cell cycle progression; however, the precise involvement of anion channels in this process is unclear. Here, Cl− currents in Ehrlich Lettre Ascites (ELA) cells were monitored during cell cycle progression, under three conditions: (i) after osmotic swelling (i.e., VRAC), (ii) after an increase in the free intracellular Ca2+ concentration (i.e., the Ca2+‐activated Cl− current, CaCC), and (iii) under steady‐state isotonic conditions. The maximal swelling‐activated VRAC current decreased in G1 and increased in early S phase, compared to that in G0. The isotonic steady‐state current, which seems to be predominantly VRAC, also decreased in G1, and increased again in early S phase, to a level similar to that in G0. In contrast, the maximal CaCC current (500 nM free Ca2+ in the pipette), was unaltered from G0 to G1, but decreased in early S phase. A novel high‐affinity anion channel inhibitor, the acidic di‐aryl‐urea NS3728, which inhibited both VRAC and CaCC, attenuated ELA cell growth, suggesting a possible mechanistic link between cell cycle progression and cell cycle‐dependent changes in the capacity for conductive Cl− transport. It is suggested that in ELA cells, entrance into the S phase requires an increase in VRAC activity and/or an increased potential for regulatory volume decrease (RVD), and at the same time a decrease in CaCC magnitude. J. Cell. Physiol. 210: 831–842, 2007.

Tuning the excitability of midbrain dopamine neurons by modulating the Ca2+ sensitivity of SK channels

Small conductance Ca2+ ‐activated K+ (SK) channels play a prominent role in modulating the spontaneous activity of dopamine (DA) neurons as well as their response to synaptically‐released glutamate. SK channel gating is dependent on Ca2+ binding to constitutively bound calmodulin, which itself is subject to endogenous and exogenous modulation. In the present study, patch‐clamp recording techniques were used to examine the relationship between the apparent Ca2+ affinity of cloned SK3 channels expressed in cultured human embryonic kidney 293 cells and the excitability of DA neurons in slices from rat substantia nigra using the positive and negative SK channel modulators, 6,7‐dichloro‐1H‐indole‐2,3‐dione‐3‐oxime and R‐N‐(benzimidazol‐2‐yl)‐1,2,3,4‐tetrohydro‐1‐naphtylamine. Increasing the apparent Ca2+ affinity of SK channels decreased the responsiveness of DA neurons to depolarizing current pulses, enhanced spike frequency adaptation and slowed spontaneous firing, effects attributable to an increase in the amplitude and duration of an apamin‐sensitive afterhyperpolarization. In contrast, decreasing the apparent Ca2+ affinity of SK channels enhanced DA neuronal excitability and changed the firing pattern from a pacemaker to an irregular or bursting discharge. Both the reduction in apparent Ca2+ affinity and the bursting associated with negative SK channel modulation were gradually surmounted by co‐application of the positive SK channel modulator. These results underscore the importance of SK channels in ‘tuning’ the excitability of DA neurons and demonstrate that gating modulation, in a manner analogous to physiological regulation of SK channels in vivo, represents a means of altering the response of DA neurons to membrane depolarization.

Synthesis and Structure−Activity Relationship Studies of 2-(N-Substituted)-aminobenzimidazoles as Potent Negative Gating Modulators of Small 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 Parkinsons 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.

Modulation of the volume-sensitive K+ current in Ehrlich ascites tumour cells by pH.

Abstract. The effects of extracellular and intracellular pH (pHo and pHi respectively) on the regulatory volume decrease (RVD) response and on the volume-sensitive K+ and Cl– currents (IK,vol and ICl,vol respectively) were studied in Ehrlich ascites tumour cells. Alkaline pHo accelerated and acidic pHo decelerated the RVD response significantly. Intra- and extracellular alkalinisation increased the amplitude of IK,vol whereas acidification had an inhibitory effect. The magnitude of ICl,vol was not affected by changes in pHi or pHo. A significant reduction in the activation time for IK,vol after hypotonic cell swelling was observed upon moderate intracellular alkalinisation (to pHi 7.9). A further increase in pHi to 8.4 resulted in the spontaneous activation of an IK under isotonic conditions which resembled IK,vol with respect to its pharmacological profile and current/voltage (I/V) relation. Noise analysis demonstrated that the increased amplitude of IK,vol at alkaline pH resulted mainly from an increase in the number of channels (N) contributing to the current. The channel open probability, Po, was largely unaffected by pH. The pH dependence and the biophysical and pharmacological properties of IK,vol are similar to those of the cloned tandem pore-domain acid-sensitive K+ (TASK) channels, and in the current study the presence of TASK-1 was confirmed in Ehrlich cells.

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 KCa2 channels

Acting as a negative gating modulator, (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) shifts the apparent Ca2+-dependence of the small-conductance Ca2+-activated K+ channels KCa2.1–2.3 to higher Ca2+ concentrations. Similar to the positive KCa 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 Ca2+ sensor calmodulin. However, by using a progressive chimeric approach, we were able to localize the site-of-action of NS8593 to the KCa2 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 wild-type KCa2.3. In contrast, when we transferred the KCa2.3 pore to KCa3.1, the channel became sensitive to NS8593. Using site-directed mutagenesis, we subsequently identified two specific residues in the inner vestibule of KCa2.3 (Ser507 and Ala532) that determined the effect of NS8593. Mutation of these residues to the corresponding residues in KCa3.1 (Thr250 and Val275) made KCa2.3 insensitive to NS8593, whereas introduction of serine and alanine into KCa3.1 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 KCa3.1 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 KCa2 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.