Modulation of KCNQ2/3 potassium channels by the novel anticonvulsant retigabine

Retigabine is a novel anticonvulsant with an unknown mechanism of action. It has recently been reported that retigabine modulates a potassium channel current in nerve growth factor-differentiated PC12 cells (), however, to date the molecular correlate of this current has not been identified. In the present study we have examined the effects of retigabine on recombinant human KCNQ2 and KCNQ3 potassium channels, expressed either alone or in combination in Xenopus oocytes. Application of 10 microM retigabine to oocytes expressing the KCNQ2/3 heteromeric channel shifted both the activation threshold and voltage for half-activation by approximately 20 mV in the hyperpolarizing direction, leading to an increase in current amplitude at test potentials between -80 mV and +20 mV. Retigabine also had a marked effect on KCNQ current kinetics, increasing the rate of channel activation but slowing deactivation at a given test potential. Similar effects of retigabine were observed in oocytes expressing KCNQ2 alone, suggesting that KCNQ2 may be the molecular target of retigabine. Membrane potential recordings in oocytes expressing the KCNQ2/3 heteromeric channel showed that application of retigabine leads to a concentration-dependent hyperpolarization of the oocyte, from a resting potential of -63 mV under control conditions to -85 mV in the presence of 100 microM retigabine (IC(50) = 5.2 microM). In control experiments retigabine had no effect on either resting membrane potential or endogenous oocyte membrane currents. In conclusion, we have shown that retigabine acts as a KCNQ potassium channel opener. Because the heteromeric KCNQ2/3 channel has recently been reported to underlie the M-current, it is likely that M-current modulation can explain the anticonvulsant actions of retigabine in animal models of epilepsy.     

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.     

Characterization of KCNQ5/Q3 potassium channels expressed in mammalian cells

Heteromeric KCNQ5/Q3 channels were stably expressed in Chinese Hamster ovary cells and characterized using the whole cell voltage-clamp technique. KCNQ5/Q3 channels were activated by the novel anticonvulsant, retigabine (EC(50) 1.4 microM) by a mechanism that involved drug-induced, leftward shifts in the voltage-dependence of channel activation (-31.8 mV by 30 microM retigabine). KCNQ5/Q3 channels were inhibited by linopirdine (IC(50) 7.7 microM) and barium (IC(50) 0.46 mM), at concentrations similar to those required to inhibit native M-currents. These findings identify KCNQ5/Q3 channels as a molecular target for retigabine and raise the possibility that activation of KCNQ5/Q3 channels may be responsible for some of the anti-convulsant activity of this agent. Furthermore, the sensitivity of KCNQ5/Q3 channels to linopirdine supports the possibility that potassium channels comprised of KCNQ5 and KCNQ3 may make a contribution to native M-currents.     

N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide (ICA-27243): a novel, selective KCNQ2/Q3 potassium channel activator

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are voltage-gated K(+) channel subunits that underlie the neuronal M current. In humans, mutations in these genes lead to a rare form of neonatal epilepsy (Biervert et al., 1998; Singh et al., 1998), suggesting that KCNQ2/Q3 channels may be attractive targets for novel antiepileptic drugs. In the present study, we have identified the compound N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide (ICA-27243) as a selective activator of the neuronal M current and KCNQ2/Q3 channels. In SH-SY5Y human neuroblastoma cells, ICA-27243 produced membrane potential hyperpolarization that could be prevented by coadministration with the M-current inhibitors 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE-991) and linopirdine. ICA-27243 enhanced both (86)Rb(+) efflux (EC(50) = 0.2 microM) and whole-cell currents in Chinese hamster ovary cells stably expressing heteromultimeric KCNQ2/Q3 channels (EC(50) = 0.4 microM). Activation of KCNQ2/Q3 channels was associated with a hyperpolarizing shift of the voltage dependence of channel activation (V((1/2)) shift of -19 mV at 10 microM). In contrast, ICA-27243 was less effective at activating KCNQ4 and KCNQ3/Q5 and was selective over a wide range of neurotransmitter receptors and ion channels such as voltage-dependent sodium channels and GABA-gated chloride channels. ICA-27243 (1-10 microM) was found to reversibly suppress seizure-like activity in an ex vivo hippocampal slice model of epilepsy and demonstrated in vivo anticonvulsant activity (ED(50) = 8.4 mg/kg) in the mouse maximal electroshock epilepsy model. In conclusion, ICA-27243 represents the first member of a novel chemical class of selective KCNQ2/Q3 activators with anticonvulsant-like activity in experimental models of epilepsy.     

Meclofenamic acid and diclofenac, novel templates of KCNQ2/Q3 potassium channel openers, depress cortical neuron activity and exhibit anticonvulsant properties

The voltage-dependent M-type potassium current (M-current) plays a major role in controlling brain excitability by stabilizing the membrane potential and acting as a brake for neuronal firing. The KCNQ2/Q3 heteromeric channel complex was identified as the molecular correlate of the M-current. Furthermore, the KCNQ2 and KCNQ3 channel alpha subunits are mutated in families with benign familial neonatal convulsions, a neonatal form of epilepsy. Enhancement of KCNQ2/Q3 potassium currents may provide an important target for antiepileptic drug development. Here, we show that meclofenamic acid (meclofenamate) and diclofenac, two related molecules previously used as anti-inflammatory drugs, act as novel KCNQ2/Q3 channel openers. Extracellular application of meclofenamate (EC(50) = 25 microM) and diclofenac (EC(50) = 2.6 microM) resulted in the activation of KCNQ2/Q3 K(+) currents, heterologously expressed in Chinese hamster ovary cells. Both openers activated KCNQ2/Q3 channels by causing a hyperpolarizing shift of the voltage activation curve (-23 and -15 mV, respectively) and by markedly slowing the deactivation kinetics. The effects of the drugs were stronger on KCNQ2 than on KCNQ3 channel alpha subunits. In contrast, they did not enhance KCNQ1 K(+) currents. Both openers increased KCNQ2/Q3 current amplitude at physiologically relevant potentials and led to hyperpolarization of the resting membrane potential. In cultured cortical neurons, meclofenamate and diclofenac enhanced the M-current and reduced evoked and spontaneous action potentials, whereas in vivo diclofenac exhibited an anticonvulsant activity (ED(50) = 43 mg/kg). These compounds potentially constitute novel drug templates for the treatment of neuronal hyperexcitability including epilepsy, migraine, or neuropathic pain.     

The anti-hyperalgesic activity of retigabine is mediated by KCNQ potassium channel activation

Retigabine (N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester) has a broad anticonvulsant spectrum and is currently in clinical development for epilepsy. The compound has an opening effect on neuronal KCNQ channels. At higher concentrations an augmentation of gamma-aminobutyric acid (GABA) induced currents as well as a weak blocking effect on sodium and calcium currents were observed. The goal of this study was to characterise the activity of retigabine in models of acute and neuropathic pain and to investigate if the potassium channel opening effect of retigabine contributes to its activity.Retigabine was tested in mice and rats in the tail flick model of acute pain and in the nerve ligation model with tight ligation of the 5th spinal nerve (L5) using both thermal and tactile stimulation. While retigabine like gabapentin had almost no analgesic effect in mice it showed some analgesic effects in rats in the tail flick model. These effects could not be antagonised with linopirdine, a selective KCNQ potassium channel blocker, indicating a different mode of action for this activity. In L5-ligated rats retigabine significantly and dose-dependently elevated the pain threshold and prolonged the withdrawal latency after tactile and thermal stimulation, respectively. In the L5 ligation model with thermal stimulation retigabine 10 mg/kg p.o. was as effective as 100 mg/kg gabapentin or 10 mg/kg tramadol. The L5 model with tactile stimulation was used to test the role of the KCNQ potassium channel opening effect of retigabine. If retigabine 10 mg/kg p.o. was administered alone it was as effective as tramadol 10 mg/kg p.o. in elevating the pain threshold. Linopirdine (1 and 3 mg/kg i.p.) had nearly no influence on neuropathic pain response. If we administered both retigabine and linopirdine the effect of retigabine was abolished or diminished depending on the dose of linopirdine used.In summary, retigabine is effective in predictive models for neuropathic pain. The activity is comparable to tramadol and is present at lower doses compared with gabapentin. Since the anti-allodynic effect can be inhibited by linopirdine we can conclude that the potassium channel opening properties of retigabine are critically involved in its ability to reduce neuropathic pain response.     

Retigabine: in partial seizures

Retigabine has anticonvulsant properties that appear to be primarily mediated by opening neuronal voltage-gated potassium channels. This action has been shown in neuronal KCNQ2/3 and KCNQ3/5 potassium channels. In addition to this unique action, retigabine also potentiates GABA-evoked currents in cortical neurons at high concentrations. When used as adjunctive therapy in patients with partial seizures, retigabine 600-1200 mg/day (200-400 mg three times daily) was associated with significant linear dose-dependent reductions in monthly seizure frequency compared with placebo in a large 16-week randomised phase II trial. Median monthly seizure frequency decreased from baseline by up to 35% among patients in the retigabine treatment arms compared with 13% in the placebo group. Retigabine 1200 mg/day was also significantly more effective than retigabine 600 mg/day. Responder rates, defined as the proportion of patients with > or = 50% reduction in seizure frequency, were significantly higher among patients in the retigabine 900 and 1200 mg/day groups than in those who received placebo. CNS-related adverse events were the most commonly reported treatment-emergent adverse events associated with retigabine in clinical trials. Across all three retigabine groups in the large phase II trial, somnolence (20.3%), dizziness (14.6%), confusion (12.3%) and speech disorder (11.3%) were the most frequent CNS-related adverse events.     

Investigations into the mechanism of action of the new anticonvulsant retigabine. Interaction with GABAergic and glutamatergic neurotransmission and with voltage gated ion channels

Retigabine (N-(2-amino-4-(4-fluorobenzylamino)phenyl) carbamic acid ethyl ester, CAS 150812-12-7, D-23129) is a novel anticonvulsant currently undergoing phase II clinical trials. The compound was shown to possess broad spectrum and potent anticonvulsant properties both in vitro and in vivo. The mechanism of action of this drug is currently not fully understood. In previous studies a potent opening effect on K+ channels and an increased release of newly synthesized gamma-aminobutyric acid (GABA) were reported. The aim of this study was to investigate the interaction of retigabine with GABA, kainate and N-methyl-D-aspartate (NMDA) induced currents as well as with voltage gated Na+ and Ca++ channels. Retigabine concentration dependently potentiated GABA induced currents in rat cortical neurones. Significant effects were only seen with concentrations of 10 mumol/l and above. The action of retigabine was not antagonised by flumazenil indicating interaction with other than benzodiazepine binding sites. In comparison with the K+ channel opening effect which can be seen at concentrations as low as 0.1 mumol/l the contribution of this mechanism to the anticonvulsant activity of retigabine may be minor. Inhibitory effects observed on voltage activated Na+ and Ca++ channels as well as on kainate induced currents were only observed at the highest concentration tested (100 mumol/l) and can be considered non specific. No significant interaction with NMDA induced currents was observed.     

The pharmacological effect of positive KCNQ (Kv7) modulators on dopamine release from striatal slices

Retigabine is an anti-epileptic drug that inhibits neuronal firing by stabilizing the membrane potential through positive modulation of voltage-dependent KCNQ potassium channels in cortical neurons and in mesencephalic dopamine (DA) neurons. The purpose of this study was to compare the effect of retigabine with other positive KCNQ modulators on the KCl-induced release of DA in rat striatal slices. Retigabine was found to inhibit KCl-dependent release of DA, and the IC(50) was estimated to be 0.7 μM. The KCNQ channel blocker XE-991 enhanced striatal DA release and completely abolished the effect of retigabine. Other compounds of the same class but with some preferences for different KCNQ subtypes such as ICA-27243, BMS-204352 and S-(1) were also tested. All three compounds produced a significant effect albeit weaker than retigabine. The potency of ICA-27243 was in the range of retigabine, and with a lower potency of BMS-204352 and S-(1). This study demonstrates that KCNQ channel openers inhibit KCl-induced DA release at relevant concentrations. The equal potency of ICA-27243 and retigabine suggests that the KCNQ2/3 isoform is likely the dominant subtype mediating this effect.     

Discovery of a retigabine derivative that inhibits KCNQ2 potassium channels

Aim:

Retigabine, an activator of KCNQ2-5 channels, is currently used to treat partial-onset seizures. The aim of this study was to explore the possibility that structure modification of retigabine could lead to novel inhibitors of KCNQ2 channels, which were valuable tools for KCNQ channel studies.

Methods:

A series of retigabine derivatives was designed and synthesized. KCNQ2 channels were expressed in CHO cells. KCNQ2 currents were recorded using whole-cell voltage clamp technique. Test compound in extracellular solution was delivered to the recorded cell using an ALA 8 Channel Solution Exchange System.

Results:

A total of 23 retigabine derivatives (HN31-HN410) were synthesized and tested electrophysiologically. Among the compounds, HN38 was the most potent inhibitor of KCNQ2 channels (its IC₅₀ value=0.10±0.05 μmol/L), and was 7-fold more potent than the classical KCNQ inhibitor XE991. Further analysis revealed that HN38 (3 μmol/L) had no detectable effect on channel activation, but accelerated deactivation at hyperpolarizing voltages. In contrast, XE991 (3 μmol/L) did not affect the kinetics of channel activation and deactivation.

Conclusion:

The retigabine derivative HN38 is a potent KCNQ2 inhibitor, which differs from XE991 in its influence on the channel kinetics. Our study provides a new strategy for the design and development of potent KCNQ2 channel inhibitors.     

Retigabine and flupirtine exert neuroprotective actions in organotypic hippocampal cultures

Retigabine and flupirtine are two structurally related molecules provided of anticonvulsant and analgesic actions. The present study has investigated the neuroprotective potential, as well as the possible underlying molecular mechanisms, exerted by retigabine and flupirtine in rat organotypic hippocampal slice cultures (OHSCs) exposed to N-methyl-D-aspartate (NMDA), oxygen and glucose deprivation followed by reoxygenation (OGD), or serum withdrawal (SW). Region-specific vulnerability of hippocampal subfields occurred with each of these injury models. Specifically, CA1 was the most susceptible region to both NMDA and OGD-induced neurodegeneration, whereas selective cell death in the dentate gyrus (DG) occurred upon OHSCs exposure to SW. The NMDA antagonist MK-801 (10-30 microM), despite blocking NMDA- and OGD-induced cell death, failed to prevent SW-induced neurodegeneration. Interestingly, retigabine (0.01-10 microM) and flupirtine (0.01-10 microM) dose-dependently prevented DG neuronal death induced by SW, with IC50 s of 0.4 microM and 0.7 microM, respectively. By contrast, retigabine and flupirtine (each at 10 microM) were less effective in counteracting NMDA- or OGD-induced toxicity in the CA1 region. Both retigabine and flupirtine (0.1-10 microM) reduced SW-induced ROS production in the DG with IC50 s of approximately 1 microM. This suggested that antioxidant actions of these compounds participated in OHSC neuroprotection during SW. By contrast, activation of KCNQ K+ channels seemed not to be involved in retigabine-induced OHSCs neuroprotection during SW, since linopirdine (20 microM) and XE-991 (10 microM), two KCNQ blockers, failed to reverse retigabine-induced neuronal rescue.     

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 [Schr?der 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.     

Antihistamine mepyramine directly inhibits KCNQ/M channel and depolarizes rat superior cervical ganglion neurons

The first-generation antihistamines are widely prescribed medications that relieve allergic reactions and urticaria by blocking the peripheral histamine H(1) receptor. Overdose of these drugs often results in serious neuronal toxic effects, including seizures, convulsions and worsening of epileptic symptoms. The KCNQ/M K(+) channel plays a crucial role in controlling neuron excitability. Here, we demonstrate that mepyramine and diphenhydramine, two structurally related first-generation antihistamines, can act as potent KCNQ/M channel blockers. Extracellular application of these drugs quickly and reversibly reduced KCNQ2/Q3 currents heterologously expressed in HEK293 cells. The current inhibition was concentration and voltage dependent. The estimated IC(50) (12.5 and 48.1 microM, respectively) is within the range of drug concentrations detected in poisoned patients (30-300 microM). Both drugs shifted the I-V curve of KCNQ2/Q3 channel to more depolarized potentials and altered channel gating properties by prolonging activation and shortening deactivation kinetics. Mepyramine also inhibited the individual homomeric KCNQ1-4 and heteromeric KCNQ3/Q5 currents. Moreover, mepyramine inhibited KCNQ2/Q3 current in an outside-out patch excised from HEK293 cells and the inhibitory effect was neither observed when it was applied intracellularly nor affected by blocking phospholipase C (PLC) activity, indicating an extracellular and direct channel blocking mechanism. Finally, in cultured rat superior cervical ganglion (SCG) neurons, mepyramine reduced the M type K(+) current in a concentration-dependent manner and led to marked membrane potential depolarization. It is likely that these effects may be involved in the adverse neuroexcitatory effects observed in patients experiencing an overdose of antihistamines.     

Retigabine strongly reduces repetitive firing in rat entorhinal cortex

Retigabine (D-23129) [N-(2-amino-4-(4-fluorobenzylamino)phenyl) carbamic acid ethyl ester] is a novel antiepileptic drug. The compound was shown to possess anticonvulsant properties both in vivo and in vitro. We investigated the effects of retigabine on neurones in the rat medial entorhinal cortex using conventional intracellular recordings in combined hippocampal-entorhinal cortex slices. Retigabine strongly reduced the number of action potentials elicited by 1 s long depolarising current injections. Both the amplitudes of monosynaptic inhibitory postsynaptic potentials/currents (IPSP/Cs) and the amplitudes of excitatory postsynaptic potentials (EPSPs) remained unaffected. The drug increased outward rectification and induced a membrane-potential hyperpolarisation in most of the tested neurones. The findings suggest that retigabine exerts its anticonvulsant effects by activation of a K(+)conductance, however it cannot be excluded from our experiments that other mechanisms may be involved in the effect of retigabine on membrane properties.     

Block of human voltage-sensitive Na+ currents in differentiated SH-SY5Y cells by lifarizine.

1. The ability of lifarizine (RS-87476) to block human voltage-sensitive Na+ channel currents was studied by use of whole cell patch clamp recording from differentiated neuroblastoma cells (SH-SY5Y). 2. The Na+ conductance in differentiated SH-SY5Y cells (24.0 +/- 2.4 nS, n = 11) was half-maximally activated by 10 ms depolarizations to -37 +/- 2 mV and was half-maximally inactivated by predepolarizing pulses of 200 ms duration to -86 +/- 3 mV (n = 11). 3. At low stimulus frequencies (0.1 to 0.33 Hz) voltage-dependent sodium currents were completely blocked, in a concentration-dependent manner, by extracellular application of either tetrodotoxin (EC50 = 4 +/- 1 nM, n = 12) or by lifarizine (EC50 = 783 +/- 67 nM, n = 9). The onset of block by lifarizine (tau = 91 +/- 14 s at 10 microM) was considerably slower than that of tetrodotoxin (tau = 16 +/- 3 s at 100 nM). 4. Lifarizine (1 microM) reduced the peak sodium conductance in each cell (from 26.4 +/- 2.0 nS to 15.1 +/- 2.7 nS, n = 4) without changing the macroscopic kinetics of sodium current activation or inactivation (V1/2 = -35 1 mV and -87 +/- 4 mV respectively, n = 4). Similarly, lifarizine (1 microM) did not affect the reversal potential of the macroscopic sodium current (+14 +/- 5 mV in control and +16 +/- 2 mV in 1 microM lifarizine; n = 4) or reactivation time-constant (tau = 14.0 +/- 4.4 ms).(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous calcium channels in human embryonic kidney (HEK293) cells.

1. We have identified endogenous calcium channel currents in HEK293 cells. Whole cell endogenous currents (ISr-HEK) were studied in single HEK293 cells with 10 mM strontium as the charge carrier by the patch clamp technique. The kinetic properties and pharmacological features of ISr-HEK were characterized and compared with the properties of a heterologously expressed chimeric L-type calcium channel construct. 2. ISr-HEK activated on depolarization to voltages positive of -40 mV. It had transient current kinetics with a time to peak of 16 +/- 1.4 ms (n = 7) and an inactivation times constant of 52 +/- 5 ms (n = 7) at a test potential of 0 mV. The voltage for half maximal activation was -19.0 +/- 1.5 mV (n = 7) and the voltage for half maximal steady-state inactivation was -39.7 +/- 2.3 mV (n = 7). 3. Block of ISr-HEK by the dihydropyridine isradipine was not stereoselective; 1 microM (+) and (-)-isradipine inhibited the current by 30 +/- 4% (n = 3) and 29 +/- 2% (n = 4) respectively. (+)-Isradipine and (-)-isradipine (10 microM) inhibited ISr-HEK by 89 +/- 4% (n = 5) and 88 +/- 8% (n = 3) respectively. The 7-bromo substituted (+/-)-isradipine (VO2605, 10 microM) which is almost inactive on L-type calcium channels also inhibited ISr-HEK (83 +/- 9%, n = 3) as was observed for 10 microM (-)-nimodipine (78 +/- 6%, n = 5). Interestingly, 10 microM (+/-)-Bay K 8644 (n = 5) had no effect on the current. ISr-HEK was only slightly inhibited by the cone snail toxins omega-CTx GVIA (1 microM, inhibition by 17 +/- 3%, n = 4) and omega-CTx MVIIC (1 microM, inhibition by 20 +/- 3%, n = 4). The funnel web spider toxin omega-Aga IVA (200 nM) inhibited ISr-HEK by 19 +/- 2%, n = 4). 4. In cells expressing ISr-HEK, maximum inward current densities of 0.24 +/- 0.03 pA/pF and 0.39 +/- 0.7 pA/ pF (at a test potential of -10 mV) were estimated in two different batches of HEK293 cells. The current density increased to 0.88 +/- 0.18 pA/pF or 1.11 +/- 0.2 pA/pF respectively, if the cells were cultured for 4 days in serum-free medium. 5. Co-expression of a chimeric L-type calcium channel construct revealed that ISr-HEK and L-type calcium channel currents could be distinguished by their different voltage-dependencies and current kinetics. The current density after heterologous expression of the L-type alpha 1 subunit chimera was estimated to be about ten times higher in serum containing medium (2.14 +/- 0.45 pA/pF) than that of ISr-HEK under the same conditions.     

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.     

Bepridil block of recombinant human cardiac IKs current shows a time-dependent unblock

Whole-cell patch-clamp techniques were employed to examine the effects of bepridil, a Ca2+ channel blocker with Vaughan Williams class III action, on a slow component of cardiac delayed rectifier K+ current (IKs), which was reconstituted in HEK293 cells by transfecting KCNQ1 and KCNE1. Micromolar bepridil inhibited tail currents carried by KCNQ1/KCNE1 channels in a concentration-dependent manner (IC50 = 5.3 +/- 0.7 microM at -40 mV from 1000 milliseconds test pulse). When the effect of the drug was examined with a short test pulse protocol (250 milliseconds), IC50 became two-fold smaller than that measured with 1000 milliseconds test pulse (2.5 +/- 0.8 microM). The envelope-of-tails protocol was used to assess how the duration of depolarizing pulse affects the drug action on the outward KCNQ1/KCNE1 channel current. The drug significantly inhibited tail currents more potently during shorter pulses (<600 milliseconds). Bepridil's block was therefore time dependent, and its binding affinity to the channel was greater in the closed state channel, as evidenced by unblocking during prolonged depolarization. These properties of channel blockade appear to underscore the mechanism of bepridil's effect on IKs current.