M-channel Opener Research Articles

Acute Treatment with the M-Channel (Kv7, KCNQ) Opener Retigabine Reduces the Long-Term Effects of Repetitive Blast Traumatic Brain Injuries.

We investigated whether pharmacological increase of "M-type" (KCNQ, Kv7) K + channel currents by the M-channel opener, retigabine (RTG), acutely after repetitive traumatic brain injuries (rTBIs), prevents or reduces their long-term detrimental effects. rTBIs were studied using a blast shock air wave mouse model. Animals were monitored by video and electroencephalogram (EEG) records for nine months after the last injury to assess the occurrence of post-traumatic seizures (PTS), post-traumatic epilepsy (PTE), sleep-wake cycle architecture alterations, and the power of the EEG signals. We evaluated the development of long-term changes in the brain associated with various neurodegenerative diseases in mice by examining transactive response DNA-binding protein 43 (TDP-43) expression and nerve fiber damage ~ 2years after the rTBIs. We observed acute RTG treatment to reduce the duration of PTS and impair the development of PTE. Acute RTG treatment also prevented post-injury hypersomnia, nerve fiber damage, and cortical TDP-43 accumulation and translocation from the nucleusto the cytoplasm. Mice that developed PTE displayed impaired rapid eye movement (REM) sleep, and there were significant correlations between seizure duration and time spent in the different stages of the sleep-wake cycle. We observed acute RTG treatment to impair injury-induced reduction of age-related increase in gamma frequency power of theEGG, which has been suggested to be necessary for a healthy aged brain. The data show that RTG, administered acutely post-TBI, is a promising, novel therapeutic option to blunt/prevent several long-term effects of rTBIs. Furthermore, our results show a direct relationship between sleep architecture and PTE.

Excitatory and inhibitory hippocampal neurons differ in their homeostatic adaptation to chronic M-channel modulation.

A large body of studies has investigated bidirectional homeostatic plasticity both in vitro and in vivo using numerous pharmacological manipulations of activity or behavioral paradigms. However, these experiments rarely explored in the same cellular system the bidirectionality of the plasticity and simultaneously on excitatory and inhibitory neurons. M-channels are voltage-gated potassium channels that play a crucial role in regulating neuronal excitability and plasticity. In cultured hippocampal excitatory neurons, we previously showed that chronic exposure to the M-channel blocker XE991 leads to adaptative compensations, thereby triggering at different timescales intrinsic and synaptic homeostatic plasticity. This plastic adaptation barely occurs in hippocampal inhibitory neurons. In this study, we examined whether this homeostatic plasticity induced by M-channel inhibition was bidirectional by investigating the acute and chronic effects of the M-channel opener retigabine on hippocampal neuronal excitability. Acute retigabine exposure decreased excitability in both excitatory and inhibitory neurons. Chronic retigabine treatment triggered in excitatory neurons homeostatic adaptation of the threshold current and spontaneous firing rate at a time scale of 4–24 h. These plastic changes were accompanied by a substantial decrease in the M-current density and by a small, though significant, proximal relocation of Kv7.3-FGF14 segment along the axon initial segment. Thus, bidirectional homeostatic changes were observed in excitatory neurons though not symmetric in kinetics and mechanisms. Contrastingly, in inhibitory neurons, the compensatory changes in intrinsic excitability barely occurred after 48 h, while no homeostatic normalization of the spontaneous firing rate was observed. Our results indicate that excitatory and inhibitory hippocampal neurons differ in their adaptation to chronic alterations in neuronal excitability induced by M-channel bidirectional modulation.

Downregulation of KCNQ channels in the CeA contributes to elevated sympathetic outflow in hypertension

Elevation of sympathetic outflow is associated with many hypertensive conditions. The central nucleus of amygdala (CeA) is crucial in the regulation of emotion, pain, and cardiovascular functions. The M‐channel is a non‐inactivating voltage‐dependent K+ channel stabilizing the membrane potential and mainly consists of heterotetrameric Kv7.2 and Kv7.3 subunits. However, the role of M channels in the CeA in heightened sympathetic vasomotor tone in hypertension remain unknown. Microinjecting of the specific M‐channel blocker XE‐991 into the CeA significantly increased arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA) but did not alter heart rate (HR) in anesthetized Wistar Kyoto rats (WKY) rats. In spontaneously hypertensive rats (SHRs), microinjection of the same dose of XE‐991 into the CeA had no effect on ABP, RSNA, or HR in SHRs. On the other hand, microinjection of the M‐channel opener QO‐58 into the CeA decreased ABP and RSNA in WKY rats but failed to alter these cardiovascular variables in SHRs. The protein and mRNA levels of Kv7.2 and Kv7.3 in the CeA were significantly lower in SHRs than in WKY rats. Lowering ABP via celiac ganglionectomy in SHRs did not altered the expression levels of Kv7.2 and Kv7.3 in the CeA. In a brain slice preparation, the basal firing activity of labeled CeA‐RVLM projection neurons was significantly greater in SHRs than in WKY rats. Blocking M‐currents with XE‐991 increased both spontaneous and current injection‐evoked firing rate of CeA‐RVLM projection neurons in WKY rats but had no such effect in SHRs. Also, the M‐currents triggered by a membrane depolarization in CeA‐RVLM neurons were significantly smaller in SHRs than in WKY rats. These data suggest that M‐channel activity is impaired and leads to hyperactivity of CeA‐RVLM projection neurons, which contributes to elevated sympathetic vasomotor activity in hypertension.Support or Funding Information SupportThis study was supported by NIH grant HL139523 and HL142133.

Functional responses of the hippocampus to hyperexcitability depend on directed, neuron-specific KCNQ2 K+ channel plasticity.

M-type (KCNQ2/3) K+ channels play dominant roles in regulation of active and passive neuronal discharge properties such as resting membrane potential, spike-frequency adaptation, and hyper-excitatory states. However, plasticity of M-channel expression and function in nongenetic forms of epileptogenesis are still not well understood. Using transgenic mice with an EGFP reporter to detect expression maps of KCNQ2 mRNA, we assayed hyperexcitability-induced alterations in KCNQ2 transcription across subregions of the hippocampus. Pilocarpine and pentylenetetrazol chemoconvulsant models of seizure induction were used, and brain tissue examined 48 hr later. We observed increases in KCNQ2 mRNA in CA1 and CA3 pyramidal neurons after chemoconvulsant-induced hyperexcitability at 48 hr, but no significant change was observed in dentate gyrus (DG) granule cells. Using chromogenic in situ hybridization assays, changes to KCNQ3 transcription were not detected after hyper-excitation challenge, but the results for KCNQ2 paralleled those using the KCNQ2-mRNA reporter mice. In mice 7 days after pilocarpine challenge, levels of KCNQ2 mRNA were similar in all regions to those from control mice. In brain-slice electrophysiology recordings, CA1 pyramidal neurons demonstrated increased M-current amplitudes 48 hr after hyperexcitability; however, there were no significant changes to DG granule cell M-current amplitude. Traumatic brain injury induced significantly greater KCNQ2 expression in the hippocampal hemisphere that was ipsilateral to the trauma. In vivo, after a secondary challenge with subconvulsant dose of pentylenetetrazole, control mice were susceptible to tonic-clonic seizures, whereas mice administered the M-channel opener retigabine were protected from such seizures. This study demonstrates that increased excitatory activity promotes KCNQ2 upregulation in the hippocampus in a cell-type specific manner. Such novel ion channel expressional plasticity may serve as a compensatory mechanism after a hyperexcitable event, at least in the short term. The upregulation described could be potentially leveraged in anticonvulsant enhancement of KCNQ2 channels as therapeutic target for preventing onset of epileptogenic seizures.

KCNQ/M-channels regulate mouse vagal bronchopulmonary C-fiber excitability and cough sensitivity.

Increased airway vagal sensory C-fiber activity contributes to the symptoms of inflammatory airway diseases. The KCNQ/Kv7/M-channel is a well-known determinant of neuronal excitability, yet whether it regulates the activity of vagal bronchopulmonary C-fibers and airway reflex sensitivity remains unknown. Here we addressed this issue using single-cell RT-PCR, patch clamp technique, extracellular recording of single vagal nerve fibers innervating the mouse lungs, and telemetric recording of cough in free-moving mice. Single-cell mRNA analysis and biophysical properties of M-current (IM) suggest that KCNQ3/Kv7.3 is the major M-channel subunit in mouse nodose neurons. The M-channel opener retigabine negatively shifted the voltage-dependent activation of IM, leading to membrane hyperpolarization, increased rheobase, and suppression of both evoked and spontaneous action potential (AP) firing in nodose neurons in an M-channel inhibitor XE991-sensitive manner. Retigabine also markedly suppressed the α,β-methylene ATP-induced AP firing in nodose C-fiber terminals innervating the mouse lungs, and coughing evoked by irritant gases in awake mice. In conclusion, KCNQ/M-channels play a role in regulating the excitability of vagal airway C-fibers at both the cell soma and nerve terminals. Drugs that open M-channels in airway sensory afferents may relieve the sufferings associated with pulmonary inflammatory diseases such as chronic coughing.

Ethanol Withdrawal Drives Anxiety-Related Behaviors by Reducing M-type Potassium Channel Activity in the Lateral Habenula.

Alcohol use disorders (AUDs) and anxiety disorders (ADs) are often seen concurrently, but their underlying cellular basis is unclear. For unclear reasons, the lateral habenula (LHb), a key brain region involved in the pathophysiology of ADs, becomes hyperactive after ethanol withdrawal. M-type K+ channels (M-channels), important regulators of neuronal activity, are abundant in the LHb, yet little is known about their role in AUDs and associated ADs. We report here that in rats at 24 h withdrawal from systemic ethanol administration (either by intraperitoneal injection, 2 g/kg, twice/day, for 7 days; or intermittent drinking 20% ethanol in a two-bottle free choice protocol for 8 weeks), the basal firing rate and the excitability of LHb neurons in brain slices was higher, whereas the amplitude of medium afterhyperpolarization and M-type K+ currents were smaller, when compared to ethanol naive rats. Concordantly, M-channel blocker (XE991)-induced increase in the spontaneous firing rate in LHb neurons was smaller. The protein expression of M-channel subunits, KCNQ2/3 in the LHb was also smaller. Moreover, anxiety levels (tested in open field, marble burying, and elevated plus maze) were higher, which were alleviated by LHb inhibition either chemogenetically or by local infusion of the M-channel opener, retigabine. Intra-LHb infusion of retigabine also reduced ethanol consumption and preference. These findings reveal an important role of LHb M-channels in the expression of AUDs and ADs, and suggest that the M-channels could be a potential therapeutic target for alcoholics.

Electrophysiological characterization of the M-current in rat hypoglossal motoneurons

The M-current (IM) is a voltage-dependent, persistent K+ current so termed because it is strongly inhibited by the cholinergic agonist muscarine. The IM main function is to limit neuronal excitability by contrasting action potential firing. Although motoneurons are sensitive to acetylcholine, the role of IM in modulating their excitability is still controversial. The aim of the present report was to examine the presence of IM in hypoglossal motoneurons (HMs) and its role in the modulation of firing properties using an in vitro model of rat brainstem slice. For this purpose, we employed the whole-cell patch-clamp technique to record HM responses upon stimulation with either a standard IM deactivation voltage protocol or depolarizing current steps. Voltage commands from depolarized potential induced inward relaxations with the common characteristics of IM, comprising inhibition by either muscarine (10μM) or the selective IM inhibitor linopirdine (30μM). IM was pharmacologically distinguished from the hyperpolarization-activated inward-rectifying current and, within the −20 to −50mV range, deactivated with >100-ms time constant. Current-clamp experiments demonstrated that IM strongly regulated HM action potential firing, since both muscarine and linopirdine increased spike frequency whereas the M-channel opener retigabine (20μM) reduced it. Conversely, IM seemed uninvolved in the generation of the medium afterhyperpolarizing potential. Our results suggest that HMs possess IM, whose pharmacological modulation is an important tool to up- or down-regulate excitability, to be explored in experimental models of neurodegeneration.

M-current preservation contributes to anticonvulsant effects of valproic acid.

Valproic acid (VPA) has been widely used for decades to treat epilepsy; however, its mechanism of action remains poorly understood. Here, we report that the anticonvulsant effects of nonacute VPA treatment involve preservation of the M-current, a low-threshold noninactivating potassium current, during seizures. In a wide variety of neurons, activation of Gq-coupled receptors, such as the m1 muscarinic acetylcholine receptor, suppresses the M-current and induces hyperexcitability. We demonstrated that VPA treatment disrupts muscarinic suppression of the M-current and prevents resultant agonist-induced neuronal hyperexcitability. We also determined that VPA treatment interferes with M-channel signaling by inhibiting palmitoylation of a signaling scaffold protein, AKAP79/150, in cultured neurons. In a kainate-induced murine seizure model, administration of a dose of an M-channel inhibitor that did not affect kainate-induced seizure transiently eliminated the anticonvulsant effects of VPA. Retigabine, an M-channel opener that does not open receptor-suppressed M-channels, provided anticonvulsant effects only when administered prior to seizure induction in control animals. In contrast, treatment of VPA-treated mice with retigabine induced anticonvulsant effects even when administered after seizure induction. Together, these results suggest that receptor-induced M-current suppression plays a role in the pathophysiology of seizures and that preservation of the M-current during seizures has potential as an effective therapeutic strategy.

Triple Cysteine Module within M-Type K+Channels Mediates Reciprocal Channel Modulation by Nitric Oxide and Reactive Oxygen Species

We have identified a new signaling role for nitric oxide (NO) in neurons from the trigeminal ganglia (TG). We show that in rat sensory neurons from the TG the NO donor, S-nitroso-N-acetyl-dl-penicillamine, inhibited M-current. This inhibitory effect was blocked by NO scavenging, while inhibition of NO synthases increased M-current, suggesting that tonic NO levels inhibit M-current in TG neurons. Moreover NO increased neuronal excitability and calcitonin gene-related peptide (CGRP) release and these effects could be prevented by perturbing M-channel function. First, NO-induced depolarization was prevented by pre-application of the M-channel blocker XE991 and second, NO-induced increase in CGRP release was prevented by incubation with the M-channel opener retigabine. We investigated the mechanism of the effects of NO on M-channels and identified a site of action of NO to be the redox modulatory site at the triplet of cysteines within the cytosolic linker between transmembrane domains 2 and 3, which is also a site of oxidative modification of M-channels by reactive oxygen species (ROS). NO and oxidative modifications have opposing effects on M-current, suggesting that a tightly controlled local redox and NO environment will exert fine control over M-channel activity and thus neuronal excitability. Together our data have identified a dynamic redox sensor within neuronal M-channels, which mediates reciprocal regulation of channel activity by NO and ROS. This sensor may play an important role in mediating excitatory effects of NO in such trigeminal disorders as headache and migraine.

M-Type K+ Channel as Plasma Membrane Nitric Oxide and Reactive Oxygen Species Sensor

M-type K+ channels control excitability of many neuron types. M channel activity is potently enhanced by oxidative modification of triple cysteine pocket within the cytosolic linker between transmembrane domains 2 and 3. Thus, in sensory neurons from dorsal root (DRG) and trigeminal (TG) ganglia M channel activity is increased by the mitochondrial release of reactive oxygen species (ROS) induced by neuromodulator substance P. Here we identified a new signalling role for nitric oxide (NO) in TG sensory neurons. We show that in rat TG neurons, the NO donor, S-Nitroso-N-acetyl-DL-penicillamine (SNAP), inhibited M-current by 53±12% (n=12). This inhibitory effect was blocked by scavenging of NO and inhibition of NO synthases increased M-current, suggesting that tonic NO levels inhibit M-current in TG neurons. NO increased release of calcitonin gene-related peptide (CGRP) from TG neurons (130±10% of control, p≤0.05), consistent with an increase in neuronal excitability. Importantly, incubation with the M-channel opener retigabine completely abolished increases in CGRP release. We further investigated the mechanism of the effects of NO on M channels and identified a site of action of NO to be the same triplet of cysteines, which is also a site of oxidative modification of M channels by ROS. We now show that the same triplet of cysteines can be S-nitrosylated in the presence of SNAP. We show that NO and oxidative modifications have opposing effects on M-current, suggesting that a tightly controlled local redox and NO environment will exert control over M-channel activity and thus neuronal excitability. Together our data have identified a dynamic redox sensor within neuronal M-channels which mediates reciprocal regulation of channel activity by S-nitrosylation and oxidative modification. This sensor may play an important role in controlling neuronal excitability by redox and NO-related mechanisms.

Role of M-Type Potassium Channels Glutamatergic Synaptic Transmission and Status Epilepticus (IN5-1.003)

Objective: Whether M-currents modulate glutamatergic synaptic transmission and whether the drugs that open these channels could terminate seizures induced by muscarinic stimulation. Background Muscarinic activation by pilocarpine and anologs result in seizures presumably by the inhibition of M-currents. Muscarinic stimulation modulates glutamatergic synaptic transmission but the mechanisms remain uncertain. Design/Methods: Whole-cell patch clamp recordings from CA3 and CA1 hippocampal pyramidal neurons and video EEG recoprdings from rats. Results: In voltage clamp recordings, XE-991 an M channel blocker increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), but had no effect on their amplitude. XE-991 and its anolog linopordine also increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) but had no effect on their amplitude. M1 receptor agonist NcN-A-343 also increased the frequency of mEPSCs. The M-channel opener flupirtine had the effect opposite of XE-991. The XE-991 enhancement of mEPSCs frequency was not observed in calcium free external ACSF. When P/Q- or N-type calcium channel was blocked, the effect of XE-991 on mEPSCs was partially prevented. These data suggest that M-channel blockage increases presynaptic calcium dependent glutamate release in CA1 neurons. Current clamp recordings performed in CA3 neurons revealed that XE-991 depolarized membrane potential and increased burst firing. McN-A-343 had similar effect on CA3 neurons as XE-991. We then tested whether M-channel opener flupirtine could terminate status epilepticus (SE). Administration of flupirtine (50 mg/kg) and diazepam (10 mg/kg) individually 10 minutes after continuous EEG activity of first grade seizure was observed did not terminate SE; however, co-administration both drugs significantly reduced the length of SE, with 80% (4/5) rats returning to normal EEG within 90 minutes of treatment. Conclusions: Cholinergic activation causes depolarization of presynaptic CA3 neurons via M-channels, which activates calcium channels to increase glutamate release. Cholinergic inhibition of M-channel contributes to seizure generation.Drugs targeting M currents can terminate status epilepticus. Supported by: Congessionaly Directed Medical Research Program of the Department of Defense. NIH. Disclosure: Dr. Kapur has received (royalty or license fee or contractual rights) payments from a patent. Dr. Sun has nothing to disclose.

Regulation of Airway Smooth Muscle Contraction by KV7 (M-Type) K+ Channels

BK-type Ca2+-activated K+ channels have established roles in regulating voltage-dependent Ca2+ influx and contraction of airway smooth muscle (ASM). Recently, Kv7 (M-type) voltage-dependent K+ channels have been uncovered as a regulator of membrane voltage of smooth muscle cell types. Here, we investigate the role of Kv7 channels in rodent trachea. We isolated Kv7 current in acutely dissociated rat ASM cells under perforated-patch voltage clamp with the BK-channel blocker, paxilline (1 μM), using 2s depolarizations from a holding potential of −60 mV. Kv7 current was quantified as the sustained current component over the last 400 ms of the step, which was almost completely blocked by the specific Kv7 blocker, XE991 (10 μM). The voltage-dependence of activation of this sustained current (V0.5 = −28±1 mV) was similar to the XE991-sensitive component (V0.5 = −31±2 mV), typical of M-type currents. The M-channel opener, flupirtine (10 μM), increased current amplitudes by two-fold and shifted the V0.5 to −36±1 mV for the total sustained current and to −40±2 mV for the flupirtine-sensitive current. In contraction experiments on mouse trachea, we found little effect of XE991 on carbachol-evoked (0.5 μM) trachea constriction, but flupertine (30 μM) caused a relaxation of trachea by 21±2%. The flupertine effect is likely via opposing voltage-dependent Ca2+ influx, since no effect of flupertine occurred in a low K+/hyperpolarizing bathing solution (1mM K+). Interestingly, although XE991 failed to affect wild type trachea, it does enhance contraction of trachea from BK channel β1 KO mice at low carbachol concentrations (i.e., 2.3 fold increased contraction in 0.1 μM carbachol) These results indicate that Kv7 channels in rodent trachea may play a secondary role to BK channels in regulating airway constriction. Nevertheless, Kv7 channel agonists may be useful in moderating airway constriction in asthma.

Role of neuronal potassium M-channels in sympathetic regulation of cardiac function

Role of neuronal potassium M-channels in sympathetic regulation of cardiac function

Functional role of M-type (KCNQ) K+channels in adrenergic control of cardiomyocyte contraction rate by sympathetic neurons

M-type (KCNQ) K⁺ channels are known to regulate excitability and firing properties of sympathetic neurons (SNs), but their role in regulating neurotransmitter release is unclear, requiring further study. We sought to use a physiological preparation in which SNs innervate primary cardiomyocytes to evaluate the direct role of M-channels in the release of noradrenaline (NA) from SNs. Co-cultures of rat SNs and mouse cardiomyocytes were prepared, and the contraction rate (CR) of the cardiomyocyte syncytium monitored by video microscopy. We excited the SNs with nicotine, acting on nicotinic acetylcholine receptors, and monitored the increase in CR in the presence or absence of the specific M-channel opener retigabine, or agonists of bradykinin B2 or purinergic P2Y receptors on the SNs. The maximal adrenergic effect on the CR was determined by application of isoproterenol (isoprenaline). To isolate the actions of B2 or P2Y receptor stimulation to the neurons, we prepared cardiomyocytes from B2 receptor or P2Y2 receptor knock-out mice, respectively. We found that co-application of retigabine strongly decreased the nicotine-induced increase in CR. Conversely, co-application of bradykinin or the P2Y-receptor agonist UTP augmented the nicotine-induced increase in CR to about half of the level produced by isoproterenol. All effects on the CR were wholly blocked by propranolol. Our data support the role of M-type K⁺ channels in the control of NA release by SNs at functional adrenergic synapses on cardiomyocytes.We conclude that physiological receptor agonists control the heart rate via the regulation of M-current in SNs.

Activation of Voltage-Gated KCNQ/Kv7 Channels by Anticonvulsant Retigabine Attenuates Mechanical Allodynia of Inflammatory Temporomandibular Joint in Rats

BackgroundTemporomandibular disorders (TMDs) are characterized by persistent orofacial pain and have diverse etiologic factors that are not well understood. It is thought that central sensitization leads to neuronal hyperexcitability and contributes to hyperalgesia and spontaneous pain. Nonsteroidal anti-inflammatory drugs (NSAIDs) are currently the first choice of drug to relieve TMD pain. NSAIDS were shown to exhibit anticonvulsant properties and suppress cortical neuron activities by enhancing neuronal voltage-gated potassium KCNQ/Kv7 channels (M-current), suggesting that specific activation of M-current might be beneficial for TMD pain.ResultsIn this study, we selected a new anticonvulsant drug retigabine that specifically activates M-current, and investigated the effect of retigabine on inflammation of the temporomandibular joint (TMJ) induced by complete Freund's adjuvant (CFA) in rats. The results show that the head withdrawal threshold for escape from mechanical stimulation applied to facial skin over the TMJ in inflamed rats was significantly lower than that in control rats. Administration of centrally acting M-channel opener retigabine (2.5 and 7.5 mg/kg) can dose-dependently raise the head withdrawal threshold of mechanical allodynia, and this analgesic effect can be reversed by the specific KCNQ channel blocker XE991 (3 mg/kg). Food intake is known to be negatively associated with TMJ inflammation. Food intake was increased significantly by the administration of retigabine (2.5 and 7.5 mg/kg), and this effect was reversed by XE991 (3 mg/kg). Furthermore, intracerebralventricular injection of retigabine further confirmed the analgesic effect of central retigabine on inflammatory TMJ.ConclusionsOur findings indicate that central sensitization is involved in inflammatory TMJ pain and pharmacological intervention for controlling central hyperexcitability by activation of neuronal KCNQ/M-channels may have therapeutic potential for TMDs.

A tale of switched functions: from cyclooxygenase inhibition to M-channel modulation in new diphenylamine derivatives.

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.

M-Channels (Kv7/KCNQ Channels) That Regulate Synaptic Integration, Excitability, and Spike Pattern of CA1 Pyramidal Cells Are Located in the Perisomatic Region

To understand how electrical signal processing in cortical pyramidal neurons is executed by ion channels, it is essential to know their subcellular distribution. M-channels (encoded by Kv7.2-Kv7.5/KCNQ2-KCNQ5 genes) have multiple important functions in neurons, including control of excitability, spike afterpotentials, adaptation, and theta resonance. Nevertheless, the subcellular distribution of these channels has remained elusive. To determine the M-channel distribution within CA1 pyramidal neurons, we combined whole-cell patch-clamp recording from the soma and apical dendrite with focal drug application, in rat hippocampal slices. Both a M-channel opener (retigabine [N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester]) and a blocker (XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-antracenone]) changed the somatic subthreshold voltage response but had no observable effect on local dendritic responses. Under conditions promoting dendritic Ca2+ spikes, local somatic but not dendritic application of M-channel blockers (linopirdine and XE991) enhanced the Ca2+ spikes. Simultaneous dendritic and somatic whole-cell recordings showed that the medium afterhyperpolarization after a burst of spikes underwent strong attenuation along the apical dendrite and was fully blocked by somatic XE991 application. Finally, by combining patch-clamp and extracellular recordings with computer simulations, we found that perisomatic M-channels reduce the summation of EPSPs. We conclude that functional M-channels appear to be concentrated in the perisomatic region of CA1 pyramidal neurons, with no detectable M-channel activity in the distal apical dendrites.

Oxidative modification of M-type K+ channels as a mechanism of cytoprotective neuronal silencing

Voltage-gated K(+) channels of the Kv7 family underlie the neuronal M current that regulates action potential firing. Suppression of M current increases excitability and its enhancement can silence neurons. We here show that three of five Kv7 channels undergo strong enhancement of their activity by oxidative modification induced by physiological concentrations of hydrogen peroxide. A triple cysteine pocket in the channel S2-S3 linker is critical for this effect. Oxidation-induced enhancement of M current produced a hyperpolarization and a dramatic reduction of action potential firing frequency in rat sympathetic neurons. As hydrogen peroxide is robustly produced during hypoxia-induced oxidative stress, we used an oxygen/glucose deprivation neurodegeneration model that showed neuronal death to be severely accelerated by M current blockade. Such blockade had no effect on survival of normoxic neurons. This work describes a novel pathway of M-channel regulation and suggests a role for M channels in protective neuronal silencing during oxidative stress.

Kv7/KCNQ/M‐channels in rat glutamatergic hippocampal axons and their role in regulation of excitability and transmitter release

M-current (I(M)) plays a key role in regulating neuronal excitability. Mutations in Kv7/KCNQ subunits, the molecular correlates of I(M), are associated with a familial human epilepsy syndrome. Kv7/KCNQ subunits are widely expressed, and I(M) has been recorded in somata of several types of neurons, but the subcellular distribution of M-channels remains elusive. By combining field-potential, whole-cell and intracellular recordings from area CA1 in rat hippocampal slices, and computational modelling, we provide evidence for functional M-channels in unmyelinated axons in the brain. Our data indicate that presynaptic M-channels can regulate axonal excitability and synaptic transmission, provided the axons are depolarized into the I(M) activation range (beyond approximately -65 mV). Here, such depolarization was achieved by increasing the extracellular K(+) concentration ([K(+)](o)). Extracellular recordings in the presence of moderately elevated [K(+)](o) (7-11 mm), showed that the specific M-channel blocker XE991 reduced the amplitude of the presynaptic fibre volley and the field EPSP in a [K(+)](o)-dependent manner, both in stratum radiatum and in stratum lacknosum moleculare. The M-channel opener, retigabine, had opposite effects. The higher the [K(+)](o), the greater the effects of XE991 and retigabine. Similar pharmacological modulation of EPSPs recorded intracellularly from CA1 pyramidal neurons, while blocking postsynaptic K(+) channels with intracellular Cs(+), confirmed that active M-channels are located presynaptically. Computational analysis with an axon model showed that presynaptic I(M) can control Na(+) channel inactivation and thereby affect the presynaptic action potential amplitude and Ca(2+) influx, provided the axonal membrane potential is sufficiently depolarized. Finally, we compared the effects of blocking I(M) on the spike after-depolarization and bursting in CA3 pyramidal neuron somata versus their axons. In standard [K(+)](o) (2.5 mm), XE991 increased the ADP and promoted burst firing at the soma, but not in the axons. However, I(M) contributed to the refractory period in the axons when spikes were broadened by a low dose 4-aminopyridine (200 microm). Our results indicate that functional Kv7/KCNQ/M-channels are present in unmyelinated axons in the brain, and that these channels may have contrasting effects on excitability depending on their subcellular localization.

Retigabine-induced population primary afferent hyperpolarisation in vitro

Retigabine is a compound of potential interest in analgesia which acts as an M-channel opener to depress neuronal excitability. Here we study the effects of retigabine and its antagonist XE-991 on populations of primary afferents. Experiments were performed using a hemisected spinal cord preparation from rat pups maintained under in vitro conditions. Recording from dorsal roots were performed using tight fitting suction electrodes coupled to AC and DC amplifiers. The adjacent dorsal root was electrically stimulated at regular intervals. The effects of the modulators on basal potential, spontaneous potentials and dorsal root-dorsal root responses were studied. Superfusion of retigabine (10 μM) produced long lasting and robust hyperpolarisation of primary afferents which persisted during superfusion of picrotoxin (20 μM) and tetrodotoxin (0.5 μM). Other effects of retigabine were (1) increase in stimulation threshold; (2) increase in size of responses to suprathreshold stimuli; and (3) increase in amplitude and decrease in frequency of spontaneous dorsal root potentials. Superfusion of XE-991 had little effect on its own but blocked all the effects of retigabine. These results indicate the presence of functional M-currents in central terminals of primary afferents and in the interneurones that mediate dorsal root potentials. The depressant effects of retigabine on the excitability of these structures may contribute to its analgesic effects after pain-inducing treatments.