Allisartan ameliorates vascular remodeling through regulation of voltage-gated potassium channels in hypertensive rats.

BACKGROUND: The objective of the present study was to determine the effect of allisartan, a new angiotensin II type 1 receptor antagonist on vascular remodeling through voltage gated potassium channels (Kv7) in hypertensive rats. METHODS: The study included a total of 47 Sprague Dawley (SD) rats. The animals were randomized to sham operation (n = 14), untreated hypertensive control group (n = 18) and allisartan treatment group (n = 15). Using renal artery stenosis, hypertension was induced in animals. Single dose of allisartan was administered intra-gastrically to animals in the allisartan treatment group and match placebo in the other 2 groups. Wire myography was used to measure the muscle tension in isolated mesenteric arteries from the animals. Real-time polymerase chain reaction was used to quantify the expression of Kv7 channel mRNA subunits. RESULTS: After 4 weeks of treatment, a significant decrease in mean arterial, systolic and diastolic blood pressure (SBP and DBP) was observed in allisartan treatment group compared to hypertension control group. The median arterial wall thickness and area/diameter ratio reduced significantly in treatment group compared to untreated hypertension group (P < 0.05). Wire myography demonstrated increased relaxation of mesenteric artery with increase in concentration of ML213. A significant up-regulation in the expression of all Kv7 mRNA subunits was observed in allisartan group compared to untreated hypertension group. CONCLUSIONS: From the results, allisartan was found to lower BP and preserve vascular remodeling through Kv7 channels.

KV7 channels are potential regulators of the exercise pressor reflex.

The exercise pressor reflex (EPR) originates in skeletal muscle and is activated by exercise-induced signals to increase arterial blood pressure and cardiac output. Muscle ischemia can elicit the EPR, which can be inappropriately activated in patients with peripheral vascular disease or heart failure to increase the incidence of myocardial infarction. We seek to better understand the receptor/channels that control excitability of group III and group IV muscle afferent fibers that give rise to the EPR. Bradykinin (BK) is released within contracting muscle and can evoke the EPR. However, the mechanism is incompletely understood. KV7 channels strongly regulate neuronal excitability and are inhibited by BK. We have identified KV7 currents in muscle afferent neurons by their characteristic activation/deactivation kinetics, enhancement by the KV7 activator retigabine, and block by KV7 specific inhibitor XE991. The blocking of KV7 current by different XE991 concentrations suggests that the KV7 current is generated by both KV7.2/7.3 (high affinity) and KV7.5 (low affinity) channels. The KV7 current was inhibited by 300 nM BK in neurons with diameters consistent with both group III and group IV afferents. The inhibition of KV7 by BK could be a mechanism by which this metabolic mediator generates the EPR. Furthermore, our results suggest that KV7 channel activators such as retigabine, could be used to reduce cardiac stress resulting from the exacerbated EPR in patients with cardiovascular disease.NEW & NOTEWORTHY KV7 channels control neuronal excitability. We show that these channels are expressed in muscle afferents and generate currents that are blocked by XE991 and bradykinin (BK). The XE991 block suggests that KV7 current is generated by KV7.2/3 and KV7.5 channels. The BK inhibition of KV7 channels may explain how BK activates the exercise pressor reflex (EPR). Retigabine can enhance KV7 current, which could help control the inappropriately activated EPR in patients with cardiovascular disease.

Potassium channels act as chemosensors for solute transporters.

Potassium channels form physical complexes with solute transporters in vivo, yet little is known about their range of possible signaling modalities and the underlying mechanisms. The KCNQ2/3 potassium channel, which generates neuronal M-current, is voltage-gated and its activity is also stimulated by binding of various small molecules. KCNQ2/3 forms reciprocally regulating complexes with sodium-coupled myo-inositol transporters (SMITs) in mammalian neurons. Here, we report that the neurotransmitter γ-aminobutyric acid (GABA) and other small molecules directly regulate myo-inositol transport in rat dorsal root ganglia, and by human SMIT1-KCNQ2/3 complexes in vitro, by inducing a distinct KCNQ2/3 pore conformation. Reciprocally, SMIT1 tunes KCNQ2/3 sensing of GABA and related metabolites. Ion permeation and mutagenesis studies suggest that SMIT1 and GABA similarly alter KCNQ2/3 pore conformation but via different KCNQ subunits and molecular mechanisms. KCNQ channels therefore act as chemosensors to enable co-assembled myo-inositol transporters to respond to diverse stimuli including neurotransmitters, metabolites and drugs.

Activation of Kv 7 channels as a novel mechanism for NO/cGMP-induced pulmonary vasodilation.

BACKGROUND AND PURPOSE: The NO/cGMP pathway represents a major physiological signalling controlling tone in pulmonary arteries (PA), and drugs activating this pathway are used to treat pulmonary arterial hypertension. Kv channels expressed in PA smooth muscle cells (PASMCs) are key determinants of vascular tone. We aimed to analyse the contribution of Kv 1.5 and Kv 7 channels in the electrophysiological and vasodilating effects evoked by NO donors and the GC stimulator riociguat in PA. EXPERIMENTAL APPROACH: Kv currents were recorded in isolated rat PASMCs using the patch-clamp technique. Vascular reactivity was assessed in a wire myograph. KEY RESULTS: The NO donors diethylamine NONOate diethylammonium (DEA-NO) and sodium nitroprusside hyperpolarized the membrane potential and induced a bimodal effect on Kv currents (augmenting the current between -40 and -10 mV and decreasing it at more depolarized potentials). The hyperpolarization and the enhancement of the current were suppressed by Kv 7 channel inhibitors and by the GC inhibitor ODQ but preserved when Kv 1.5 channels were inhibited. Additionally, DEA-NO enhanced Kv 7.5 currents in COS7 cells expressing the KCNQ5 gene. Riociguat increased Kv currents at all potentials ≥-40 mV and induced membrane hyperpolarization. Both effects were prevented by Kv 7 inhibition. Likewise, PA relaxation induced by NO donors and riociguat was attenuated by Kv 7 inhibitors. CONCLUSIONS AND IMPLICATIONS: NO donors and riociguat enhance Kv 7 currents, leading to PASMC hyperpolarization. This mechanism contributes to NO/cGMP-induced PA vasodilation. Our study identifies Kv 7 channels as a novel mechanism of action of vasodilator drugs used in the treatment of pulmonary arterial hypertension.

Centipede KCNQ Inhibitor SsTx Also Targets KV1.3.

It was recently discovered that Ssm Spooky Toxin (SsTx) with 53 residues serves as a key killer factor in red-headed centipede's venom arsenal, due to its potent blockage of the widely expressed KCNQ channels to simultaneously and efficiently disrupt cardiovascular, respiratory, muscular, and nervous systems, suggesting that SsTx is a basic compound for centipedes' defense and predation. Here, we show that SsTx also inhibits KV1.3 channel, which would amplify the broad-spectrum disruptive effect of blocking KV7 channels. Interestingly, residue R12 in SsTx extends into the selectivity filter to block KV7.4, however, residue K11 in SsTx replaces this ploy when toxin binds on KV1.3. Both SsTx and its mutant SsTx_R12A inhibit cytokines production in T cells without affecting the level of KV1.3 expression. The results further suggest that SsTx is a key molecule for defense and predation in the centipedes' venoms and it evolves efficient strategy to disturb multiple physiological targets.

Investigating the Role of G Protein ßγ in Kv7-Dependent Relaxations of the Rat Vasculature.

Objective- In renal arteries, inhibitors of G protein ßγ subunits (Gßγ) reduce Kv7 activity and inhibit Kv7-dependent receptor-mediated vasorelaxations. However, the mechanisms underlying receptor-mediated relaxation are artery specific. Consequently, the aim of this study was to ascertain the role of Gßγ in Kv7-dependent vasorelaxations of the rat vasculature. Approach and Results- Isometric tension recording was performed in isolated rat renal, mesenteric, and cerebral arteries to study isoproterenol and calcitonin gene-related peptide relaxations. Kv7.4 was knocked down via morpholino transfection while inhibition of Gßγ was investigated with gallein and M119K. Proximity ligation assay was performed on isolated myocytes to study the association between Kv7.4 and G protein ß subunits or signaling intermediaries. Isoproterenol or calcitonin gene-related peptide-induced relaxations were attenuated by Kv7.4 knockdown in all arteries studied. Inhibition of Gßγ with gallein or M119K had no effect on isoproterenol-mediated relaxations in mesenteric artery but had a marked effect on calcitonin gene-related peptide-induced responses in mesenteric artery and cerebral artery and isoproterenol responses in renal artery. Isoproterenol increased association with Kv7.4 and Rap1a in mesenteric artery which were not sensitive to gallein, whereas in renal artery, isoproterenol increased Kv7.4-AKAP (A-kinase anchoring protein) associations in a gallein-sensitive manner. Conclusions- The Gßγ-Kv7 relationship differs between vessels and is an essential requirement for AKAP, but not Rap-mediated regulation of the channel.

Direct neurotransmitter activation of voltage-gated potassium channels.

Voltage-gated potassium channels KCNQ2-5 generate the M-current, which controls neuronal excitability. KCNQ2-5 subunits each harbor a high-affinity anticonvulsant drug-binding pocket containing an essential tryptophan (W265 in human KCNQ3) conserved for >500 million years, yet lacking a known physiological function. Here, phylogenetic analysis, electrostatic potential mapping, in silico docking, electrophysiology, and radioligand binding assays reveal that the anticonvulsant binding pocket evolved to accommodate endogenous neurotransmitters including γ-aminobutyric acid (GABA), which directly activates KCNQ5 and KCNQ3 via W265. GABA, and endogenous metabolites ß-hydroxybutyric acid (BHB) and γ-amino-ß-hydroxybutyric acid (GABOB), competitively and differentially shift the voltage dependence of KCNQ3 activation. Our results uncover a novel paradigm: direct neurotransmitter activation of voltage-gated ion channels, enabling chemosensing of the neurotransmitter/metabolite landscape to regulate channel activity and cellular excitability.

4-Aminopyridine: a pan voltage-gated potassium channel inhibitor that enhances Kv 7.4 currents and inhibits noradrenaline-mediated contraction of rat mesenteric small arteries.

BACKGROUND AND PURPOSE: Kv 7.4 and Kv 7.5 channels are regulators of vascular tone. 4-Aminopyridine (4-AP) is considered a broad inhibitor of voltage-gated potassium (KV ) channels, with little inhibitory effect on Kv 7 family members at mmol concentrations. However, the effect of 4-AP on Kv 7 channels has not been systematically studied. The aim of this study was to investigate the pharmacological activity of 4-AP on Kv 7.4 and Kv 7.5 channels and characterize the effect of 4-AP on rat resistance arteries. EXPERIMENTAL APPROACH: Voltage clamp experiments were performed on Xenopus laevis oocytes injected with cRNA encoding KCNQ4 or KCNQ5, HEK cells expressing Kv 7.4 channels and on rat, freshly isolated mesenteric artery smooth muscle cells. The effect of 4-AP on tension, membrane potential, intracellular calcium and pH was assessed in rat mesenteric artery segments. KEY RESULTS: 4-AP increased the Kv 7.4-mediated current in oocytes and HEK cells but did not affect Kv 7.5 current. 4-AP also enhanced native mesenteric artery myocyte K+ current at sub-mmol concentrations. When applied to NA-preconstricted mesenteric artery segments, 4-AP hyperpolarized the membrane, decreased [Ca2+ ]i and caused concentration-dependent relaxations that were independent of 4-AP-mediated changes in intracellular pH. Application of the Kv 7 channel blocker XE991 and BKCa channel blocker iberiotoxin attenuated 4-AP-mediated relaxation. 4-AP also inhibited the NA-mediated signal transduction to elicit a relaxation. CONCLUSIONS AND IMPLICATIONS: These data show that 4-AP is able to relax NA-preconstricted rat mesenteric arteries by enhancing the activity of Kv 7.4 and BKCa channels and attenuating NA-mediated signalling.

Kv7 potassium channels as signal transduction intermediates in the control of microvascular tone.

Potassium channels are recognized as important regulators of cellular functions in most, if not all cell types. These cellular proteins assemble to form gated pores in the plasma membrane, which serve to regulate the flow of potassium ions (K+ ) from the cytosol to the extracellular space. In VSMCs, the open state of potassium channels enables the efflux of K+ and thereby establishes a negative resting voltage across the plasma membrane that inhibits the opening of VSCCs. Under these conditions, cytosolic Ca2+ concentrations are relatively low and Ca2+ -dependent contraction is inhibited. Recent research has identified Kv7 family potassium channels as important contributors to resting membrane voltage in VSMCs, with much of the research focusing on the effects of drugs that specifically activate or block these channels to produce corresponding effects on VSMC contraction and vascular tone. Increasingly, evidence is emerging that these channels are not just good drug targets-they are also essential intermediates in vascular signal transduction, mediating vasoconstrictor or vasodilator responses to a variety of physiological stimuli. This review will summarize recent research findings that support a crucial function of Kv7 channels in both positive (vasoconstrictive) and negative (vasorelaxant) regulation of microvascular tone.

EAG channels expressed in microvillar photoreceptors are unsuited to diurnal vision.

KEY POINTS: The principles underlying the evolutionary selection of ion channels for expression in sensory neurons are unclear. Photoreceptor depolarization in the diurnal Drosophila melanogaster is predominantly provided by light-activated transient receptor potential (TRP) channels, whereas repolarization is mediated by sustained voltage-gated K+ channels of the Shab family. In the present study, we show that phototransduction in the nocturnal cockroach Periplaneta americana is predominantly mediated by TRP-like channels, whereas membrane repolarization is based on EAG channels. Although bright light stimulates Shab channels in Drosophila, further restricting depolarization and improving membrane bandwidth, it strongly suppresses EAG conductance in Periplaneta. This light-dependent inhibition (LDI) is caused by calcium and is abolished by chelating intracellular calcium or suppressing eag gene expression. LDI increases membrane resistance, augments gain and reduces the signalling bandwidth. This makes EAG unsuitable for light response conditioning during the day and might have resulted in the evolutionary replacement of EAG by other delayed rectifiers in diurnal insects. ABSTRACT: The principles underlying evolutionary selection of ion channels for expression in sensory neurons are unclear. Among species possessing microvillar photoreceptors, the major ionic conductances have only been identified in Drosophila melanogaster. In Drosophila, depolarization is provided by light-activated transient receptor potential (TRP) channels with a minor contribution from TRP-like (TRPL) channels, whereas repolarization is mediated by sustained voltage-gated K+ (Kv) channels of the Shab family. Bright light stimulates Shab channels, further restricting depolarization and improving membrane bandwidth. In the present study, data obtained using a combination of electrophysiological, pharmacological and molecular knockdown techniques strongly suggest that in photoreceptors of the nocturnal cockroach Periplaneta americana the major excitatory channel is TRPL, whereas the predominant delayed rectifier is EAG, a ubiquitous but enigmatic Kv channel. By contrast to the diurnal Drosophila, bright light strongly suppresses EAG conductance in Periplaneta. This light-dependent inhibition (LDI) is caused by calcium entering the cytosol and is amplified following inhibition of calcium extrusion, and it can also be abolished by chelating intracellular calcium or suppressing eag gene expression by RNA interference. LDI increases membrane resistance, augments gain and reduces the signalling bandwidth, impairing information transfer. LDI is also observed in the nocturnal cricket Gryllus integer, whereas, in the diurnal water strider Gerris lacustris, the delayed rectifier is up-regulated by light. Although LDI is not expected to reduce delayed rectifier current in the normal illumination environment of nocturnal cockroaches and crickets, it makes EAG unsuitable for light response conditioning during the day, and might have resulted in the evolutionary replacement of EAG by other delayed rectifiers in diurnal insects.

Acute stress diminishes M-current contributing to elevated activity of hypothalamic-pituitary-adrenal axis.

Acute stress stimulates corticotrophin-releasing hormone (CRH)-expressing neurons in the hypothalamic paraventricular nucleus (PVN), which is an essential component of hypothalamic-pituitary-adrenal (HPA) axis. However, the cellular and molecular mechanisms remain unclear. The M-channel is a voltage-dependent K+ channel involved in stabilizing the neuronal membrane potential and regulating neuronal excitability. In this study, we tested our hypothesis that acute stress suppresses expression of Kv7 channels to stimulate PVN-CRH neurons and the HPA axis. Rat PVN-CRH neurons were identified by expressing enhanced green fluorescent protein driven by Crh promoter. Acute restraint stress attenuated the excitatory effect of Kv7 blocker XE-991 on the firing activity of PVN-CRH neurons and blunted the increase in plasma corticosterone (CORT) levels induced by microinjection of XE-991 into the PVN. Furthermore, acute stress significantly decreased the M-currents in PVN-CRH neurons and reduced PVN expression of Kv7.3 subunit in the membrane. In addition, acute stress significantly increased phosphorylated AMP-activated protein kinase (AMPK) levels in the PVN tissue. Intracerebroventricular injection of the AMPK inhibitor dorsomorphin restored acute stress-induced elevation of CORT levels and reduction of membrane Kv7.3 protein level in the PVN. Dorsomorphin treatment increased the M-currents and reduced the firing activity of PVN-CRH neurons in acutely stressed rats. Collectively, these data suggest that acute stress diminishes Kv7 channels to stimulate PVN-CRH neurons and the HPA axis potentially via increased AMPK activity.

KV 7/M channels as targets for lipopolysaccharide-induced inflammatory neuronal hyperexcitability.

KEY POINTS: Neuroinflammation associated with CNS insults leads to neuronal hyperexcitability, which may culminate in epileptiform discharges. Application of the endotoxin lipopolysaccharide (LPS) to brain tissue initiates a neuroinflammatory cascade, providing an experimental model to study the mechanisms of neuroinflammatory neuronal hyperexcitability. Here we show that LPS application to hippocampal slices markedly enhances the excitability of CA1 pyramidal cells by inhibiting a specific potassium current, the M-current, generated by KV 7/M channels, which controls the excitability of almost every neuron in the CNS. The LPS-induced M-current inhibition is triggered by sequential activation of microglia, astrocytes and pyramidal cells, mediated by metabotropic purinergic and glutamatergic transmission, leading to blockade of KV 7/M channels by calcium released from intracellular stores. The identification of the downstream molecular target of neuroinflammation, namely the KV 7/M channel, potentially has far reaching implications for the understanding and treatment of many acute and chronic brain disorders. ABSTRACT: Acute brain insults and many chronic brain diseases manifest an innate inflammatory response. The hallmark of this response is glia activation, which promotes repair of damaged tissue, but also induces structural and functional changes that may lead to an increase in neuronal excitability. We have investigated the mechanisms involved in the modulation of neuronal activity by acute inflammation. Initiating inflammatory responses in hippocampal tissue rapidly led to neuronal depolarization and repetitive firing even in the absence of active synaptic transmission. This action was mediated by a complex metabotropic purinergic and glutamatergic glia-to-neuron signalling cascade, leading to the blockade of neuronal KV 7/M channels by Ca2+ released from internal stores. These channels generate the low voltage-activating, non-inactivating M-type K+ current (M-current) that controls intrinsic neuronal excitability, and its inhibition was the predominant cause of the inflammation-induced hyperexcitability. Our discovery that the ubiquitous KV 7/M channels are the downstream target of the inflammation-induced cascade, has far reaching implications for the understanding and treatment of many acute and chronic brain disorders.

Nonreciprocal mechanisms in up- and downregulation of spinal motoneuron excitability by modulators of KCNQ/Kv7 channels.

KCNQ/Kv7 channels form a slow noninactivating K+ current, also known as the M current. They activate in the subthreshold range of membrane potentials and regulate different aspects of excitability in neurons of the central nervous system. In spinal motoneurons (MNs), KCNQ/Kv7 channels have been identified in the somata, axonal initial segment, and nodes of Ranvier, where they generate a slow, noninactivating, K+ current sensitive to both muscarinic receptor-mediated inhibition and KCNQ/Kv7 channel blockers. In this study, we thoroughly reevaluated the function of up- and downregulation of KCNQ/Kv7 channels in mouse immature spinal MNs. Using electrophysiological techniques together with specific pharmacological modulators of the activity of KCNQ/Kv7 channels, we show that enhancement of the activity of these channels decreases the excitability of spinal MNs in mouse neonates. This action on MNs results from a combination of hyperpolarization of the resting membrane potential, a decrease in the input resistance, and depolarization of the voltage threshold. On the other hand, the effect of inhibition of KCNQ/Kv7 channels suggested that these channels play a limited role in regulating basal excitability. Computer simulations confirmed that pharmacological enhancement of KCNQ/Kv7 channel activity decreases excitability and also suggested that the effects of inhibition of KCNQ/Kv7 channels on the excitability of spinal MNs do not depend on a direct effect in these neurons but likely on spinal cord synaptic partners. These results indicate that KCNQ/Kv7 channels have a fundamental role in the modulation of the excitability of spinal MNs acting both in these neurons and in their local presynaptic partners.

Effects of novel subtype selective M-current activators on spinal reflexes in vitro: Comparison with retigabine.

The activation of Kv7 channels and the resulting M-current is a powerful mechanism to control neuronal excitability with profound effects in pain pathways. Despite the lack of specific data on the expression and role of these channels in nociceptive processing, much attention has been paid at exploring their potential value as targets for analgesia. Here we have characterized the spinal actions of two novel subunit selective Kv7 activators, ICA-069673 and ML213, and compared their effects to those of retigabine that acts with similar affinity on all neuronal Kv7 channels. Spinal reflexes were recorded in a mouse spinal cord in vitro preparation to allow the testing of the compounds on native spinal pathways at known concentrations. As retigabine, novel compounds depressed spinal segmental transmission with particularly strong effects on wind up, showing an adequate pro-analgesic profile. ML213 presented the highest potency. In contrast to retigabine, the effects of ICA-069673 and ML213 were blocked by XE-991 even at the highest concentrations used, suggesting specific effect on Kv7 channels. In addition, the effects of ICA-069673 on repetitive stimulation are consistent with a mode of action involving state or activity dependent interaction with the channels. Compared to retigabine, novel Kv7 openers maintain strong depressant effects on spinal nociceptive transmission showing an improved specificity on Kv7 channels. The differential effects obtained with these Kv7 openers may indicate the existence of several Kv7 conformations in spinal circuits.

A biophysical model examining the role of low-voltage-activated potassium currents in shaping the responses of vestibular ganglion neurons.

The vestibular nerve is characterized by two broad groups of neurons that differ in the timing of their interspike intervals; some fire at highly regular intervals, whereas others fire at highly irregular intervals. Heterogeneity in ion channel properties has been proposed as shaping these firing patterns (Highstein SM, Politoff AL. Brain Res 150: 182-187, 1978; Smith CE, Goldberg JM. Biol Cybern 54: 41-51, 1986). Kalluri et al. (J Neurophysiol 104: 2034-2051, 2010) proposed that regularity is controlled by the density of low-voltage-activated potassium currents (IKL). To examine the impact of IKL on spike timing regularity, we implemented a single-compartment model with three conductances known to be present in the vestibular ganglion: transient sodium (gNa), low-voltage-activated potassium (gKL), and high-voltage-activated potassium (gKH). Consistent with in vitro observations, removing gKL depolarized resting potential, increased input resistance and membrane time constant, and converted current step-evoked firing patterns from transient (1 spike at current onset) to sustained (many spikes). Modeled neurons were driven with a time-varying synaptic conductance that captured the random arrival times and amplitudes of glutamate-driven synaptic events. In the presence of gKL, spiking occurred only in response to large events with fast onsets. Models without gKL exhibited greater integration by responding to the superposition of rapidly arriving events. Three synaptic conductance were modeled, each with different kinetics to represent a variety of different synaptic processes. In response to all three types of synaptic conductance, models containing gKL produced spike trains with irregular interspike intervals. Only models lacking gKL when driven by rapidly arriving small excitatory postsynaptic currents were capable of generating regular spiking.

KCNQ potassium channels in sensory system and neural circuits.

M channels, an important regulator of neural excitability, are composed of four subunits of the Kv7 (KCNQ) K(+) channel family. M channels were named as such because their activity was suppressed by stimulation of muscarinic acetylcholine receptors. These channels are of particular interest because they are activated at the subthreshold membrane potentials. Furthermore, neural KCNQ channels are drug targets for the treatments of epilepsy and a variety of neurological disorders, including chronic and neuropathic pain, deafness, and mental illness. This review will update readers on the roles of KCNQ channels in the sensory system and neural circuits as well as discuss their respective mechanisms and the implications for physiology and medicine. We will also consider future perspectives and the development of additional pharmacological models, such as seizure, stroke, pain and mental illness, which work in combination with drug-design targeting of KCNQ channels. These models will hopefully deepen our understanding of KCNQ channels and provide general therapeutic prospects of related channelopathies.

δ Subunit-containing GABAA receptors are preferred targets for the centrally acting analgesic flupirtine.

BACKGROUND AND PURPOSE: The Kv 7 channel activator flupirtine is a clinical analgesic characterized as 'selective neuronal potassium channel opener'. Flupirtine was found to exert comparable actions at GABAA receptors and Kv 7 channels in neurons of pain pathways, but not in hippocampus. EXPERIMENTAL APPROACH: Expression patterns of GABAA receptors were explored in immunoblots of rat dorsal root ganglia, dorsal horns and hippocampi using antibodies for 10 different subunits. Effects of flupirtine on recombinant and native GABAA receptors were investigated in patch clamp experiments and compared with the actions on Kv 7 channels. KEY RESULTS: Immunoblots pointed towards α2, α3, ß3 and γ2 subunits as targets, but in all γ2-containing receptors the effects of flupirtine were alike: leftward shift of GABA concentration-response curves and diminished maximal amplitudes. After replacement of γ2S by δ, flupirtine increased maximal amplitudes. Currents through α1ß2δ receptors were more enhanced than those through Kv 7 channels. In hippocampal neurons, flupirtine prolonged inhibitory postsynaptic currents, left miniature inhibitory postsynaptic currents (mIPSCs) unaltered and increased bicuculline-sensitive tonic currents; penicillin abolished mIPSCs, but not tonic currents; concentration-response curves for GABA-induced currents were shifted to the left by flupirtine without changes in maximal amplitudes; in the presence of penicillin, maximal amplitudes were increased; GABA-induced currents in the presence of penicillin were more sensitive towards flupirtine than K(+) currents. In dorsal horn neurons, currents evoked by the δ-preferring agonist THIP (gaboxadol) were more sensitive towards flupirtine than K(+) currents. CONCLUSIONS AND IMPLICATIONS: Flupirtine prefers δ-containing GABAA receptors over γ-containing ones and over Kv 7 channels.

KV7 Channels Regulate Firing during Synaptic Integration in GABAergic Striatal Neurons.

Striatal projection neurons (SPNs) process motor and cognitive information. Their activity is affected by Parkinson's disease, in which dopamine concentration is decreased and acetylcholine concentration is increased. Acetylcholine activates muscarinic receptors in SPNs. Its main source is the cholinergic interneuron that responds with a briefer latency than SPNs during a cortical command. Therefore, an important question is whether muscarinic G-protein coupled receptors and their signaling cascades are fast enough to intervene during synaptic responses to regulate synaptic integration and firing. One of the most known voltage dependent channels regulated by muscarinic receptors is the KV7/KCNQ channel. It is not known whether these channels regulate the integration of suprathreshold corticostriatal responses. Here, we study the impact of cholinergic muscarinic modulation on the synaptic response of SPNs by regulating KV7 channels. We found that KV7 channels regulate corticostriatal synaptic integration and that this modulation occurs in the dendritic/spines compartment. In contrast, it is negligible in the somatic compartment. This modulation occurs on sub- and suprathreshold responses and lasts during the whole duration of the responses, hundreds of milliseconds, greatly altering SPNs firing properties. This modulation affected the behavior of the striatal microcircuit.