Selective inhibition of A-fiber-mediated excitatory transmission underlies the analgesic effects of KCNQ channel opening in the spinal dorsal horn.

Neuronal voltage-gated KCNQ (Kv7) channels, expressed centrally and peripherally, mediate low-threshold and non-inactivating M-currents responsible for the control of tonic excitability of mammalian neurons. Pharmacological opening of KCNQ channels has been reported to generate analgesic effects in animal models of neuropathic pain. Here, we examined the possible involvement of central KCNQ channels in the analgesic effects of retigabine, a KCNQ channel opener. Behaviorally, intraperitoneally applied retigabine exerted analgesic effects on thermal and mechanical hypersensitivity in male mice developing neuropathic pain after partial sciatic nerve ligation, which was antagonized by the KCNQ channel blocker XE991 preadministered intraperitoneally and intrathecally. Intrathecally applied retigabine also exerted analgesic effects that were inhibited by intrathecally injected XE991. We then explored the synaptic mechanisms underlying the analgesic effects of retigabine in the spinal dorsal horn. Whole-cell recordings were made from dorsal horn neurons in spinal slices with attached dorsal roots from adult male mice developing neuropathic pain, and the effects of retigabine on miniature and afferent-evoked postsynaptic currents were examined. Retigabine reduced the amplitude of A-fiber-mediated EPSCs without affecting C-fiber-mediated excitatory synaptic transmission. A-fiber-mediated EPSCs remained unaltered by retigabine in the presence of XE991, consistently with the behavioral findings. The frequency and amplitude of mEPSCs were not affected by retigabine. Thus, opening of KCNQ channels in the central terminals of primary afferent A-fibers inhibits excitatory synaptic transmission in the spinal dorsal horn, most likely contributing to the analgesic effect of retigabine.

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.

Effective Activation by Kynurenic Acid and Its Aminoalkylated Derivatives on M-Type K+ Current.

Kynurenic acid (KYNA, 4-oxoquinoline-2-carboxylic acid), an intermediate of the tryptophan metabolism, has been recognized to exert different neuroactive actions; however, the need of how it or its aminoalkylated amide derivative N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-oxo-1,4-dihydroquinoline-2-carboxamide (KYNA-A4) exerts any effects on ion currents in excitable cells remains largely unmet. In this study, the investigations of how KYNA and other structurally similar KYNA derivatives have any adjustments on different ionic currents in pituitary GH3 cells and hippocampal mHippoE-14 neurons were performed by patch-clamp technique. KYNA or KYNA-A4 increased the amplitude of M-type K+ current (IK(M)) and concomitantly enhanced the activation time course of the current. The EC50 value required for KYNA- or KYNA-A4 -stimulated IK(M) was yielded to be 18.1 or 6.4 µM, respectively. The presence of KYNA or KYNA-A4 shifted the relationship of normalized IK(M)-conductance versus membrane potential to more depolarized potential with no change in the gating charge of the current. The voltage-dependent hysteretic area of IK(M) elicited by long-lasting triangular ramp pulse was observed in GH3 cells and that was increased during exposure to KYNA or KYNA-A4. In cell-attached current recordings, addition of KYNA raised the open probability of M-type K+ channels, along with increased mean open time of the channel. Cell exposure to KYNA or KYNA-A4 mildly inhibited delayed-rectifying K+ current; however, neither erg-mediated K+ current, hyperpolarization-activated cation current, nor voltage-gated Na+ current in GH3 cells was changed by KYNA or KYNA-A4. Under whole-cell, current-clamp recordings, exposure to KYNA or KYNA-A4 diminished the frequency of spontaneous action potentials; moreover, their reduction in firing frequency was attenuated by linopirdine, yet not by iberiotoxin or apamin. In hippocampal mHippoE-14 neurons, the addition of KYNA also increased the IK(M) amplitude effectively. Taken together, the actions presented herein would be one of the noticeable mechanisms through which they modulate functional activities of excitable cells occurring in vivo.

Functional and behavioral signatures of Kv7 activator drug subtypes.

OBJECTIVE: Voltage-gated potassium channels of the KCNQ (Kv7) family are targeted by a variety of activator compounds with therapeutic potential for treatment of epilepsy. Exploration of this drug class has revealed a variety of effective compounds with diverse mechanisms. In this study, we aimed to clarify functional criteria for categorization of Kv7 activator compounds, and to compare the effects of prototypical drugs in a zebrafish larvae model. METHODS: In vitro electrophysiological approaches with recombinant ion channels were used to highlight functional properties important for classification of drug mechanisms. We also benchmarked the effects of representative antiepileptic Kv7 activator drugs using behavioral seizure assays of zebrafish larvae and in vivo Ca2+ imaging with the ratiometric Ca2+ sensor CaMPARI. RESULTS: Drug effects on channel gating kinetics, and drug sensitivity profiles to diagnostic channel mutations, were used to highlight properties for categorization of Kv7 activator drugs into voltage sensor-targeted or pore-targeted subtypes. Quantifying seizures and ratiometric Ca2+ imaging in freely swimming zebrafish larvae demonstrated that while all Kv7 activators tested lead to suppression of neuronal excitability, pore-targeted activators (like ML213 and retigabine) strongly suppress seizure behavior, whereas ICA-069673 triggers a seizure-like hypermotile behavior. SIGNIFICANCE: This study suggests criteria to categorize antiepileptic Kv7 activator drugs based on their underlying mechanism. We also establish the use of in vivo CaMPARI as a tool for screening effects of anticonvulsant drugs on neuronal excitability in zebrafish. In summary, despite a shared ability to suppress neuronal excitability, our findings illustrate how mechanistic differences between Kv7 activator subtypes influence their effects on heteromeric channels and lead to vastly different in vivo outcomes.

Antinociceptive Efficacy of Retigabine and Flupirtine for Gout Arthritis Pain.

INTRODUCTION: Gout arthritis is an inflammatory disease characterized by severe acute pain. The goal of pharmacological gout arthritis treatments is to reduce pain, and thereby increase the patient's quality of life. The Kv7/M channel activators retigabine and flupirtine show analgesic efficacy in animal models of osteoarthritic pain. We hypothesized that these drugs may also alleviate gout arthritis pain. OBJECTIVE: To determine the effects of retigabine and flupirtine on pain behavior associated with monosodium urate (MSU)-induced gout arthritis. METHODS: The gout arthritis model was established with an intra-articular injection of MSU into the right ankle joint, animals were treated with retigabine or flupirtine, and pain-related behaviors were assessed. RESULTS: Retigabine and flupirtine significantly increased the mechanical threshold and prolonged the paw withdrawal latency in a rat model of gout arthritis pain in a dose-dependent manner. The antinociceptive effects of retigabine and flupirtine were fully antagonized by the Kv7/M channel blocker XE991. CONCLUSION: Retigabine and flupirtine showed antinociceptive effects for MSU-induced gout pain at different times during pain development.

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,ß-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.

Treatment of myotonia congenita with retigabine in mice.

Patients with myotonia congenita suffer from muscle stiffness caused by muscle hyperexcitability. Although loss-of-function mutations in the ClC-1 muscle chloride channel have been known for 25 years to cause myotonia congenita, this discovery has led to little progress on development of therapy. Currently, treatment is primarily focused on reducing hyperexcitability by blocking Na+ current. However, other approaches such as increasing K+ currents might also be effective. For example, the K+ channel activator retigabine, which opens KCNQ channels, is effective in treating epilepsy because it causes hyperpolarization of the resting membrane potential in neurons. In this study, we found that retigabine greatly reduced the duration of myotonia in vitro. Detailed study of its mechanism of action revealed that retigabine had no effect on any of the traditional measures of muscle excitability such as resting potential, input resistance or the properties of single action potentials. Instead it appears to shorten myotonia by activating K+ current during trains of action potentials. Retigabine also greatly reduced the severity of myotonia in vivo, which was measured using a muscle force transducer. Despite its efficacy in vivo, retigabine did not improve motor performance of mice with myotonia congenita. There are a number of potential explanations for the lack of motor improvement in vivo including central nervous system side effects. Nonetheless, the striking effectiveness of retigabine on muscle itself suggests that activating potassium currents is an effective method to treat disorders of muscle hyperexcitability.

Four drug-sensitive subunits are required for maximal effect of a voltage sensor-targeted KCNQ opener.

KCNQ2-5 (Kv7.2-Kv7.5) channels are strongly influenced by an emerging class of small-molecule channel activators. Retigabine is the prototypical KCNQ activator that is thought to bind within the pore. It requires the presence of a Trp side chain that is conserved among retigabine-sensitive channels but absent in the retigabine-insensitive KCNQ1 subtype. Recent work has demonstrated that certain KCNQ openers are insensitive to mutations of this conserved Trp, and that their effects are instead abolished or attenuated by mutations in the voltage-sensing domain (VSD). In this study, we investigate the stoichiometry of a VSD-targeted KCNQ2 channel activator, ICA-069673, by forming concatenated channel constructs with varying numbers of drug-insensitive subunits. In homomeric WT KCNQ2 channels, ICA-069673 strongly stabilizes an activated channel conformation, which is reflected in the pronounced deceleration of deactivation and leftward shift of the conductance-voltage relationship. A full complement of four drug-sensitive subunits is required for maximal sensitivity to ICA-069673-even a single drug-insensitive subunit leads to significantly weakened effects. In a companion article (see Yau et al. in this issue), we demonstrate very different stoichiometry for the action of retigabine on KCNQ3, for which a single retigabine-sensitive subunit enables near-maximal effect. Together, these studies highlight fundamental differences in the site and mechanism of activation between retigabine and voltage sensor-targeted KCNQ openers.

Ancient and modern anticonvulsants act synergistically in a KCNQ potassium channel binding pocket.

Epilepsy has been treated for centuries with herbal remedies, including leaves of the African shrub Mallotus oppositifolius, yet the underlying molecular mechanisms have remained unclear. Voltage-gated potassium channel isoforms KCNQ2-5, predominantly KCNQ2/3 heteromers, underlie the neuronal M-current, which suppresses neuronal excitability, protecting against seizures. Here, in silico docking, mutagenesis and cellular electrophysiology reveal that two components of M. oppositifolius leaf extract, mallotoxin (MTX) and isovaleric acid (IVA), act synergistically to open neuronal KCNQs, including KCNQ2/3 channels. Correspondingly, MTX and IVA combine to suppress pentylene tetrazole-induced tonic seizures in mice, whereas individually they are ineffective. Co-administering MTX and IVA with the modern, synthetic anticonvulsant retigabine creates a further synergy that voltage independently locks KCNQ2/3 open. Leveraging this synergy, which harnesses ancient and modern medicines to exploit differential KCNQ isoform preferences, presents an approach to developing safe yet effective anticonvulsants.

Activation of KCNQ Channels Suppresses Spontaneous Activity in Dorsal Root Ganglion Neurons and Reduces Chronic Pain after Spinal Cord Injury.

A majority of people who have sustained spinal cord injury (SCI) experience chronic pain after injury, and this pain is highly resistant to available treatments. Contusive SCI in rats at T10 results in hyperexcitability of primary sensory neurons, which contributes to chronic pain. KCNQ channels are widely expressed in nociceptive dorsal root ganglion (DRG) neurons, are important for controlling their excitability, and their activation has proven effective in reducing pain in peripheral nerve injury and inflammation models. The possibility that activators of KCNQ channels could be useful for treating SCI-induced chronic pain is strongly supported by the following findings. First, SCI, unlike peripheral nerve injury, failed to decrease the functional or biochemical expression of KCNQ channels in DRG as revealed by electrophysiology, real-time quantitative polymerase chain reaction, and Western blot; therefore, these channels remain available for pharmacological targeting of SCI pain. Second, treatment with retigabine, a specific KCNQ channel opener, profoundly decreased spontaneous activity in primary sensory neurons of SCI animals both in vitro and in vivo without changing the peripheral mechanical threshold. Third, retigabine reversed SCI-induced reflex hypersensitivity, adding to our previous demonstration that retigabine supports the conditioning of place preference after SCI (an operant measure of spontaneous pain). In contrast to SCI animals, naïve animals showed no effects of retigabine on reflex sensitivity or conditioned place preference by pairing with retigabine, indicating that a dose that blocks chronic pain-related behavior has no effect on normal pain sensitivity or motivational state. These results encourage the further exploration of U.S. Food and Drug Administration-approved KCNQ activators for treating SCI pain, as well as efforts to develop a new generation of KCNQ activators that lack central side effects.

KV7 channels in the human detrusor: channel modulator effects and gene and protein expression.

Voltage-gated type 7 K+ (KV7 or KCNQ) channels regulate the contractility of various smooth muscles. With this study, we aimed to assess the role of KV7 channels in the regulation of human detrusor contractility, as well as the gene and protein expression of KV7 channels in this tissue. For these purposes, the isolated organ technique, RT-qPCR, and Western blot were used, respectively. XE-991, a selective KV7 channel blocker, concentration-dependently contracted the human detrusor; mean EC50 and Emax of XE-991-induced concentration-response curve were 14.1 µM and 28.8 % of the maximal bethanechol-induced contraction, respectively. Flupirtine and retigabine, selective KV7.2-7.5 channel activators, induced concentration-dependent relaxations of bethanechol-precontracted strips, with maximal relaxations of 51.6 and 51.8 % of the precontraction, respectively. XE-991 blocked the relaxations induced by flupirtine and retigabine. All five KCNQ genes were found to be expressed in the detrusor with KCNQ4 being the most expressed among them. Different bands, having sizes similar to some of reported KV7.1, 7.4, and 7.5 channel subunit isoforms, were detected in the detrusor by Western blot with the KV7.4 band being the most intense among them. In conclusion, KV7 channels contribute to set the basal tone of the human detrusor. In addition, KV7 channel activators significantly relax the detrusor. The KV7.4 channels are probably the most important KV7 channels expressed in the human detrusor. These data suggest that selective KV7.4 channel activators might represent new pharmacological tools for inducing therapeutic relaxation of the detrusor.

Kcne4 Deletion Sex-Dependently Alters Vascular Reactivity.

Voltage-gated potassium (Kv) channels formed by Kv7 (KCNQ) α-subunits are recognized as crucial for vascular smooth muscle function, in addition to their established roles in the heart (Kv7.1) and the brain (Kv7.2-5). In vivo, Kv7 α-subunits are often regulated by KCNE subfamily ancillary (ß) subunits. We investigated the effects of targeted germline Kcne4 deletion on mesenteric artery reactivity in adult male and female mice. Kcne4 deletion increased mesenteric artery contractility in response to α-adrenoceptor agonist methoxamine, and decreased responses to Kv7.2-7.5 channel activator ML213, in male but not female mice. In contrast, Kcne4 deletion markedly decreased vasorelaxation in response to isoprenaline in both male and female mice. Kcne4 expression was 2-fold lower in the female versus the male mouse mesenteric artery, and Kcne4 deletion elicited only moderate changes of other Kcne transcripts, with no striking sex-specific differences. However, Kv7.4 protein expression in females was twice that in males, and was reduced in both sexes by Kcne4 deletion. Our findings confirm a crucial role for KCNE4 in regulation of Kv7 channel activity to modulate vascular tone, and provide the first known molecular mechanism for sex-specificity of this modulation that has important implications for vascular reactivity and may underlie sex-specific susceptibility to cardiovascular diseases.

Involvement of Potassium Channels and Calcium-Independent Mechanisms in Hydrogen Sulfide-Induced Relaxation of Rat Mesenteric Small Arteries.

Endogenous hydrogen sulfide (H2S) is involved in the regulation of vascular tone. We hypothesized that the lowering of calcium and opening of potassium (K) channels as well as calcium-independent mechanisms are involved in H2S-induced relaxation in rat mesenteric small arteries. Amperometric recordings revealed that free [H2S] after addition to closed tubes of sodium hydrosulfide (NaHS), Na2S, and GYY4137 [P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid] were, respectively, 14%, 17%, and 1% of added amount. The compounds caused equipotent relaxations in isometric myographs, but based on the measured free [H2S], GYY4137 caused more relaxation in relation to released free H2S than NaHS and Na2S in rat mesenteric small arteries. Simultaneous measurements of [H2S] and tension showed that 15 µM of free H2S caused 61% relaxation in superior mesenteric arteries. Simultaneous measurements of smooth muscle calcium and tension revealed that NaHS lowered calcium and caused relaxation of NE-contracted arteries, while high extracellular potassium reduced NaHS relaxation without corresponding calcium changes. In NE-contracted arteries, NaHS (1 mM) lowered the phosphorylation of myosin light chain, while phosphorylation of myosin phosphatase target subunit 1 remained unchanged. Protein kinase A and G, inhibitors of guanylate cyclase, failed to reduce NaHS relaxation, whereas blockers of voltage-gated KV7 channels inhibited NaHS relaxation, and blockers of mitochondrial complex I and III abolished NaHS relaxation. Our findings suggest that low micromolar concentrations of free H2S open K channels followed by lowering of smooth muscle calcium, and by another mechanism involving mitochondrial complex I and III leads to uncoupling of force, and hence vasodilation.

Atomic basis for therapeutic activation of neuronal potassium channels.

Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2-5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators.

Kv7 channels critically determine coronary artery reactivity: left-right differences and down-regulation by hyperglycaemia.

AIMS: Voltage-gated potassium channels encoded by KCNQ genes (Kv7 channels) are emerging as important regulators of vascular tone. In this study, we analysed the contribution of Kv7 channels to the vasodilation induced by hypoxia and the cyclic AMP pathway in the coronary circulation. We also assessed their regional distribution and possible impairment by diabetes. METHODS AND RESULTS: We examined the effects of Kv7 channel modulators on K+ currents and vascular reactivity in rat left and right coronary arteries (LCAs and RCAs, respectively). Currents from LCA were more sensitive to Kv7 channel inhibitors (XE991, linopirdine) and activators (flupirtine, retigabine) than those from RCA. Accordingly, LCAs were more sensitive than RCAs to the relaxation induced by Kv7 channel enhancers. Likewise, relaxation induced by the adenylyl cyclase activator forskolin and hypoxia, which were mediated through Kv7 channel activation, were greater in LCA than in RCA. KCNQ1 and KCNQ5 expression was markedly higher in LCA than in RCA. After incubation with high glucose (HG, 30 mmol/L), myocytes from LCA, but not from RCA, were more depolarized and showed reduced Kv7 currents. In HG-incubated LCA, the effects of Kv7 channel modulators and forskolin were diminished, and the expression of KCNQ1 and KCNQ5 was reduced. Finally, vascular responses induced by Kv7 channel modulators were impaired in LCA, but not in RCA, from type 1 diabetic rats. CONCLUSION: Our results reveal that the high expression and function of Kv7 channels in the LCA and their down-regulation by diabetes critically determine the sensitivity to key regulators of coronary tone.

Diabetes attenuates urothelial modulation of detrusor contractility and spontaneous activity.

OBJECTIVES: To investigate the effect of diabetes on urothelial modulation of bladder contractility. METHODS: Bladder strips (urothelium intact or denuded) were prepared from 8-week-old streptozotocin-induced diabetic (n = 19) and non-diabetic control rats (n = 10). The effect of modulators of MaxiK (iberiotoxin and tetraethylammonium) and Kv7 (XE991 and retigabine) potassium channel activity were investigated for their effects on both carbachol-induced force generation and spontaneous contractile activity. RESULTS: In bladder strips from non-diabetic animals, the presence of the urothelium resulted in marked sensitivity to carbachol-induced force generation by modulators of MaxiK and Kv7 channel activity, whereas in the diabetic animal urothelial sensitivity to these agents was significantly diminished. Urothelial-intact bladder strips from non-diabetic animals were more sensitive to modulators of Kv7 activity in reducing the amplitude of spontaneous phasic contractions than urothelial-denuded bladder strips, whereas in diabetic animals the presence or absence of the urothelium did not alter the sensitivity to modulators of Kv7 activity. Spontaneous activity in the presence of tetraethylammonium was not affected by the urothelium in bladder strips from either diabetic or non-diabetic animals. CONCLUSIONS: The presence of the urothelium in bladders from non-diabetic animals modulates the activity of potassium blockers to affect bladder contractility, whereas in the diabetic bladder this effect is attenuated. These findings could help to explain the lack of success of pharmaceutical treatments targeting potassium channels to treat bladder pathology in patients with diseases imparing urothelial function.

Functional assembly of Kv7.1/Kv7.5 channels with emerging properties on vascular muscle physiology.

OBJECTIVE: Voltage-dependent K(+) (Kv) channels from the Kv7 family are expressed in blood vessels and contribute to cardiovascular physiology. Although Kv7 channel blockers trigger muscle contractions, Kv7 activators act as vasorelaxants. Kv7.1 and Kv7.5 are expressed in many vessels. Kv7.1 is under intense investigation because Kv7.1 blockers fail to modulate smooth muscle reactivity. In this study, we analyzed whether Kv7.1 and Kv7.5 may form functional heterotetrameric channels increasing the channel diversity in vascular smooth muscles. APPROACH AND RESULTS: Kv7.1 and Kv7.5 currents elicited in arterial myocytes, oocyte, and mammalian expression systems suggest the formation of heterotetrameric complexes. Kv7.1/Kv7.5 heteromers, exhibiting different pharmacological characteristics, participate in the arterial tone. Kv7.1/Kv7.5 associations were confirmed by coimmunoprecipitation, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching experiments. Kv7.1/Kv7.5 heterotetramers were highly retained at the endoplasmic reticulum. Studies in HEK-293 cells, heart, brain, and smooth and skeletal muscles demonstrated that the predominant presence of Kv7.5 stimulates release of Kv7.1/Kv7.5 oligomers out of lipid raft microdomains. Electrophysiological studies supported that KCNE1 and KCNE3 regulatory subunits further increased the channel diversity. Finally, the analysis of rat isolated myocytes and human blood vessels demonstrated that Kv7.1 and Kv7.5 exhibited a differential expression, which may lead to channel diversity. CONCLUSIONS: Kv7.1 and Kv7.5 form heterotetrameric channels increasing the diversity of structures which fine-tune blood vessel reactivity. Because the lipid raft localization of ion channels is crucial for cardiovascular physiology, Kv7.1/Kv7.5 heteromers provide efficient spatial and temporal regulation of smooth muscle function. Our results shed light on the debate about the contribution of Kv7 channels to vasoconstriction and hypertension.

M1-muscarinic receptors promote fear memory consolidation via phospholipase C and the M-current.

Neuromodulators released during and after a fearful experience promote the consolidation of long-term memory for that experience. Because overconsolidation may contribute to the recurrent and intrusive memories of post-traumatic stress disorder, neuromodulatory receptors provide a potential pharmacological target for prevention. Stimulation of muscarinic receptors promotes memory consolidation in several conditioning paradigms, an effect primarily associated with the M1 receptor (M1R). However, neither inhibiting nor genetically disrupting M1R impairs the consolidation of cued fear memory. Using the M1R agonist cevimeline and antagonist telenzepine, as well as M1R knock-out mice, we show here that M1R, along with ß2-adrenergic (ß2AR) and D5-dopaminergic (D5R) receptors, regulates the consolidation of cued fear memory by redundantly activating phospholipase C (PLC) in the basolateral amygdala (BLA). We also demonstrate that fear memory consolidation in the BLA is mediated in part by neuromodulatory inhibition of the M-current, which is conducted by KCNQ channels and is known to be inhibited by muscarinic receptors. Manipulating the M-current by administering the KCNQ channel blocker XE991 or the KCNQ channel opener retigabine reverses the effects on consolidation caused by manipulating ß2AR, D5R, M1R, and PLC. Finally, we show that cAMP and protein kinase A (cAMP/PKA) signaling relevant to this stage of consolidation is upstream of these neuromodulators and PLC, suggesting an important presynaptic role for cAMP/PKA in consolidation. These results support the idea that neuromodulatory regulation of ion channel activity and neuronal excitability is a critical mechanism for promoting consolidation well after acquisition has occurred.

The interaction potential of retigabine (ezogabine) with other antiepileptic drugs.

BACKGROUND: Retigabine is an antiepileptic drug (AED) that reduces neuronal excitability by enhancing neuronal KCNQ (Kv7) potassium channel activity. METHODS: This manuscript provides an overview of the drug-drug interaction potential of retigabine with other AEDs, using data collated from both in vitro work and clinical studies, either previously published or from relevant information collated during the development of retigabine. RESULTS: Retigabine is not a substrate for the major CYP enzymes and at clinically relevant concentrations there is little or no potential for retigabine to inhibit or induce the CYP enzymes or to inhibit the major renal drug transporters. The addition of retigabine to a range of existing AEDs showed little or no effect on the AED trough concentrations apart from a 20% decrease in lamotrigine concentrations. Results from a small phase II study showed that co-administration of valproic acid and topiramate had no impact on the PK of retigabine whereas carbamazepine and phenytoin increased the clearance of retigabine by approximately 27% and 36%, respectively. Conversely, a population PK analysis of combined data from phase I, II and III studies showed that none of the coadministered AEDs affected retigabine clearance apart from lamotrigine which lowered retigabine clearance by 6.7%. CONCLUSION: Retigabine is not metabolized by CYP isozymes and does not induce or inhibit these isozymes at clinically relevant concentrations. Therefore, retigabine is associated with a low potential for PK interactions with other drugs via CYP450. Overall, there was little or no potential for retigabine to interact with other available AEDs. Although some PK interactions were observed with lamotrigine, these are unlikely to be clinically relevant.

KV 7 channels are involved in hypoxia-induced vasodilatation of porcine coronary arteries.

BACKGROUND AND PURPOSE: Hypoxia causes vasodilatation of coronary arteries, but the underlying mechanisms are poorly understood. We hypothesized that hypoxia reduces intracellular Ca(2+) concentration ([Ca(2+)](i)) by opening of K channels and release of H2S. EXPERIMENTAL APPROACH: Porcine coronary arteries without endothelium were mounted for measurement of isometric tension and [Ca(2+)](i), and the expression of voltage-gated K channels K(V)7 channels (encoded by KCNQ genes) and large-conductance calcium-activated K channels (K(Ca)1.1) was examined. Voltage clamp assessed the role of K(V)7 channels in hypoxia. KEY RESULTS: Gradual reduction of oxygen concentration from 95 to 1% dilated the precontracted coronary arteries and this was associated with reduced [Ca(2+)](i) in PGF(2α) (10 µM)-contracted arteries whereas no fall in [Ca(2+)](i) was observed in 30 mM K-contracted arteries. Blockers of ATP-sensitive voltage-gated potassium channels and K(Ca)1.1 inhibited hypoxia-induced dilatation in PGF2α -contracted arteries; this inhibition was more marked in the presence of the K(v)7 channel blockers, XE991 and linopirdine, while a K(V)7.1 blocker, failed to change hypoxic vasodilatation. XE991 also inhibited H2S- and adenosine-induced vasodilatation. PCR revealed the expression of K(V)7.1, K(V)7.4, K(V)7.5 and K(Ca)1.1 channels, and K(Ca)1.1, K(V)7.4 and K(V)7.5 were also identified by immunoblotting. Voltage clamp studies showed the XE991-sensitive current was more marked in hypoxic conditions. CONCLUSION: The K(V)7.4 and K(V)7.5 channels, which we identified in the coronary arteries, appear to have a major role in hypoxia-induced vasodilatation. The voltage clamp results further support the involvement of K(V)7 channels in this vasodilatation. Activation of these K(V)7 channels may be induced by H2S and adenosine.