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 ␣ 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 ϭ 25 M) and diclofenac (EC 50 ϭ 2.6 M) resulted in the activation of KCNQ2/Q3 K ϩ currents, heterologously expressed in Chinese hamster ovary cells. Both openers activated KCNQ2/Q3 channels by causing a hyperpolarizing shift of the voltage activation curve (Ϫ23 and Ϫ15 mV, respectively) and by markedly slowing the deactivation kinetics. The effects of the drugs were stronger on KCNQ2 than on KCNQ3 channel ␣ subunits. In contrast, they did not enhance KCNQ1 K ϩ 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 ϭ 43 mg/kg). These compounds potentially constitute novel drug templates for the treatment of neuronal hyperexcitability including epilepsy, migraine, or neuropathic pain.Voltage-dependent K ϩ (Kv) channels play a major role in brain excitability through the regulation of action potential generation and propagation, the tuning of neuronal firing patterns, or the modulation of neurotransmitter release. The M-type K ϩ channel generates a subthreshold, voltage-gated K ϩ current (M-current) that plays an important role in controlling neuronal excitability. Brown and Adams (1980) first identified the M-current in frog sympathetic neurons as a slowly activating, noninactivating, voltage-sensitive K ϩ current, which was inhibited by muscarinic acetylcholine receptor stimulation (Brown and Adams, 1980). M-currents were also characterized in hippocampal and cortical neurons (Brown, 1988;Marrion, 1997;Cooper and Jan, 2003). Modulation of the M-current has profound effects on brain excitability because this noninactivating K ϩ channel exhibits significant conductance in the voltage range of action potential initiation. The low-threshold gating and the slow activation and deactivation of the M-current act as a brake for repetitive firing and neuronal excitability (

ion channel ͉ membrane protein ͉ structure ͉ acetylcholine

The M-type K ϩ current (M-current), encoded by Kv7.2/3 (KCNQ2/3) K ϩ channels, plays a critical role in regulating neuronal excitability because it counteracts subthreshold depolarizations. Here we have characterized the functions of pre-and postsynaptic M-channels using a novel Kv7.2/3 channel opener, NH6, which we synthesized as a new derivative of N-phenylanthranilic acid. NH6 exhibits a good selectivity as it does not affect Kv7.1 and I KS K ϩ currents as well as NR1/NR2B, AMPA, and GABA A receptor-mediated currents. Superfusion of NH6 increased recombinant Kv7.2/3 current amplitude (EC 50 ϭ 18 M) by causing a hyperpolarizing shift of the voltage activation curve and by markedly slowing the deactivation kinetics. Activation of native M-currents by NH6 robustly reduced the number of evoked and spontaneous action potentials in cultured cortical, hippocampal and dorsal root ganglion neurons. In hippocampal slices, NH6 decreased somatically evoked spike afterdepolarization of CA1 pyramidal neurons and induced regular firing in bursting neurons. Activation of M-channels by NH6, potently reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents. Activation of M-channels also decreased the frequency of miniature excitatory (mEPSC) and inhibitory (mIPSC) postsynaptic currents without affecting their amplitude and waveform, thus suggesting that Mchannels presynaptically inhibit glutamate and GABA release. Our results suggest a role of presynaptic M-channels in the release of glutamate and GABA. They also indicate that M-channels act pre-and postsynaptically to dampen neuronal excitability.

Inactivation is an inherent property of most voltage-gated K(+) channels. While fast N-type inactivation has been analyzed in biophysical and structural details, the mechanisms underlying slow inactivation are yet poorly understood. Here, we characterized a slow inactivation mechanism in various KCNQ1 pore mutants, including L273F, which hinders entry of external Ba(2+) to its deep site in the pore and traps it by slowing its egress. Kinetic studies, molecular modeling, and dynamics simulations suggest that this slow inactivation involves conformational changes that converge to the outer carbonyl ring of the selectivity filter, where the backbone becomes less flexible. This mechanism involves acceleration of inactivation kinetics and enhancement of Ba(2+) trapping at elevated external K(+) concentrations. Hence, KCNQ1 slow inactivation considerably differs from C-type inactivation where vacation of K(+) from the filter was invoked. We suggest that trapping of K(+) at s(1) due to filter rigidity and hindrance of the dehydration-resolvation transition underlie the slow inactivation of KCNQ1 pore mutants.

To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluCl␤ receptor of Caenorhabditis elegans and the vertebrate cationic ␣7-acetylcholine receptor and monitored anionic/cationic permeability ratios. Complete conversion of the ion charge selectivity in a set of receptor microchimeras indicates that the selectivity filter of the GluCl␤ receptor is created by a sequence connecting the first with the second transmembrane segments. A single substitution of a negatively charged residue within this sequence converted the selectivity of the GluCl␤ receptor's pore from anionic to cationic. Unexpectedly, elimination of the charge of each basic residue of the selectivity filter, one at a time or concomitantly, moderately reduced the P Cl /P Na ratios, but the GluCl␤ receptor's mutants retained high capacity to select Cl The invertebrate GluCl 2 receptor channels are pentameric transmembrane receptors belonging to a wide superfamily of Cys-loop receptors activated by various neurotransmitters such as acetylcholine (ACh), serotonin (5-hydroxtryptamine, 5HT), ␥-aminobutyric acid (GABA), Gly, Glu, or histamine (Fig. 1A) (1-8). This superfamily consists of cationic channels permeable to Na ϩ , K ϩ , and, in many subunit combinations, to Ca 2ϩ ions, as well as of anionic channels selective to Cl Ϫ ions (reviewed by Keramidas et al. (9)). Structural similarities shared by Cys-loop receptors enabled swapping of pore sequences between cationic and anionic channels so as to assess the involvement of specific amino acids in ion charge selectivity. It was previously shown that concomitant replacement of the residues at positions Ϫ2Ј, Ϫ1Ј, and 13Ј ( Fig. 1, B and C) of cationic receptors by the residues found at the homologous positions of anionic receptors, and vice versa, lead to conversion of ion charge selectivity (10 -13).Further mutagenesis studies led to the recognition that the different capacities of cationic versus anionic Cys-loop receptors to distinguish between the charge of ions rely on the differences in the amino acid composition at positions Ϫ1Ј and Ϫ2Ј (Fig. 1C) (13-16). The conserved pore-facing Glu residue at position Ϫ1Ј of cationic Cys-loop receptors was further inferred to form, around the axis of ion conduction, a negatively charged ring that plays the key role in cationic selectivity by interacting with cations and repulsing anions (12,13,15,16). Conversely, a conserved arginine at position 0Ј of anionic Cys-loop receptors was inferred to interact with anions and repulse cations. A basic residue at position 0Ј is also typical of all cationic Cys-loop receptors (Fig. 1C and the ligand-gated ion channels data base), but it was suggested that local conformational differences in the M1-M2 connecting segment (M1-M2 loop) orient this basic residue to the pore lumen only in anionic Cys-loop receptors (9, 15). These local conformational differences have been attributed to a proline residue, which is present exclusively at pos...

The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ channels are poorly understood. Here we used external barium to investigate the permeation characteristics of homomeric KCNQ1 channels. We assessed the Ba2+ binding kinetics and the concentration and voltage dependence of Ba2+ steady-state block. Our results indicate that extracellular Ba2+ exerts a series of complex effects, including a voltage-dependent pore blockade as well as unique gating alterations. External barium interacts with the permeation pathway of KCNQ1 at two discrete and nonsequential sites. (a) A slow deep Ba2+ site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba2+ site that barely contributes to channel block and mostly affects channel gating by shifting rightward the voltage dependence of activation, slowing activation, speeding up deactivation kinetics, and inhibiting channel inactivation. A model of voltage-dependent block cannot predict the complex impact of Ba2+ on channel gating in low external K+ solutions. Ba2+ binding to this superficial site likely modifies the gating transitions states of KCNQ1. Both sites appear to reside in the permeation pathway as high external K+ attenuates Ba2+ inhibition of channel conductance and abolishes its impact on channel gating. Our data suggest that despite the high degree of homology of the pore region among the various K+ channels, KCNQ1 channels display significant structural and functional uniqueness.