Bo Skaaning Jensen

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.

The anticonvulsant retigabine attenuates nociceptive behaviours in rat models of persistent and neuropathic pain.

We have tested for anti-nociceptive effects of the anticonvulsant KCNQ channel opener, N-(2-amino-4-(4-fluorobenzylamino)-phenyl)carbamic acid ethyl ester (retigabine), in rat models of experimental pain. In the chronic constriction injury and spared nerve models of neuropathic pain, injection of retigabine (5 and 20 mg/kg, p.o.) significantly attenuated (P<0.05) mechanical hypersensitivity in response to pin prick stimulation of the injured hindpaw. In contrast, retigabine had no effect on mechanical hypersensitivity to von Frey stimulation of the injured hindpaw in either model. Cold sensitivity in response to ethyl chloride was only attenuated (P<0.05) in the chronic constriction injury model. In the formalin test, retigabine (20 mg/kg, p.o.) attenuated flinching behaviour in the second phase compared with vehicle (P<0.05), and this effect was completely reversed by the KCNQ channel blocker 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991; 3 mg/kg, i.p.). Neither retigabine nor XE-991 administration affected the latency to respond to noxious thermal stimulation of the tail in control animals. These results suggest that retigabine may prove to be effective in the treatment of neuropathic pain.

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.

Pharmacological modulation of SK3 channels.

Small-conductance, calcium-activated K+ channels (SK channels) are voltage-insensitive channels that have been identified molecularly within the last few years. As SK channels play a fundamental role in most excitable cells and participate in afterhyperpolarization (AHP) and spike-frequency adaptation, pharmacological modulation of SK channels may be of significant clinical importance. Here we report the functional expression of SK3 in HEK293 and demonstrate a broad pharmacological profile for these channels. Brain slice studies commonly employ 4-aminopyridine (4-AP) to block voltage-dependent K+ channels or a methyl derivative of bicuculline, a blocker of gamma-aminobutyric acid (GABA)-gated Cl- channels, in order to investigate the role of various synapses in specialized neural networks. However, in this study both 4-AP and bicuculline are shown to inhibit SK3 channels (IC50 values of 512 microM and 6 microM, respectively) at concentrations lower than those used for brain slice recordings. Riluzole, a potent neuroprotective drug with anti-ischemic, anticonvulsant and sedative effects currently used in the treatment of amyotrophic lateral sclerosis, activates SK3 channels at concentrations of 3 microM and above. Amitriptyline, a tricyclic antidepressive widely used clinically, inhibits SK3 channels with an IC50 of 39.1 +/- 10 microM (n=6).

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.

Na+-K+-2Cl−cotransport in Ehrlich cells: regulation by protein phosphatases and kinases

To identify protein kinases (PK) and phosphatases (PP) involved in regulation of the Na+-K+-2Cl-cotransporter in Ehrlich cells, the effect of various PK and PP inhibitors was examined. The PP-1, PP-2A, and PP-3 inhibitor calyculin A (Cal-A) was a potent activator of Na+-K+-2Cl-cotransport (EC50 = 35 nM). Activation by Cal-A was rapid (<1 min) but transient. Inactivation is probably due to a 10% cell swelling and/or the concurrent increase in intracellular Cl- concentration. Cell shrinkage also activates the Na+-K+-2Cl-cotransport system. Combining cell shrinkage with Cal-A treatment prolonged the cotransport activation compared with stimulation with Cal-A alone, suggesting PK stimulation by cell shrinkage. Shrinkage-induced cotransport activation was pH and Ca2+/calmodulin dependent. Inhibition of myosin light chain kinase by ML-7 and ML-9 or of PKA by H-89 and KT-5720 inhibited cotransport activity induced by Cal-A and by cell shrinkage, with IC50 values similar to reported inhibition constants of the respective kinases in vitro. Cell shrinkage increased the ML-7-sensitive cotransport activity, whereas the H-89-sensitive activity was unchanged, suggesting that myosin light chain kinase is a modulator of the Na+-K+-2Cl-cotransport activity during regulatory volume increase.

Activation of protein kinase C during cell volume regulation in Ehrlich mouse ascites tumor cells

We have previously demonstrated that in Ehrlich cells a bumetanide-sensitive Na+,K+,2Cl- cotransporter is activated during regulatory volume decrease after cell shrinkage (hypertonic conditions) as well as during the late phase of regulatory volume decrease (hypotonic conditions). It is, however, quiescent under isotonic conditions. Using a protein kinase C assay system (Amersham, UK) it is here demonstrated that hypertonic cell shrinkage results in an increase in protein kinase C activity to 174% within the first minute, concomitant with the activation of the Na+,K+,2Cl- cotransporter. Hypotonic cell swelling results in a late activation of protein kinase C concomitant with a late activation of the Na+,K+,2Cl- cotransporter. The activation of protein kinase C during hypertonic as well as hypotonic conditions is inhibited by H-7. The more specific protein kinase C inhibitor chelerythrine inhibited protein kinase C as well as the Na+,K+,2Cl- cotransporter to the same extent as did H-7. These results indicate the involvement of protein kinase C in the regulation of the Na+,K+,2Cl- cotransporter in Ehrlich ascites tumor cells during cell volume regulation.

Activation of KCNQ5 channels stably expressed in HEK293 cells by BMS-204352.

The novel anti-ischemic compound, BMS-204352 ((3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indol-2-one)), strongly activates the voltage-gated K+ channel KCNQ5 in a concentration-dependent manner with an EC50 of 2.4 microM. At 10 microM, BMS-204352 increased the steady state current at -30 mV by 12-fold, in contrast to the 2-fold increase observed for the other KCNQ channels [Schrøder et al., 2001]. Retigabine ((D-23129; N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester) induced a smaller, yet qualitatively similar effect on KCNQ5. Furthermore, BMS-204352 (10 microM) did not significantly shift the KCNQ5 activation curves (threshold and potential for half-activation, V1/2), as observed for the other KCNQ channels. In the presence of BMS-204352, the activation and deactivation kinetics of the KCNQ5 currents were slowed as the slow activation time constant increased up to 10-fold. The M-current blockers, linopirdine (DuP 996; 3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one) and XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone), inhibited the activation of the KCNQ5 channel induced by the BMS-204352. Thus, BMS-204352 appears to be an efficacious KCNQ channels activator, and the pharmacological properties of the compound on the KCNQ5 channel seems to be different from what has been obtained on the other KCNQ channels.

Increased production of α-amylase from Thermomyces lanuginosus by the addition of Tween 80

The thermophilic fungus Thermomyces lanuginosus was cultivated in shake flasks for up to 120 h with low molecular weight dextran as carbon source supplemented with either Tween 80 or Triton X-100. Addition of Tween 80 to the growth medium gave a 2.7-fold increase in maximum α-amylase activity as compared with controls. Triton X-100 did not affect α-amylase production. In the presence of Tween 80, the α-amylase secretion was stimulated from the third day of cultivation whereas the general protein secretion was increased after only 24 h of cultivation. The amount of biomass produced increased slightly by high concentrations of Tween 80 but decreased by the addition of Triton X-100. Maximum total extracellular protein increased more than three-fold with increasing concentrations of Tween 80 in the medium. Neither the hyphal growth unit length (G) nor the hyphal diameter were affected by Tween 80. Tween 80 did not change the degree of glycosylation of the α-amylase. The addition of Tween 80 appears to give a general stimulation of protein secretion.

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

SummaryNet Cl− uptake as well as unidirectional36Cl influx during regulatory volume increase (RVI) require external K+. Half-maximal rate of bumetanide-sensitive36Cl uptake is attained at about 3.3mm external K+. The bumetanide-sensitive K+ influx found during RVI is strongly dependent on both Na+ and Cl−. The bumetanide-sensitive unidirectional Na+ influx during RVI is dependent on K+ as well as on Cl−. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na+, K+ and Cl−. In the presence of ouabain and Ba+ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na+, 0.8 K+, 2.0 Cl− or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K+ and Cl− flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na+, K+, 2Cl− cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K+ influx is activated. During hypotonic conditions a small bumetanide-sensitive K+ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca2+ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K+ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na+, K+, Cl− cotransport involves both Ca2+/calmodulin and protein kinase C.