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.

KCNQ2/KCNQ3 channels are the molecular correlates of the neuronal M-channels, which play a major role in the control of neuronal excitability. Notably, they differ from homomeric KCNQ2 channels in their distribution pattern within neurons, with unique expression of KCNQ2 in axons and nerve terminals. Here, combined reciprocal coimmunoprecipitation and two-electrode voltage clamp analyses in Xenopus oocytes revealed a strong association of syntaxin 1A, a major component of the exocytotic SNARE complex, with KCNQ2 homomeric channels resulting in a ∼2-fold reduction in macroscopic conductance and ∼2-fold slower activation kinetics. Remarkably, the interaction of KCNQ2/Q3 heteromeric channels with syntaxin 1A was significantly weaker and KCNQ3 homomeric channels were practically resistant to syntaxin 1A. Analysis of different KCNQ2 and KCNQ3 chimeras and deletion mutants combined with in-vitro binding analysis pinpointed a crucial C-terminal syntaxin 1A-association domain in KCNQ2. Pull-down and coimmunoprecipitation analyses in hippocampal and cortical synaptosomes demonstrated a physical interaction of brain KCNQ2 with syntaxin 1A, and confocal immunofluorescence microscopy showed high colocalization of KCNQ2 and syntaxin 1A at presynaptic varicosities. The selective interaction of syntaxin 1A with KCNQ2, combined with a numerical simulation of syntaxin 1A's impact in a firing-neuron model, suggest that syntaxin 1A's interaction is targeted at regulating KCNQ2 channels to fine-tune presynaptic transmitter release, without interfering with the function of KCNQ2/3 channels in neuronal firing frequency adaptation.

Cyclooxygenase (COX) enzymes are molecular targets of nonsteroidal anti-inflammatory drugs (NSAIDs), the most used medication worldwide. However, the COX enzymes are not the sole molecular targets of NSAIDs. Recently, we showed that two NSAIDs, diclofenac and meclofenamate, also act as openers of Kv7.2/3 K+ channels underlying the neuronal M-current. Here we designed new derivatives of diphenylamine carboxylate to dissociate the M-channel opener property from COX inhibition. The carboxylate moiety was derivatized into amides or esters and linked to various alkyl and ether chains. Powerful M-channel openers were generated, provided that the diphenylamine moiety and a terminal hydroxyl group are preserved. In transfected CHO cells, they activated recombinant Kv7.2/3 K+ channels, causing a hyperpolarizing shift of current activation as measured by whole-cell patch-clamp recording. In sensory dorsal root ganglion and hippocampal neurons, the openers hyperpolarized the membrane potential and robustly depressed evoked spike discharges. They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents. In vivo, the openers exhibited anti-convulsant activity, as measured in mice by the maximal electroshock seizure model. Conversion of the carboxylate function into amide abolished COX inhibition but preserved M-channel modulation. Remarkably, the very same template let us generating potent M-channel blockers. Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition. They also provide a structural framework for designing novel M-channel modulators, including openers and blockers.

The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and β subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds. By electrophysiological characterization of these mutants, we found heteromeric assemblies with two equivalent Glu-binding sites at β/α intersubunit interfaces, where the GluClβ and GluClα subunits, respectively, contribute the “principal” and “complementary” components of the putative Glu-binding pockets. We identified a mutation in the IVM-binding site (far away from the Glu-binding sites), which significantly increased the sensitivity of the heteromeric mutant receptor to both Glu and IVM, and improved the receptor subunits’ cooperativity. We further characterized this heteromeric GluClR mutant as a receptor having a third Glu-binding site at an α/α intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three α and two β subunits arranged in a counterclockwise β-α-β-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces.

Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that bind neurotransmitters to open an intrinsic transmembrane ion channel pore. The recent crystal structure of a prokaryotic pLGIC from the cyanobacterium Gloeobacter violaceus (GLIC) revealed that it naturally lacks an N-terminal extracellular ␣ helix and an intracellular domain that are typical of eukaryotic pLGICs. GLIC does not respond to neurotransmitters acting at eukaryotic pLGICs but is activated by protons. To determine whether the structural differences account for functional differences, we used a eukaryotic chimeric acetylcholineglutamate pLGIC that was modified to carry deletions corresponding to the sequences missing in the prokaryotic homolog GLIC. Deletions made in the N-terminal extracellular ␣ helix did not prevent the expression of receptor subunits and the appearance of receptor assemblies on the cell surface but abolished the capability of the receptor to bind ␣-bungarotoxin (a competitive antagonist) and to respond to the neurotransmitter. Other truncated chimeric receptors that lacked the intracellular domain did bind ligands; displayed robust acetylcholine-elicited responses; and shared with the full-length chimeric receptor similar anionic selectivity, effective open pore diameter, and unitary conductance. We suggest that the integrity of the N-terminal ␣ helix is crucial for ligand accommodation because it stabilizes the intersubunit interfaces adjacent to the neurotransmitter-binding pocket(s). We also conclude that the intracellular domain of the chimeric acetylcholine-glutamate receptor does not modulate the ion channel conductance and is not involved in positioning of the pore-lining helices in the conformation necessary for coordinating a Cl ؊ ion within the intracellular vestibule of the ion channel pore.Cys-loop receptors constitute a superfamily of cell surface oligomers that bind neurotransmitters such as acetylcholine (ACh), 3 serotonin (5-hydroxytryptamine (5HT)), ␥-aminobutyric acid (GABA), glycine (Gly), glutamate (Glu), and histamine to open an intrinsic transmembrane ion channel (for reviews, see Refs. 1-8). Hence, Cys-loop receptors mediate the rapid flow of ions such as Na ϩ , K ϩ , Ca 2ϩ , and Cl Ϫ across the cell membrane down their electrochemical gradients to alter the membrane potential or enable Ca 2ϩ influxes and signaling. Cys-loop receptors are known also as pentameric ligand-gated ion channels (pLGICs). The subunits of pLGICs are radially aligned around an axis of 5-fold symmetry, which is the axis of the ion permeation pathway. Each subunit traverses the membrane four times. Upon receptor assembly, the N-terminal extracellular segment forms a ligand-binding domain (LigBD) having neurotransmitterbinding pockets at intersubunit interfaces. The ion channel domain is formed by 20 transmembrane helices, M1-M4 of each subunit (see Fig. 1, A and B, numbered 1-4). All eukaryotic pLGICs have a long intracellular sequence that connects M3 with M4. This sequence (the so-called M3-M4 loop or linker) f...

ion that effectively acts as a "foot in the door." We infer that, upon deactivation, the cytoplasmic side of the pore of the AChserotonin receptor chimera constricts to close the channel. Eukaryotic pLGICs3 (also called Cys-loop receptors) constitute a superfamily of transmembrane receptors located at the cell surface of excitable neuronal and muscle cells. There, they mediate rapid transport of ions, such as Nadown their electrochemical gradients to alter the membrane potential or to enable a rise in intracellular Ca 2ϩ . The five subunits of pLGICs are radially aligned around the axis of ion permeation pathway to form a channel activatable by neurotransmitters such as ACh, serotonin, ␥-aminobutyric acid, glycine, glutamate, or histamine that bind to an extracellular ligandbinding domain (LigBD) (Fig. 1A) (1-7).Although it is well accepted that loops located at the LigBDchannel interface mediate movements of the pore-lining helices (M2s) (8 -16), the specific M2 motions that open and close pLGICs are widely debated. Cryo-electron microscopy images of open and closed conformations of the nicotinic ACh receptor (nAChR) from Torpedo marmorata gave rise to the concept of gating motions, in which rotations along the longitudinal axis of the M2 segments open or close a mid-pore hydrophobic barrier (17, 18). State-dependent accessibilities of cysteines, histidines, or lysines substituted along the M2s to methanethiosulfonates, Zn 2ϩ or protons, have excluded channel gating via rotations (19 -23). It was further suggested that rigid body tilting (21) or small scale dilation (23) motions of the M2s gate the pore of an ACh-serotonin receptor chimera or a muscle nAChR, respectively. Several computational simulations suggested that the opening and closing of pLGICs depend on "global quaternary twist" motions (24, 25), rotations (26, 27), or rotations combined with either tilting (28) or bending vibrations (29) of the M2s. In contrast to these computational simulations, recent x-ray crystal structures of two different prokaryotic pLGICs, one displaying a closed pore conformation (30) and another with a potentially open pore conformation (31, 32), suggest that the pore-lining helices rigidly tilt to gate the channel. Yet, a major difference between the tilting gating motions suggested for the prokaryotic channels and those suggested for a eukaryotic pLGIC (21) emerges. In the case of the prokaryotic channels, the putative gating process involves the opening of a barrier to ions at the extracellular side of the pore (30 -32), whereas functional studies in eukaryotic pLGICs indicate that activation involves opening of a barrier located at the cytoplasmic side of the pore (19 -21, 33).Here, we used eukaryotic pLGICs that have an engineered capacity to coordinate a metal ion at the cytoplasmic side of their pore (21). In a previous study the accessibility of the engineered histidines to Zn 2ϩ ions was probed before or after activation (21), but in this study we detect Zn 2ϩ ion trapping inside the pore upon agonist dissociation....

Ivermectin (IVM) is a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. By activating invertebrate pentameric glutamate-gated chloride channels (GluCl receptors; GluClRs), IVM induces sustained chloride influx and long-lasting membrane hyperpolarization that inhibit neural excitation in nematodes. Although IVM activates the C. elegans heteromeric GluClα/β receptor, it cannot activate a homomeric receptor composed of the C. elegans GluClβ subunits. To understand this incapability, we generated a homopentameric α7-GluClβ chimeric receptor that consists of an extracellular ligand-binding domain of an α7 nicotinic acetylcholine receptor known to be potentiated by IVM, and a chloride-selective channel domain assembled from GluClβ subunits. Application of IVM prior to acetylcholine inhibited the responses of the chimeric α7-GluClβR. Adding IVM to activated α7-GluClβRs, considerably accelerated the decline of ACh-elicited currents and stabilized the receptors in a non-conducting state. Determination of IVM association and dissociation rate constants and recovery experiments suggest that, following initial IVM binding to open α7-GluClβRs, the drug induces a conformational change and locks the ion channel in a closed state for a long duration. We further found that IVM also inhibits the activation by glutamate of a homomeric receptor assembled from the C. elegans full-length GluClβ subunits.

The neuromuscular acetylcholine receptor (AChR) alternatively switches between an inactive (closed) and active (open) conformation. during the gating isomerization (R4R*) process. The 'gate' region is formed by the equatorial residues (9'-16'). We and others have speculated that channel gating involves