KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are voltage-gated K ϩ 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 ϩ efflux (EC 50 ϭ 0.2 M) and whole-cell currents in Chinese hamster ovary cells stably expressing heteromultimeric KCNQ2/Q3 channels (EC 50 ϭ 0.4 M). Activation of KCNQ2/Q3 channels was associated with a hyperpolarizing shift of the voltage dependence of channel activation (V 1/2 shift of Ϫ19 mV at 10 M). 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 voltagedependent sodium channels and GABA-gated chloride channels. ICA-27243 (1-10 M) 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 ϭ 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.
Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxinsensitive (TTX-S) resurgent currents have been described in many different neuron populations, including cerebellar and dorsal root ganglia (DRG) neurons. In most cases, sodium channel Nav1.6 is the major contributor to these TTX-S resurgent currents. Here we report a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, but not all. As with TTX-S resurgent currents, they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the 4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages, and are sensitive to the Nav1.8 blocker A803467. Moreover, coimmunoprecipitation experiments from rat DRG lysates indicate the endogenous sodium channel 4 subunits associate with Nav1.8 in DRG neurons. These results suggest that slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. We also show that both TTX-S and TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators. Furthermore, the 4 peptide increased excitability of small DRG neurons in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain.
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