Elsevier

Epilepsy Research

Volume 35, Issue 2, June 1999, Pages 99-107
Epilepsy Research

Characterization of the K+ channel opening effect of the anticonvulsant retigabine in PC12 cells

https://doi.org/10.1016/S0920-1211(98)00131-4Get rights and content

Abstract

Retigabine (D-23129) is a new anticonvulsant compound which acts as a K+ channel opener in neuronal cells. The aim of the present study was to further characterize the retigabine induced K+ current. In nerve growth factor treated PC12 cells and in rat cortical neurones the application of retigabine activated a K+ current. In contrast, however, no K+ current activation was observed in untreated PC12 and in glial cells which were cultivated together with the neuronal cells. To characterise the retigabine activated K+ current, K+ channel blockers were used. The retigabine induced current was not affected by 1 and 10 mM 4-aminopyridine (4AP). Ba2+ 1 mM resulted in a reduction of 88.6±3.0% (n=5); 10 mM abolished the current. Tetraetylamonium (TEA), 1 and 10 mM, reduced the current by 23.6±3.1 and 61.6±3.7%, respectively. To investigate the current/voltage (I/V) relation of the current initiated by retigabine (10 μM), cells were clamped to a holding potential of −80 mV and a ramp stimulation protocol (−120 to +60 mV in 5 s) was applied prior to and during application of retigabine. Subtraction of the two traces yielded the current induced by retigabine. A nearly linear relationship was determined between −120 and −40 mV. At potentials positive to −40 mV, the response was variable. This was due to the additionally observed weak blocking effect of retigabine on delayed rectifier (Kdr) currents. If the ramp was applied in the presence of 10 mM 4AP to block Kdr, a nearly linear I/V-relationship was present from −120 to +60 mV. The comparison of the I/V relation and pharmacology with published K+ channel subtypes gives evidence that an unknown neuronal K+ channel subtype may be involved.

Introduction

Epilepsy is a chronic disease which is characterized by a condition of recurrent paroxysmal seizures of cerebral origin. The prevalence of epilepsy is found to be 0.5–1% of the world’s population (over 50 million people) which makes it one of the most common neurological disorders (Porter, 1993).

A large percentage (20–30%) of patients diagnosed as having epilepsy are not adequately controlled by presently available medications (Dam, 1986, Loiseau, 1988, Schmidt et al., 1992). In many cases, seizure control is limited by occurrence of severe side effects directly related to the antiepileptic drug treatment. There is therefore a need to develop novel, more effective and less toxic pharmacological agents rather than variations based on currently available but inadequate therapeutic regimes to increase control of seizures in currently drug resistant patients and to reduce side effects in patients which are only seizure free at the expense of chronic side effects.

Retigabine (D-23129) is a new anticonvulsant currently undergoing phase 2 clinical trials. The drug is chemically unrelated to currently marketed anticonvulsants. It exhibited pronounced anticonvulsant activity in a broad range of in vivo and in vitro seizure models and in genetic models of epileptic syndromes (Dailey et al., 1995, Kapetanovic and Rundfeldt, 1996, Rostock et al., 1996). In a model discussed to be the most predictive for complex partial seizures, retigabine was particularly effective in increasing the threshold (Tober et al., 1996). While in previous studies direct and indirect facilitating effects on GABAergic neurotransmission and weak blocking effects on neuronal sodium channels were reported (Kapetanovic et al., 1995, Rundfeldt et al., 1995, Kapetanovic and Rundfeldt, 1996), we recently found that retigabine acts as a potent opener of K+ channels in neuronal cells (Rundfeldt, 1997). Several lines of evidence can be found that the opening of K+ channels may be promising strategy for new anticonvulsant drug therapy (Doupnik et al., 1995, Sah, 1996, Meldrum, 1997). Opening K+ channels tends to hyperpolarize cells and decreases the effect of excitatory input. In both animal models of epilepsy and epileptic patients, mutations of K+ channels were reported as possible cause (Patil et al., 1995, Beck et al., 1996, Janigro et al., 1997, Charlier et al., 1998); an in vivo virus mediated transfection of CNS neurones with a voltage gated K+ channel resulted in a reduction in seizure susceptibility (Kirkby et al., 1996) and K+ channel openers of ATP sensitive K+ channels (given intracerebroventricularly) provide anticonvulsant action in some models (Gandolfo et al., 1989, Popoli et al., 1991). However, currently known K+ channel openers are hampered by their pronounced effects on blood pressure and other peripheral organ systems (Weston and Edwards, 1992).

The present work was aimed at characterizing the K+ channel opening effect of retigabine. Part of the work was already published in abstract form (Rundfeldt and Dost, 1997).

Section snippets

Cell culture

Experiments were performed in PC12 cells and in cultured cortical neurons.

The pheochromocytoma cell line PC12 was obtained from the European Collection of Cell Cultures (ECACC, Porton Down, UK) and cultivated as described elsewhere (Grene et al., 1982). Cells were grown at 37°C and 5% CO2 in RPMI medium containing 5% heat inactivated fetal calf serum (FCS) and 10% heat inactivated horse serum, 4 mM glutamine, penicillin (10 000 U/ml) and streptomycin (10 000 μg/ml), (Gibco Life Technologies,

Effects of nerve growth factor on current activation in PC12 cells

PC12 cells were treated with NGF to induce the neuronal like differentiation (Grene et al., 1982). If these cells were clamped at −40 mV to shift the membrane potential away from the K+ reversal potential, application of retigabine using a fast application system induced an outward K+ current which is comparable to the current induced in the cell line NG 108-15 (Rundfeldt, 1997).

As has been shown in this cell line, a rise in extracellular K+ concentration and hyperpolarization, respectively,

Selectivity of the K+ channel opening effect for neuronal cells

Some K+ channel openers were reported to exert anticonvulsant activity (Gandolfo et al., 1989, Popoli et al., 1991). However, these openers of ATP sensitive K+ channels are hampered by their pronounced effects on blood pressure and other peripheral organ systems (Weston and Edwards, 1992). The results obtained in PC12 and neuronal cells indicate, that retigabine selectively activates a K+ channel in neurones. While the data are not sufficient to exclude the possibility that retigabine may also

References (48)

  • A. Wei et al.

    Eight potassium channel families revealed by the C. elegans genome project

    Neuropharmacology

    (1996)
  • A.H. Weston et al.

    Recent progress in potassium channel opener pharmacology

    Biochem. Pharmacol.

    (1992)
  • Anonymous, 1997. D-23129, Retigabine, Drugs Fut. 22,...
  • H. Beck et al.

    Properties of a delayed rectifier potassium current in dentate granule cells isolated from the hippocampus of patients with chronic temporal lobe epilepsy

    Epilepsia

    (1996)
  • K.G. Chandy et al.

    Voltage-gated potassium channel genes

  • C. Charlier et al.

    A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family

    Nat. Genet.

    (1998)
  • L. Conforti et al.

    Selective inhibition of a slow-inactivating voltage-dependent K+ channel in rat PC12 cells by hypoxia

    J. Physiol. Lond.

    (1997)
  • M. Dam

    Intractable epilepsy: introduction

  • S.S. Garber et al.

    Regulation of ionic currents in pheochromocytoma cells by nerve growth factor and dexamethasone

    J. Neurosci.

    (1989)
  • S.A. Goldstein et al.

    ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae

    Proc. Natl. Acad. Sci. USA

    (1996)
  • L.A. Grene et al.

    PC12 pheochromocytoma cultures in neurobiological reseach

    Adv. Cell Neurobiol.

    (1982)
  • O.P. Hamill et al.

    Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches

    Pflügers Arch.

    (1981)
  • Hille, B., 1993. Ionic Channels of Excitable Membranes, 2nd ed. Sinauer, Sunderland,...
  • Q. Hu et al.

    Characterization of an inward-rectifying potassium current in NG108-15 neuroblastoma x glioma cells

    Pflügers Arch.

    (1997)
  • Cited by (46)

    • A medium-throughput functional assay of KCNQ2 potassium channels using rubidium efflux and atomic absorption spectrometry

      2003, Analytical Biochemistry
      Citation Excerpt :

      Thus, the known pharmacological profile of KCNQ2 defined by electrophysiological studies is faithfully reproduced in the Rb+ efflux assay. Retigabine is an anticonvulsant compound, capable of increasing potassium conductance in numerous cell types of neuronal origin [26,27]. It has been shown to enhance currents through heterologously expressed monomeric and heteromeric channels of the KCNQ family including KCNQ2 and KCNQ2/3 [28–31].

    • Retigabine

      2007, Neurotherapeutics
      Citation Excerpt :

      Because neuronal potassium channels, including Kv7 channels, are discussed as potential targets for neuroprotection, retigabine was evaluated in three models of neurodegeneration. It was found to reduce infarct size in a model of permanent medial cerebral artery occlusion; it reversed functional deficits induced by unilateral carotid artery occlusion24; and in a model of kainic acid-induced status epilepticus, retigabine partially prevented the severe neurodegeneration resulting from the prolonged status.31 Kv7.3 and Kv7.5 subunits are expressed in bladder and in intestinal smooth muscle.

    View all citing articles on Scopus
    View full text