xe-991--anthracenone and linopirdine

The ionic mechanisms controlling the resting membrane potential (RMP) in superior cervical ganglion (SCG) neurons have been widely studied and the M-current (I

Topics: Adaptation, Physiological; Animals; Anthracenes; HEK293 Cells; Humans; Indoles; Ion Channel Gating; KCNQ Potassium Channels; Membrane Potentials; Mice; Neurons; Potassium Channels, Tandem Pore Domain; Pyridines; Riluzole; Superior Cervical Ganglion; Sympathetic Nervous System; Temperature; Tetraethylammonium; Tetrahydronaphthalenes; Tetrazoles

M-channel inhibitors, especially XE991, are being used increasingly in animal experiments; however, insufficient characterization of XE991 at times confounds the interpretation of results when using this compound. Here, we demonstrate that XE991 and linopirdine are state-dependent inhibitors that favor the activated-subunit of neuronal Kv7/KCNQ channels. We performed patch-clamp experiments on homomeric Kv7.2 or heteromeric Kv7.2/3 channels expressed in Chinese hamster ovary cells to characterize XE991 and linopirdine. Neither inhibitor was efficacious around the resting membrane potential of cells in physiologic conditions. Inhibition of Kv7.2 and Kv7.2/3 channels by XE991 was closely related with channel activation. When the voltage dependence of activation was left-shifted by retigabine or right-shifted by the mutation, Kv7.2(R214D), the shift in half-activation voltage proportionally coincided with the shift in the half-effective potential for XE991 inhibition. Inhibition kinetics during XE991 wash-in was facilitated at depolarized potentials. Ten-minute washout of XE991 resulted in ∼30% current recovery, most of which was attributed to surface transport of Kv7.2 channels. Linopirdine also exhibited similar inhibition characteristics, with the exception of near- complete current recovery after washout at depolarized potentials. Inhibition kinetics of both XE991 and linopirdine was not as sensitive to changes in voltage as would be predicted by open- channel inhibition. Instead, they were well explained by binding to a single activated subunit. The characteristics of XE991 and linopirdine should be taken into account when these M-channel inhibitors are used in experiments.

Topics: Animals; Anthracenes; Carbamates; CHO Cells; Cricetinae; Cricetulus; Indoles; KCNQ1 Potassium Channel; KCNQ2 Potassium Channel; Kinetics; Membrane Potentials; Mutation; Patch-Clamp Techniques; Phenylenediamines; Potassium Channel Blockers; Protein Subunits; Pyridines; Rats

The voltage-gated KV7 (KCNQ) potassium channels are activated by ischemia and involved in hypoxic vasodilatation. We investigated the effect of KV7 channel modulation on cardiac ischemia and reperfusion injury and its interaction with cardioprotection by ischemic preconditioning (IPC). Reverse-transcription polymerase chain reaction revealed expression of KV7.1, KV7.4, and KV7.5 in the left anterior descending rat coronary artery and all KV7 subtypes (KV7.1-KV7.5) in the left and right ventricles of the heart. Isolated hearts were subjected to no-flow global ischemia and reperfusion with and without IPC. Infarct size was quantified by 2,3,5-triphenyltetrazolium chloride staining. Two blockers of KV7 channels, XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone] (10 µM) and linopirdine (10 µM), reduced infarct size and exerted additive infarct reduction to IPC. An opener of KV7 channels, flupirtine (10 µM) abolished infarct size reduction by IPC. Hemodynamics were measured using a catheter inserted in the left ventricle and postischemic left ventricular recovery improved in accordance with reduction of infarct size and deteriorated with increased infarct size. XE991 (10 µM) reduced coronary flow in the reperfusion phase and inhibited vasodilatation in isolated small branches of the left anterior descending coronary artery during both simulated ischemia and reoxygenation. KV7 channels are expressed in rat coronary arteries and myocardium. Inhibition of KV7 channels exerts cardioprotection and opening of KV7 channels abrogates cardioprotection by IPC. Although safety issues should be further addressed, our findings suggest a potential role for KV7 blockers in the treatment of ischemia-reperfusion injury.

Topics: Aminopyridines; Animals; Anthracenes; Coronary Circulation; Coronary Vessels; Indoles; Ischemic Preconditioning, Myocardial; KCNQ Potassium Channels; Male; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Potassium Channel Blockers; Pyridines; Rats; Rats, Wistar; Vasodilation

There is strong evidence that M-currents modulate peripheral sensory afferent excitability and that altered M-current efficacy may underpin aspects of pain-induced nociceptor sensitization. Less clear is the role of the M-current in regulating central excitability within spinal dorsal horn nociceptive circuitry. In this study, an in vitro model of central hyperexcitability that uses the potassium channel blocker 4-aminopyridine (4-AP) to induce large amplitude population spikes and 4-12Hz oscillatory activity within rat spinal substantia gelatinosa (SG) has been used to determine the impact of pharmacological modulation of the M-current on central excitability. The M-current enhancers Retigabine (10 and 30μM) and Flupirtine (30μM) had a depressant effect on 4-AP-induced excitation in SG such that the frequency of large amplitude population spikes and the power of 4-12Hz oscillatory activity were both significantly reduced. In contrast, the M-current blockers XE911 (5μM) or Linopirdine (20μM) significantly potentiated 4-12Hz oscillatory activity as evidenced by significant increases in the parameters of power amplitude and power area but had no effect on large amplitude population spikes. These data indicate that pharmacological modulation of the M-current can influence excitability of nociceptive circuitry especially under conditions of central hyperexcitability, as may occur in chronic pain conditions. It is not clear whether these effects reflect a direct effect on interneurones localized to SG or indirectly via sensory afferent terminals. Nonetheless, these central actions should be taken into account alongside peripheral actions in terms of evaluating the potential therapeutic analgesic potency of novel M-current enhancers.

Topics: 4-Aminopyridine; Action Potentials; Aminopyridines; Analgesics; Animals; Animals, Newborn; Anthracenes; Biological Clocks; Biophysical Phenomena; Carbamates; Dose-Response Relationship, Drug; Drug Interactions; Female; In Vitro Techniques; Indoles; Male; Membrane Transport Modulators; Nerve Net; Neurons; Phenylenediamines; Potassium Channel Blockers; Pyridines; Rats; Rats, Wistar; Substantia Gelatinosa

KCNQ channels have been identified in arterial smooth muscle. However, their role in vasoregulation and chronic vascular diseases remains elusive. We tested the hypothesis that KCNQ channels contribute to periadventitial vasoregulation in peripheral skeletal muscle arteries by perivascular adipose tissue and that they represent novel targets to rescue periadventitial vascular dysfunction. Two models, spontaneously hypertensive rats and New Zealand obese mice, were studied using quantitative polymerase chain reaction, the patch-clamp technique, membrane potential measurements, myography of isolated vessels, and blood pressure telemetry. In rat Gracilis muscle arteries, anticontractile effects of perivascular fat were inhibited by the KCNQ channel blockers XE991 and linopirdine but not by other selective K(+) channel inhibitors. Accordingly, XE991 and linopirdine blocked noninactivating K(+) currents in freshly isolated Gracilis artery smooth muscle cells. mRNAs of several KCNQ channel subtypes were detected in those arteries, with KCNQ4 channels being dominant. In spontaneously hypertensive rats, the anticontractile effect of perivascular fat in Gracilis muscle arteries was largely reduced compared with Wistar rats. However, the vasodilator effects of KCNQ channel openers and mRNA expression of KCNQ channels were normal. Furthermore, KCNQ channel openers restored the diminished anticontractile effects of perivascular fat in spontaneously hypertensive rats. Moreover, KCNQ channel openers reduced arterial blood pressure in both models of hypertension independent of ganglionic blockade. Thus, our data suggest that KCNQ channels play a pivotal role in periadventitial vasoregulation of peripheral skeletal muscle arteries, and KCNQ channel opening may be an effective mechanism to improve impaired periadventitial vasoregulation and associated hypertension.

Topics: Adipose Tissue; Animals; Anthracenes; Arterial Pressure; Arteries; Indoles; Isometric Contraction; KCNQ Potassium Channels; Male; Membrane Potentials; Mice; Muscle, Skeletal; Potassium Channel Blockers; Pyridines; Rats; Rats, Inbred SHR; Rats, Wistar

Members of the K(v)7 family generate a subthreshold potassium current, termed M-current, that regulates the excitability of principal central neurons. Mutations in two members of this family, K(v)7.2 (KCNQ2) and K(v)7.3 (KCNQ3) are associated with a neurological disorder known as benign familial neonatal convulsion (BFNC). Despite their importance in normal and pathological brain function, developmental expression and function of these channels remains relatively unexplored. Here, we examined the temporal expression of K(v)7 channel subunits in zebrafish larvae using a real-time quantitative PCR approach. Spatial expression in the larval zebrafish brain was assessed using whole-mount in situ hybridization. The mRNA for three members of the K(v)7 family (KCNQ2, 3 and 5) is reported in zebrafish between two and seven days post-fertilization (dpf). Using electrophysiological techniques, we show that inhibitors of K(v)7 channels (linopirdine and XE991) induce burst discharge activity in immature zebrafish between 3 and 7 dpf. This abnormal electrical activity is blocked by a K(v)7 channel opener (retigabine) and was also shown to evoke convulsive behaviors in freely swimming zebrafish. Using morpholino oligonucleotides directed against KCNQ3, we confirmed a role for KCNQ channels in generation of electrical burst discharges. These results indicate that functional K(v)7 channels are expressed in the larval zebrafish nervous system and could play a direct role in generation of seizure activity.

Topics: Action Potentials; Analysis of Variance; Animals; Anthracenes; Anticonvulsants; Brain; Carbamates; Dose-Response Relationship, Drug; Electrophysiology; Gene Expression Regulation, Developmental; Indoles; KCNQ Potassium Channels; Larva; Locomotion; Morpholinos; Phenylenediamines; Potassium Channel Blockers; Pyridines; RNA, Messenger; Swimming; Zebrafish

Previous studies have suggested that muscarinic receptor activation modulates glutamatergic transmission. M-type potassium channels mediate the effects of muscarinic activation in the hippocampus, and it has been proposed that they modulate glutamatergic synaptic transmission. We tested whether M1 muscarinic receptor activation enhances glutamatergic synaptic transmission via the inhibition of the M-type potassium channels that are present in Schaffer collateral axons and terminals. Miniature excitatory postsynaptic currents (mEPSCs) were recorded from CA1 pyramidal neurons. The M1 receptor agonist, NcN-A-343, increased the frequency of mEPSCs, but did not alter their amplitude. The M-channel blocker XE991 and its analogue linopirdine also increased the frequency of mEPSCs. Flupirtine, which opens M-channels, had the opposite effect. XE991 did not enhance mEPSCs frequency in a calcium-free external medium. Blocking P/Q- and N-type calcium channels abolished the effect of XE991 on mEPSCs. These data suggested that the inhibition of M-channels increases presynaptic calcium-dependent glutamate release in CA1 pyramidal neurons. The effects of these agents on the membrane potentials of presynaptic CA3 pyramidal neurons were studied using current clamp recordings; activation of M1 receptors and blocking M-channels depolarized neurons and increased burst firing. The input resistance of CA3 neurons was increased by the application of McN-A-343 and XE991; these effects were consistent with the closure of M-channels. Muscarinic activation inhibits M-channels in CA3 pyramidal neurons and its efferents – Schaffer collateral, which causes the depolarization, activates voltage-gated calcium channels, and ultimately elevates the intracellular calcium concentration to increase the release of glutamate on CA1 pyramidal neurons.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Action Potentials; Aminopyridines; Animals; Anthracenes; Calcium; Calcium Channels, L-Type; Glutamic Acid; Indoles; Male; Neurons; Potassium Channel Blockers; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Synaptic Transmission

Voltage-gated K(+) channels are key regulators of neuronal excitability, playing major roles in setting resting membrane potential, repolarizing the cell membrane after action potentials and affecting transmitter release. The M-type channel or M-channel is a unique voltage- and ligand-regulated K(+) channel. It is composed of the molecular counterparts KCNQ2 and KCNQ3 (also named Kv7.2 and Kv7.3) channels and expressed in the soma and dendrites of neurons. The present investigation examined the hypothesis that KCNQ2/3 channels played a regulatory role in neuronal differentiation and maturation. In cultured mouse embryonic stem (ES) cells undergoing neuronal differentiation and primary embryonic (E15-17) hippocampal cultures, KCNQ2 and KCNQ3 channels and underlying M-currents were identified. Blocking of KCNQ channels in these cells for 5 days using the specific channel blocker XE991 (10 μM) or linopirdine (30 μM) significantly decreased synaptophysin and syntaxin expression without affecting cell viability. Chronic KCNQ2/3 channel block reduced the expression of vesicular GABA transporter (v-GAT), but not vesicular glutamate transporter (v-GluT). Enhanced ERK1/2 phosphorylation was observed in XE991- and linopirdine-treated neural progenitor cells. In electrophysiological recordings, cells undergoing chronic block of KCNQ2/3 channels showed normal amplitude of mPSCs while the frequency of mPSCs was reduced. On the other hand, KCNQ channel opener N-Ethylmaleimide (NEM, 2 μM) increased mPSC frequency. Fluorescent imaging using fluorescent styryl-dye FM4-64 revealed that chronic blockade of KCNQ2/3 channels decreased endocytosis but facilitated exocytosis. These data indicate that KCNQ2/3 channels participate in the regulation of neuronal differentiation and show a tonic regulation on pre-synaptic transmitter release and recycling in developing neuronal cells.

Topics: Analysis of Variance; Animals; Anthracenes; Blotting, Western; Cell Survival; Cells, Cultured; Electrophysiology; Endocytosis; Enzyme Inhibitors; Ethylmaleimide; Exocytosis; Hippocampus; Immunohistochemistry; Immunoprecipitation; Indoles; KCNQ Potassium Channels; Membrane Potentials; Mice; Miniature Postsynaptic Potentials; Neural Stem Cells; Neurogenesis; Potassium Channel Blockers; Pyridines; Qa-SNARE Proteins; Synaptic Vesicles; Synaptophysin

Striatal cholinergic interneurons show tonic spiking activity in the intact and sliced brain, which stems from intrinsic mechanisms. Because of it, they are also known as "tonically active neurons" (TANs). Another hallmark of TAN electrophysiology is a pause response to appetitive and aversive events and to environmental cues that have predicted these events during learning. Notably, the pause response is lost after the degeneration of dopaminergic neurons in animal models of Parkinson's disease. Moreover, Parkinson's disease patients are in a hypercholinergic state and find some clinical benefit in anticholinergic drugs. Current theories propose that excitatory thalamic inputs conveying information about salient sensory stimuli trigger an intrinsic hyperpolarizing response in the striatal cholinergic interneurons. Moreover, it has been postulated that the loss of the pause response in Parkinson's disease is related to a diminution of I(sAHP), a slow outward current that mediates an afterhyperpolarization following a train of action potentials. Here we report that I(sAHP) induces a marked spike-frequency adaptation in adult rat striatal cholinergic interneurons, inducing an abrupt end of firing during sustained excitation. Chronic loss of dopaminergic neurons markedly reduces I(sAHP) and spike-frequency adaptation in cholinergic interneurons, allowing them to fire continuously and at higher rates during sustained excitation. These findings provide a plausible explanation for the hypercholinergic state in Parkinson's disease. Moreover, a reduction of I(sAHP) may alter synchronization of cholinergic interneurons with afferent inputs, thus contributing to the loss of the pause response in Parkinson's disease.

Topics: Acetylcholine; Action Potentials; Analysis of Variance; Animals; Anthracenes; Apamin; Ascorbic Acid; Barium; Computer Simulation; Corpus Striatum; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; In Vitro Techniques; Indoles; Interneurons; Male; Models, Neurological; Neuroprotective Agents; Oxidopamine; Parkinsonian Disorders; Pyridines; Rats; Rats, Sprague-Dawley; Substantia Nigra

After block of Kv1- and Kv2-mediated K(+) currents in acutely dissociated neocortical pyramidal neurons from layers II/III of rat somatosensory and motor cortex, the remaining current is slowly activating and persistent. We used whole cell voltage clamp to show that the Kv7 blockers linopirdine and XE-991 blocked a current with similar kinetics to the current remaining after combined block of Kv1 and Kv2 channels. This current was sensitive to low doses of linopirdine and activated more slowly and at more negative potentials than Kv1- or Kv2-mediated current. The Kv7-mediated current decreased in amplitude with time in whole cell recordings, but in most cells the current was stable for several minutes. Current in response to a traditional M-current protocol was blocked by muscarine, linopirdine, and XE-991. Whole cell slice recordings revealed that the Q₁₀ for channel deactivation was ∼2.5. Sharp electrode current-clamp recordings from adult pyramidal cells demonstrated that block of Kv7-mediated current with XE-991 reduced rheobase, shortened the latency to firing to near rheobase current, induced more regular firing at low current intensity, and increased the rate of firing to a given current injection. XE-991 did not affect single action potentials or spike frequency adaptation. Application of XE-991 also eliminated subthreshold voltage oscillations and increased gain for low-frequency inputs (<10 Hz) without affecting gain for higher frequency inputs. These data suggest important roles for Kv7 channels in subthreshold regulation of excitability, generation of theta-frequency subthreshold oscillations, regulation of interspike intervals, and biasing selectivity toward higher frequency inputs.

Topics: Action Potentials; Animals; Anthracenes; Indoles; Ion Channel Gating; KCNQ Potassium Channels; Motor Cortex; Patch-Clamp Techniques; Potassium; Potassium Channel Blockers; Pyramidal Cells; Pyridines; Rats; Rats, Sprague-Dawley; Sensory Thresholds; Somatosensory Cortex; Subliminal Stimulation

Of the five human KCNQ (Kv7) channels, KCNQ1 with auxiliary subunit KCNE1 mediates the native cardiac I(Ks) current with mutations causing short and long QT cardiac arrhythmias. KCNQ4 mutations cause deafness. KCNQ2/3 channels form the native M-current controlling excitability of most neurons, with mutations causing benign neonatal febrile convulsions. Drosophila contains a single KCNQ (dKCNQ) that appears to serve alone the functions of all the duplicated mammalian neuronal and cardiac KCNQ channels sharing roughly 50-60% amino acid identity therefore offering a route to investigate these channels. Current information about the functional properties of dKCNQ is lacking therefore we have investigated these properties here. Using whole cell patch clamp electrophysiology we compare the biophysical and pharmacological properties of dKCNQ with the mammalian neuronal and cardiac KCNQ channels expressed in HEK cells. We show that Drosophila KCNQ (dKCNQ) is a slowly activating and slowly-deactivating K(+) current open at sub-threshold potentials that has similar properties to neuronal KCNQ2/3 with some features of the cardiac KCNQ1/KCNE1 accompanied by conserved sensitivity to a number of clinically relevant KCNQ blockers (chromanol 293B, XE991, linopirdine) and opener (zinc pyrithione). We also investigate the molecular basis of the differential selectivity of KCNQ channels to the opener retigabine and show a single amino acid substitution (M217W) can confer sensitivity to dKCNQ. We show dKCNQ has similar electrophysiological and pharmacological properties as the mammalian KCNQ channels, allowing future study of physiological and pathological roles of KCNQ in Drosophila and whole organism screening for new modulators of KCNQ channelopathies.

Topics: Animals; Anthracenes; Carbamates; Cell Line; Chromans; Drosophila; Drosophila Proteins; Electrophysiology; Humans; Indoles; KCNQ Potassium Channels; KCNQ1 Potassium Channel; KCNQ2 Potassium Channel; KCNQ3 Potassium Channel; Organometallic Compounds; Patch-Clamp Techniques; Phenylenediamines; Pyridines; Sulfonamides

There is consensus that muscarinic and nicotinic receptors expressed in vestibular hair cells and afferent neurons are involved in the efferent modulation of the electrical activity of the afferent neurons. However the underlying mechanisms of postsynaptic control in neurons are not well understood. In our work we show that the activation of muscarinic receptors in the vestibular neurons modulates the potassium M-current modifying the activity of afferent neurons. Whole-cell patch-clamp recordings were made on vestibular-afferent neurons isolated from Wistar rats (postnatal days 7-10) and held in primary culture (18-24 h). The M-current was studied during its deactivation after depolarizing voltage-clamp pulses. In 68% of the cells studied, those of larger capacitance, the M-current antagonists linopirdine and XE-991 reduced the amplitude of the M-current by 54%+/-7% and 50%+/-3%. The muscarinic-receptor agonist oxotremorine-M also significantly reduced the M-current by 58%+/-12% in the cells. The action of oxotremorine-M was blocked by atropine, thus indicating its cholinergic nature. The erg-channel blocker E-4031 did not significantly modify the M-current amplitude. In current-clamp experiments, linopirdine, XE-991, and oxotremorine-M modified the discharge response to current pulses from single spike to multiple spiking, reducing the adaptation of the electrical discharge. Our results indicate that large soma-size cultured vestibular-afferent neurons (most probably calyx-bearing neurons) express the M-current and that the modulation of this current by activation of muscarinic-receptor reduces its spike-frequency adaptation.

Topics: Action Potentials; Animals; Animals, Newborn; Anthracenes; Atropine; Biophysical Phenomena; Calcium Channel Blockers; Cells, Cultured; Indoles; Muscarinic Agonists; Muscarinic Antagonists; Neurons, Afferent; Oxotremorine; Patch-Clamp Techniques; Piperidines; Potassium Channel Blockers; Potassium Channels; Pyridines; Rats; Rats, Wistar; Receptors, Muscarinic; Vestibule, Labyrinth

Members of the K(v)7 voltage-gated K(+) channel family are important determinants of cardiac and neuronal membrane excitability. Recently, we and others have shown that K(v)7 channels are also crucial regulators of smooth muscle activity. The aim of the present study was to assess the K(v)7 expression in different parts of the murine gastrointestinal (GI) tract and to assess their functional roles by use of pharmacological agents. Of KCNQ/K(v)7 members, both KCNQ4/K(v)7.4 and KCNQ5/K(v)7.5 genes and proteins were the most abundantly expressed K(v)7 channels in smooth muscles throughout the GI tract. Immunohistochemical staining also revealed that K(v)7.4 and K(v)7.5 but not K(v)7.1 were expressed in the circular muscle layer of the colon. In segments of distal colon circular muscle exhibiting spontaneous phasic contractions, the nonselective K(v)7 blockers XE991 and linopirdine increased the integral of tension. Increases in the integral of tension were also observed under conditions of neuronal blockade. Similar effects, although less marked, were observed in the proximal colon. As expected, the K(v)7.1-selective blocker chromanol 293B had no effect in either type of segment. These data show that K(v)7.x especially K(v)7.4 and K(v)7.5 are expressed in different regions of the murine gastrointestinal tract and blockers of K(v)7 channels augment inherent contractile activity. Drugs that selectively block K(v)7.4/7.5 might be promising therapeutics for the treatment of motility disorders such as constipation associated with irritable bowel syndrome.

Topics: Animals; Anthracenes; Blotting, Western; Carbamates; Chromans; Colon; Gastrointestinal Tract; Immunohistochemistry; Indoles; KCNQ Potassium Channels; KCNQ1 Potassium Channel; Mice; Mice, Inbred BALB C; Muscle Contraction; Muscle, Smooth; Myography; Phenylenediamines; Potassium Channel Blockers; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides

While the synaptic mechanisms involved in the generation of in vitro network oscillations have been widely studied, little is known about the importance of voltage-gated currents during such activity. Here we study the role of the M-current (I(M)) in the modulation of network oscillations in the gamma-frequency range (20-80 Hz). Kv7/KCNQ subunits, the molecular correlates of I(M), are abundantly expressed in CA1 and CA3 pyramidal neurons, and I(M) is an important modulator of pyramidal neuron firing. Using hippocampal slices, we recorded field activity and pyramidal neuron action potential timing during kainate-induced gamma oscillations. Application of the specific I(M) blocker XE991 causes a significant reduction of gamma oscillation amplitude with no significant change in oscillation frequency. Concomitant CA3 pyramidal neuron recordings show a significant increase in action potential frequency during ongoing gamma oscillations after the application of XE991. This increase is associated with a significant loss of periodicity of pyramidal neuron action potentials relative to the phase of the gamma oscillations. Using dynamic clamp, we show that I(M) acts to improve the periodicity of action potential timing and to decrease action potential frequency. We further validate these results in a compartmental model of a pyramidal neuron. Our work suggests that I(M) modulates gamma oscillations by regulating the phasing of action potential firing in pyramidal neurons.

Topics: Action Potentials; Animals; Animals, Newborn; Anthracenes; Barium Compounds; Biological Clocks; Biophysics; Chlorides; Dose-Response Relationship, Drug; Electric Stimulation; Electroencephalography; Excitatory Amino Acid Agonists; Fourier Analysis; Hippocampus; In Vitro Techniques; Indoles; Kainic Acid; KCNQ Potassium Channels; Models, Neurological; Patch-Clamp Techniques; Potassium Channel Blockers; Pyramidal Cells; Pyridines; Rats; Rats, Sprague-Dawley

Within the developing Xenopus spinal cord, voltage-gated potassium (Kv) channel genes display different expression patterns, many of which occur in opposing dorsal-ventral gradients. Regional differences in Kv gene expression would predict different patterns of potassium current (I(Kv)) regulation. However, during the first 24 h of postmitotic differentiation, all primary spinal neurons undergo a temporally coordinated upregulation of I(Kv) density that shortens the duration of the action potential. Here, we tested whether spinal neurons demonstrate regional differences in I(Kv) regulation subsequent to action potential maturation. We show that two types of neurons, I and II, can be identified in culture on the basis of biophysical and pharmacological properties of I(Kv) and different firing patterns. Chronic increases in extracellular potassium, a signature of high neuronal activity, do not alter excitability properties of either neuron type. However, elevating extracellular potassium acutely after the period of action potential maturation leads to different changes in membrane properties of the two types of neurons. I(Kv) of type I neurons gains sensitivity to the blocker XE991, whereas type II neurons increase I(Kv) density and fire fewer action potentials. Moreover, by recording from neurons in vivo, we found that primary spinal neurons can be identified as either type I or type II. Type I neurons predominate in dorsal regions, whereas type II neurons localize to ventral regions. The findings reveal a dorsal-ventral gradient for I(Kv) regulation and a novel form of neuronal plasticity in spinal cord neurons.

Topics: Animals; Anthracenes; Body Patterning; Dose-Response Relationship, Radiation; Electric Stimulation; Embryo, Nonmammalian; Excitatory Amino Acid Antagonists; Gene Expression Regulation, Developmental; In Vitro Techniques; Indoles; Magnesium; Membrane Potentials; Neuronal Plasticity; Neurons; Patch-Clamp Techniques; Potassium; Potassium Channel Blockers; Potassium Channels, Voltage-Gated; Pyridines; Spinal Cord; Time Factors; Xenopus

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.

Topics: Action Potentials; Animals; Anthracenes; Calcium; Carbamates; Cerebral Cortex; Computer Simulation; Dendrites; Electrophysiology; Excitatory Postsynaptic Potentials; Extracellular Space; In Vitro Techniques; Indoles; KCNQ Potassium Channels; Male; Models, Neurological; Patch-Clamp Techniques; Phenylenediamines; Potassium Channel Blockers; Pyramidal Cells; Pyridines; Rats; Rats, Wistar; Synapses; Tissue Distribution

In neuronal tissue, KCNQ2-5 channels conduct the physiologically important M-current. In some neurones, the M-current may in addition be conducted partly by ERG potassium channels, which have widely overlapping expression with the KCNQ channel subunits. XE991 and linopiridine are known to be standard KCNQ potassium channel blockers. These compounds have been used in many different tissues as specific pharmacological tools to discern native currents conducted by KCNQ channels from other potassium currents. In this article, we demonstrate that ERG1-2 channels are also reversibly inhibited by XE991 in the micromolar range (EC(50) 107 microM for ERG1). The effect has been characterized in Xenopus laevis oocytes expressing ERG1-2 and in the mammalian HEK293 cell line stably expressing ERG1 channels. The IC(50) values for block of KCNQ channels by XE991 range 1-65 microM. In conclusion, great care should be taken when choosing the concentration of XE991 to use for experiments on native potassium channels or animal studies in order to be able to conclude on selective KCNQ channel-mediated effects.

Topics: Acetylcholine; Animals; Anthracenes; Carbamates; Cell Line; Chromans; Dose-Response Relationship, Drug; Electrophysiology; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Gene Expression; Indoles; Oocytes; Patch-Clamp Techniques; Phenylenediamines; Potassium Channel Blockers; Potassium Channels, Voltage-Gated; Pyridines; Xenopus laevis

This study represents a novel characterisation of KCNQ-encoded potassium channels in the vasculature using a variety of pharmacological and molecular tools to determine their role in contractility.. Reverse transcriptase polymerase chain reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse aorta, carotid artery, femoral artery and mesenteric artery using primers specific for all known KCNQ genes. RNA isolated from mouse heart and brain were used as positive controls. Pharmacological experiments were undertaken on segments from the same blood vessels to determine channel functionality. Immunocytochemical experiments were performed on isolated myocytes from thoracic aorta.. All blood vessels expressed KCNQ1, 4 and 5 with hitherto 'neuronal' KCNQ4 being, surprisingly, the most abundant. The correlated proteins K(v)7.1, K(v)7.4 and K(v)7.5 were identified in the cell membranes of aortic myocytes by immunocytochemistry. Application of three compounds known to activate K(v)7 channels, retigabine (2 -20 microM), flupirtine (20 microM) and meclofenamic acid (20 microM), relaxed vessels precontracted by phenylephrine or 1 mM 4-aminopyridine but had no effect on contractions produced by 60 mM KCl or the K(v)7 channel blocker XE991 (10 microM). All vessels tested contracted upon application of the K(v)7 channel blockers XE991 and linopirdine (0.1-10 microM).. Murine blood vessels exhibit a distinctive KCNQ expression profile with 'neuronal' KCNQ4 dominating. The ion channels encoded by KCNQ genes have a crucial role in defining vascular reactivity as K(v)7 channel blockers produced marked contractions whereas K(v)7 channel activators were effective vasorelaxants.

Topics: Aminopyridines; Animals; Anthracenes; Carbamates; Dose-Response Relationship, Drug; Gene Expression Profiling; Immunohistochemistry; Indoles; Isometric Contraction; KCNQ Potassium Channels; KCNQ1 Potassium Channel; Meclofenamic Acid; Mice; Mice, Inbred BALB C; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phenylenediamines; Potassium Channel Blockers; Potassium Channels; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; RNA

The M-current (current through Kv7 channels) is a low-threshold noninactivating potassium current that is suppressed by muscarinic agonists. Recent studies have shown its role in spike burst generation and intrinsic subthreshold theta resonance, both of which are important for memory function. However, little is known about its role in principal cells of the entorhinal cortex (EC). In this study, using whole cell patch recording techniques in a rat EC slice preparation, we have examined the effects of the M-current blockers linopirdine and XE991 on the membrane dynamics of principal cells in the EC. When the M-current was blocked, layer II nonstellate cells (non-SCs) and layer III cells switched from tonic discharge to intermittent firing mode, during which layer II non-SCs showed high-frequency short-duration spike bursts due to increased fast spike afterdepolarization (ADP). When three spikes were elicited at 50 Hz, these two types of cells reacted with a slow ADP that drove delayed firing. In contrast, layer II stellate cells (SCs) and layer V cells never displayed intermittent firing, bursting behavior, or delayed firing. Under the M-current block, intrinsic excitability increased significantly in layer III and layer V cells but not in layer II SCs and non-SCs. The M-current block also had contrasting effects on the subthreshold excitability, greatly suppressing the subthreshold membrane potential oscillations in layer V cells but not in layer II SCs. Modulation of the M-current thus shifts the firing behavior, intrinsic excitability, and subthreshold membrane potential oscillations of EC principal cells in a cell-type-dependent manner.

Topics: Action Potentials; Analysis of Variance; Animals; Animals, Newborn; Anthracenes; Dose-Response Relationship, Radiation; Electric Stimulation; Entorhinal Cortex; In Vitro Techniques; Indoles; Neurons; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels; Pyridines; Rats; Rats, Long-Evans

Synchronous neuronal firing can be induced in hippocampal slices in the absence of synaptic transmission by lowering extracellular Ca2+ and raising extracellular K+. However, the ionic mechanisms underlying this nonsynaptic synchronous firing are not well understood. In this study we have investigated the role of KCNQ/Kv7 channels in regulating this form of nonsynaptic bursting activity. Incubation of rat hippocampal slices in reduced (<0.2 mM) [Ca2+]o and increased (6.3 mM) [K+]o, blocked synaptic transmission, increased neuronal firing, and led to the development of spontaneous periodic nonsynaptic epileptiform activity. This activity was recorded extracellularly as large (4.7 +/- 1.9 mV) depolarizing envelopes with superimposed high-frequency synchronous population spikes. These intraburst population spikes initially occurred at a high frequency (about 120 Hz), which decayed throughout the burst stabilizing in the gamma-frequency band (30-80 Hz). Further increasing [K+]o resulted in an increase in the interburst frequency without altering the intraburst population spike frequency. Application of retigabine (10 microM), a Kv7 channel modulator, completely abolished the bursts, in an XE-991-sensitive manner. Furthermore, application of the Kv7 channel blockers, linopirdine (10 microM) or XE-991 (10 microM) alone, abolished the gamma frequency, but not the higher-frequency population spike firing observed during low Ca2+/high K+ bursts. These data suggest that Kv7 channels are likely to play a role in the regulation of synchronous population firing activity.

Topics: Action Potentials; Animals; Anthracenes; Calcium; Carbamates; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Hippocampus; In Vitro Techniques; Indoles; KCNQ Potassium Channels; Male; Neurons; Periodicity; Phenylenediamines; Potassium; Potassium Channel Blockers; Pyridines; Rats; Synaptic Transmission

KCNQ channels have been widely studied in the nervous system, heart and inner ear, where they have important physiological functions. Recent reports indicate that KCNQ channels may also be expressed in portal vein where they are suggested to influence spontaneous contractile activity. The biophysical properties of K+ currents mediated by KCNQ channels resemble a current underlying the resting K+ conductance and resting potential of pulmonary artery smooth muscle cells. We therefore investigated a possible role of KCNQ channels in regulating the function of pulmonary arteries by determining the ability of the selective KCNQ channel blockers, linopirdine and XE991, to promote pulmonary vasoconstriction.. The tension developed by rat and mouse intrapulmonary or mesenteric arteries was measured using small vessel myography. Contractile responses to linopirdine and XE991 were measured in intact and endothelium denuded vessels. Experiments were also carried out under conditions that prevent the contractile effects of nerve released noradrenaline or ATP, or block various Ca2+ influx pathways, in order to investigate the mechanisms underlying contraction.. Linopirdine and XE991 both contracted rat and mouse pulmonary arteries but had little effect on mesenteric arteries. In each case the maximum contraction was almost as large as the response to 50 mM K+. Linopirdine had an EC50 of around 1 microM and XE991 was almost 10-fold more potent. Neither removal of the endothelium nor exposure to phentolamine or alpha,beta-methylene ATP, to block alpha1-adrenoceptors or P2X receptors, respectively, affected the contraction. Contraction was abolished in Ca2+-free solution and in the presence of 1 microM nifedipine or 10 microM levcromakalim.. The KCNQ channel blockers are potent and powerful constrictors of pulmonary arteries. This action may be selective for the pulmonary circulation as mesenteric arteries showed little response. The results imply that the drugs act directly on smooth muscle cells and contraction requires voltage-dependent Ca2+ influx. It is concluded that the drugs probably act by blocking KCNQ channels in pulmonary artery myocytes, leading to membrane depolarization and Ca2+ influx through L-type Ca2+ channels. This implies a functional role for KCNQ channels in regulating the resting membrane potential of pulmonary artery myocytes.

Topics: Animals; Anthracenes; Endothelium, Vascular; In Vitro Techniques; Indoles; KCNQ Potassium Channels; Male; Mice; Mice, Inbred BALB C; Muscle Contraction; Muscle, Smooth; Potassium Channel Blockers; Pulmonary Artery; Pyridines; Rats; Rats, Sprague-Dawley; Vasoconstriction

The M-current is a slowly activating, non-inactivating potassium current that has been shown to be present in numerous cell types. In this study, KCNQ2, Q3 and Q5, the molecular correlates of M-current in neurons, were identified in the visceral sensory neurons of the nodose ganglia from rats through immunocytochemical studies. All neurons showed expression of each of the three proteins. In voltage clamp studies, the cognition-enhancing drug linopirdine (1-50 microM) and its analogue, XE991 (10 microM), quickly and irreversibly blocked a small, slowly activating current that had kinetic properties similar to KCNQ/M-currents. This current activated between -60 and -55 mV, had a voltage-dependent activation time constant of 208 +/- 12 ms at -20 mV, a deactivation time constant of 165 +/- 24 ms at -50 mV and V1/2 of -24 +/- 2 mV, values which are consistent with previous reports for endogenous M-currents. In current clamp studies, these drugs also led to a depolarization of the resting membrane potential at values as negative as -60 mV. Flupirtine (10-20 microM), an M-current activator, caused a 3-14 mV leftward shift in the current-voltage relationship and also led to a hyperpolarization of resting membrane potential. These data indicate that the M-current is present in nodose neurons, is activated at resting membrane potential and that it is physiologically important in regulating excitability by maintaining cells at negative voltages.

Topics: Aminopyridines; Animals; Anthracenes; Cells, Cultured; Dose-Response Relationship, Drug; Indoles; KCNQ Potassium Channels; KCNQ2 Potassium Channel; KCNQ3 Potassium Channel; Membrane Potentials; Neurons, Afferent; Nodose Ganglion; Potassium; Potassium Channel Blockers; Pyridines; Rats; Rats, Sprague-Dawley; Visceral Afferents

The intrinsic firing modes of adult CA1 pyramidal cells vary along a continuum of "burstiness" from regular firing to rhythmic bursting, depending on the ionic composition of the extracellular milieu. Burstiness is low in neurons exposed to a normal extracellular Ca(2+) concentration ([Ca(2+)](o)), but is markedly enhanced by lowering [Ca(2+)](o), although not by blocking Ca(2+) and Ca(2+)-activated K(+) currents. We show, using intracellular recordings, that burstiness in low [Ca(2+)](o) persists even after truncating the apical dendrites, suggesting that bursts are generated by an interplay of membrane currents at or near the soma. To study the mechanisms of bursting, we have constructed a conductance-based, one-compartment model of CA1 pyramidal neurons. In this neuron model, reduced [Ca(2+)](o) is simulated by negatively shifting the activation curve of the persistent Na(+) current (I(NaP)) as indicated by recent experimental results. The neuron model accounts, with different parameter sets, for the diversity of firing patterns observed experimentally in both zero and normal [Ca(2+)](o). Increasing I(NaP) in the neuron model induces bursting and increases the number of spikes within a burst but is neither necessary nor sufficient for bursting. We show, using fast-slow analysis and bifurcation theory, that the M-type K(+) current (I(M)) allows bursting by shifting neuronal behavior between a silent and a tonically active state provided the kinetics of the spike generating currents are sufficiently, although not extremely, fast. We suggest that bursting in CA1 pyramidal cells can be explained by a single compartment "square bursting" mechanism with one slow variable, the activation of I(M).

Topics: Action Potentials; Animals; Anthracenes; Calcium; Electrophysiology; Indoles; Male; Models, Neurological; Models, Theoretical; Neurons; Phenytoin; Potassium Channel Blockers; Potassium Channels; Pyramidal Cells; Pyridines; Rats; Rats, Inbred Strains; Riluzole; Sodium Channel Blockers; Sodium Channels

The M-current (I(M)), comprised of Kv7 channels, is a voltage-activated K+ conductance that plays a key role in the control of cell excitability. In hippocampal principal cells, I(M) controls action potential (AP) accommodation and contributes to the medium-duration afterhyperpolarization, but the role of I(M) in control of interneuron excitability remains unclear. Here, we investigated I(M) in hippocampal stratum oriens (SO) interneurons, both from wild-type and transgenic mice in which green fluorescent protein (GFP) was expressed in somatostatin-containing interneurons. Somatodendritic expression of Kv7.2 or Kv7.3 subunits was colocalized in a subset of GFP+ SO interneurons, corresponding to oriens-lacunosum moleculare (O-LM) cells. Under voltage clamp (VC) conditions at -30 mV, the Kv7 channel antagonists linopirdine/XE-991 abolished the I(M) amplitude present during relaxation from -30 to -50 mV and reduced the holding current (I(hold)). In addition, 0.5 mM tetraethylammonium reduced I(M), suggesting that I(M) was composed of Kv7.2-containing channels. In contrast, the Kv7 channel opener retigabine increased I(M) amplitude and I(hold). When strongly depolarized in VC, the linopirdine-sensitive outward current activated rapidly and comprised up to 20% of the total current. In current-clamp recordings from GFP+ SO cells, linopirdine induced depolarization and increased AP frequency, whereas retigabine induced hyperpolarization and arrested firing. In multicompartment O-LM interneuron models that incorporated I(M), somatodendritic placement of Kv7 channels best reproduced experimentally measured I(M). The models suggest that Kv3- and Kv7-mediated channels both rapidly activate during single APs; however, Kv3 channels control rapid repolarization of the AP, whereas Kv7 channels primarily control the interspike interval.

Topics: Action Potentials; Animals; Anthracenes; Carbamates; Cell Line; Computer Simulation; Dendrites; Dose-Response Relationship, Radiation; Drug Interactions; Electric Stimulation; Green Fluorescent Proteins; Hippocampus; Humans; Immunohistochemistry; In Vitro Techniques; Indoles; Interneurons; KCNQ Potassium Channels; Membrane Potentials; Mice; Mice, Transgenic; Models, Neurological; Patch-Clamp Techniques; Phenylenediamines; Potassium Channel Blockers; Pyridines; Tetraethylammonium; Transfection

Linopirdine was developed as a cognitive enhancing molecule and demonstrated to specifically block the potassium current generated by the brain specific KCNQ2-KCNQ3 proteins (M-channel). In this study we investigated the relevance of [(3)H]linopirdine binding in rat brain extracts to the interaction with the M-channel proteins. Our results confirm the presence of a high affinity site for [(3)H]linopirdine in rat brain tissues (KD = 10 nM) but we also identified a high affinity binding site for [(3)H]linopirdine in rat liver tissues (KD = 9 nM). Competition experiments showed that [(3)H]linopirdine is displaced by unlabelled linopirdine with comparable affinities from its binding sites on rat brain and rat liver membranes. [(3)H]linopirdine was completely displaced by a set of cytochrome P450 (CYP450) ligands suggesting that [(3)H]linopirdine binding to rat brain and liver membranes is linked to CYP450 interaction. The testing of CYP450 ligands on the M-channel activity, using a Rb(+) efflux assay on cells expressing the KCNQ2-KCNQ3 proteins, demonstrated that [(3)H]linopirdine binding results cannot be correlated to M-channel inhibition. The results obtained in this study demonstrate that [(3)H]linopirdine binding to rat brain and rat liver membranes is representative for CYP450 interaction and not relevant for the binding to the M-channel proteins.

Topics: Animals; Anthracenes; Binding, Competitive; Brain; Cell Membrane; Chlorides; CHO Cells; Cricetinae; Cricetulus; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Indoles; KCNQ3 Potassium Channel; Ketoconazole; Kinetics; Liver; Male; Miconazole; Potassium Channels, Voltage-Gated; Pyridines; Pyrilamine; Quinidine; Radioligand Assay; Rats; Rats, Sprague-Dawley; Rubidium; Tritium

The effect of the KCNQ channel blockers XE991, chromanol 293B and linopirdine, was studied on voltage-dependent K+ currents in smooth muscle cells dissociated freshly from mouse portal vein (mPV) and isometric tension recordings from whole mPV. Voltage clamp experiments showed XE991 inhibited an outward current in a concentration-dependent manner with an IC50 of 5.8 microM. Block was voltage independent. Chromanol 293B and linopirdine also blocked the voltage-dependent K+ current but were less potent than XE991. At least two components--a linear (I(linear)) and an outward relaxation (I(out))--contributed to the XE991-sensitive conductance. XE991-sensitive currents were sustained at all test potentials and XE991 inhibited the enhanced holding current at -60 mV produced by bathing cells in an external solution containing 36 mM KCl. Current clamp experiments in the perforated-patch configuration showed XE991 and linopirdine depolarised the resting membrane potential and augmented the evoked response in a concentration-dependent manner. In functional experiments the spontaneous contractile activity of the mPV was increased significantly by XE991 and linopirdine. The stimulatory effect of XE991 was not affected by the presence of 4-AP, glibenclamide nor paxilline. These data provide evidence for an important role for KCNQ channels in governing cellular excitability in mPV smooth muscle cells.

Topics: 4-Aminopyridine; Animals; Anthracenes; Chromans; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Indoles; KCNQ Potassium Channels; Kinetics; Membrane Potentials; Mice; Mice, Inbred BALB C; Muscle Contraction; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Portal Vein; Potassium; Potassium Channel Blockers; Pyridines; Sulfonamides

KCNQ channel subunits are widely expressed in peripheral and central neurons, where they give rise to a muscarinic-sensitive, subthreshold, and noninactivating K+ current (M-current). It is generally agreed that activation of KCNQ/M channels contributes to spike frequency adaptation during sustained depolarizations but is too slow to influence the repolarization of solitary spikes. This concept, however, is based mainly on experiments with muscarinic agonists, the multiple effects on membrane conductances of which may overshadow the distinctive effects of KCNQ/M channel block. Here, we have used selective modulators of KCNQ/M channels to investigate their role in spike electrogenesis in CA1 pyramidal cells. Solitary spikes were evoked by brief depolarizing current pulses injected into the neurons. The KCNQ/M channel blockers linopirdine and XE991 markedly enhanced the spike afterdepolarization (ADP) and, in most neurons, converted solitary ("simple") spikes to high-frequency bursts of three to seven spikes ("complex" spikes). Conversely, the KCNQ/M channel opener retigabine reduced the spike ADP and induced regular firing in bursting neurons. Selective block of BK or SK channels had no effect on the spike ADP or firing mode in these neurons. We conclude that KCNQ/M channels activate during the spike ADP and limit its duration, thereby precluding its escalation to a burst. Consequently, down-modulation of KCNQ/M channels converts the neuronal firing pattern from simple to complex spiking, whereas up-modulation of these channels exerts the opposite effect.

Topics: Action Potentials; Animals; Anthracenes; Carbamates; Hippocampus; In Vitro Techniques; Indoles; Neurons; Phenylenediamines; Potassium; Potassium Channel Blockers; Potassium Channels; Pyramidal Cells; Pyridines; Rats

The M-current (I(K(M))) is believed to modulate neuronal excitability by producing spike frequency adaptation (SFA). Inhibitors of M-channels, such as linopirdine and 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE991), enhance depolarization-induced transmitter release and improve learning performance in animal models. As such, they are currently being tested for their therapeutic potential for treating Alzheimer's disease. The activity of these blockers has been associated with the reduction of SFA and the depolarization of the membrane observed when I(K(M)) is inhibited. To test whether this is the case, the perforated patch technique was used to investigate the capacity of I(K(M)) inhibitors to alter the resting membrane potential and to reduce SFA in mouse superior cervical ganglion neurons in culture. Linopirdine and XE991 both proved to be potent blockers of I(K(M)) when the membrane potential was held at -30 mV (IC(50) 2.56 and 0.26 microM, respectively). However, their potency gradually declined upon membrane hyperpolarization and was almost null when the membrane potential was kept at -70 mV, indicating that their blocking activity was voltage dependent. Nevertheless, I(K(M)) could be inhibited at these hyperpolarized voltages by other inhibitors such as oxotremorine-methiodide and barium. Under current-clamp conditions, neither linopirdine (10 microM) nor XE991 (3 microM) was effective in reducing the SFA and both provoked only a small slowly developed depolarization of the membrane (2.27 and 3.0 mV, respectively). In contrast, both barium (1 mM) and oxotremorine-methiodide (10 microM) depolarized mouse superior cervical ganglion neurons by about 10 mV and reduced the SFA. In contrast to classical I(K(M)) inhibitors, the activity of linopirdine and XE991 on the I(K(M)) is voltage dependent and, thus, these newly developed I(K(M)) blockers do not reduce the SFA. These results may shed light on the mode of action of these putative cognition enhancers in vivo.

Topics: Action Potentials; Adaptation, Physiological; Animals; Animals, Newborn; Anthracenes; Barium; Cells, Cultured; Dose-Response Relationship, Drug; Indoles; Ion Channel Gating; Membrane Potentials; Mice; Muscarinic Agonists; Neural Inhibition; Neurons; Oxotremorine; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels; Pyridines; Rats; Rats, Sprague-Dawley; Superior Cervical Ganglion

Heterologous expression of KCNQ2 (Kv7.2) results in the formation of a slowly activating, noninactivating, voltage-gated potassium channel. Using a cell line that stably expresses KCNQ2, we developed a rubidium flux assay to measure the functional activity and pharmacological modulation of this ion channel. Rubidium flux was performed in a 96-well microtiter plate format; rubidium was quantified using an automated atomic absorption spectrometer to enable screening of 1000 data points/day. Cells accumulated rubidium at 37 degrees C in a monoexponential manner with t(1/2)=40min. Treating cells with elevated extracellular potassium caused membrane depolarization and stimulation of rubidium efflux through KCNQ2. The rate of rubidium efflux increased with increasing extracellular potassium: the t(1/2) at 50mM potassium was 5.1 min. Potassium-stimulated efflux was potentiated by the anticonvulsant drug retigabine (EC(50)=0.5 microM). Both potassium-induced and retigabine-facilitated efflux were blocked by TEA (IC(50)s=0.4 and 0.3mM, respectively) and the neurotransmitter release enhancers and putative cognition enhancers linopirdine (IC(50)s=2.3 and 7.1 microM, respectively) and XE991 (IC(50)s=0.3 and 0.9 microM, respectively). Screening a collection of ion channel modulators revealed additional inhibitors including clofilium (IC(50) = 27 microM). These studies extend the pharmacological profile of KCNQ2 and demonstrate the feasibility of using this assay system to rapidly screen for compounds that modulate the function of KCNQ2.

Topics: Anthracenes; Carbamates; Cell Line; Clone Cells; Dose-Response Relationship, Drug; Humans; Indoles; KCNQ2 Potassium Channel; Kinetics; Phenylenediamines; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Voltage-Gated; Potassium Chloride; Pyridines; Quaternary Ammonium Compounds; Rubidium; Spectrophotometry, Atomic; Tetraethylammonium; Transfection

Neuronal hyperexcitability is a feature of epilepsy and both inflammatory and neuropathic pain. M currents [IK(M)] play a key role in regulating neuronal excitability, and mutations in neuronal KCNQ2/3 subunits, the molecular correlates of IK(M), have previously been linked to benign familial neonatal epilepsy. Here, we demonstrate that KCNQ/M channels are also present in nociceptive sensory systems. IK(M) was identified, on the basis of biophysical and pharmacological properties, in cultured neurons isolated from dorsal root ganglia (DRGs) from 17-d-old rats. Currents were inhibited by the M-channel blockers linopirdine (IC50, 2.1 microm) and XE991 (IC50, 0.26 microm) and enhanced by retigabine (10 microm). The expression of neuronal KCNQ subunits in DRG neurons was confirmed using reverse transcription-PCR and single-cell PCR analysis and by immunofluorescence. Retigabine, applied to the dorsal spinal cord, inhibited C and Adelta fiber-mediated responses of dorsal horn neurons evoked by natural or electrical afferent stimulation and the progressive "windup" discharge with repetitive stimulation in normal rats and in rats subjected to spinal nerve ligation. Retigabine also inhibited responses to intrapaw application of carrageenan in a rat model of chronic pain; this was reversed by XE991. It is suggested that IK(M) plays a key role in controlling the excitability of nociceptors and may represent a novel analgesic target.

Topics: Animals; Anthracenes; Anura; Carbamates; Cells, Cultured; CHO Cells; Cricetinae; Disease Models, Animal; Ganglia, Spinal; Hyperalgesia; Indoles; Male; Neurons, Afferent; Oocytes; Pain; Pain Management; Pain Measurement; Patch-Clamp Techniques; Phenylenediamines; Potassium Channel Blockers; Potassium Channels; Pyridines; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Transfection

We investigated the neuroprotective properties of two M-type K+ channel blockers, linopirdine and its analog XE991, in rat sympathetic neurons deprived of nerve growth factor (NGF). Linopirdine and XE991 promoted sympathetic neuronal survival 48-72 hr after NGF withdrawal in a concentration-dependent manner. Both drugs prevented neuronal apoptosis by blocking the pathway leading to the release of cytochrome c and development of "competence-to-die" after NGF deprivation. Fura-2 Ca2+ imaging showed no significant difference in intracellular free Ca2+ ([Ca2+]i) in the presence or absence of NGF; linopirdine and XE991, on the other hand, caused membrane depolarization and increases in [Ca2+]i. Whole-cell recordings showed that linopirdine and XE991 selectively blocked the M current at neuroprotective concentrations, although they additionally inhibited other K+ currents at high concentrations. Membrane depolarization and [Ca2+]i increases induced by linopirdine and XE991 were blocked by the Na+ channel blocker tetrodotoxin (TTX) or by the L-type Ca2+ channel antagonist nifedipine. TTX and nifedipine also prevented the neuroprotection elicited by linopirdine or XE991. We propose that Na+ channel activation amplifies the membrane depolarization produced by M channel blockade and is essential for subsequent Ca2+ entry via the L-type Ca2+ channel. The interaction of these three classes of ion channels highlights an integrated anti-apoptosis mechanism in sympathetic neurons.

Topics: Animals; Anthracenes; Apoptosis; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Cell Survival; Cells, Cultured; Cytochrome c Group; Dose-Response Relationship, Drug; Indoles; Membrane Potentials; Mice; Nerve Growth Factor; Neurons; Neuroprotective Agents; Potassium Channel Blockers; Potassium Channels; Protein Transport; Pyridines; Rats; Sodium Channel Blockers; Sodium Channels; Superior Cervical Ganglion; Tetrodotoxin

The novel anti-ischemic compound, BMS-204352 ((3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indol-2-one)), strongly activates the voltage-gated K+ channel KCNQ5 in a concentration-dependent manner with an EC50 of 2.4 microM. At 10 microM, BMS-204352 increased the steady state current at -30 mV by 12-fold, in contrast to the 2-fold increase observed for the other KCNQ channels [Schrøder et al., 2001]. Retigabine ((D-23129; N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester) induced a smaller, yet qualitatively similar effect on KCNQ5. Furthermore, BMS-204352 (10 microM) did not significantly shift the KCNQ5 activation curves (threshold and potential for half-activation, V1/2), as observed for the other KCNQ channels. In the presence of BMS-204352, the activation and deactivation kinetics of the KCNQ5 currents were slowed as the slow activation time constant increased up to 10-fold. The M-current blockers, linopirdine (DuP 996; 3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one) and XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone), inhibited the activation of the KCNQ5 channel induced by the BMS-204352. Thus, BMS-204352 appears to be an efficacious KCNQ channels activator, and the pharmacological properties of the compound on the KCNQ5 channel seems to be different from what has been obtained on the other KCNQ channels.

Topics: Anthracenes; Carbamates; Cell Line; Dose-Response Relationship, Drug; Gene Expression; Humans; Indoles; KCNQ Potassium Channels; Membrane Potentials; Phenylenediamines; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Voltage-Gated; Pyridines

Human cloned KCNQ4 channels were stably expressed in HEK-293 cells and characterized with respect to function and pharmacology. Patch-clamp measurements showed that the KCNQ4 channels conducted slowly activating currents at potentials more positive than -60 mV. From the Boltzmann function fitted to the activation curve, a half-activation potential of -32 mV and an equivalent gating charge of 1.4 elementary charges was determined. The instantaneous current-voltage relationship revealed strong inward rectification. The KCNQ4 channels were blocked in a voltage-independent manner by the memory-enhancing M current blockers XE-991 and linopirdine with IC(50) values of 5.5 and 14 microM, respectively. The antiarrhythmic KCNQ1 channel blocker bepridil inhibited KCNQ4 with an IC(50) value of 9.4 microM, whereas clofilium was without significant effect at 100 microM. The KCNQ4-expressing cells exhibited average resting membrane potentials of -56 mV in contrast to -12 mV recorded in the nontransfected cells. In conclusion, the activation and pharmacology of KCNQ4 channels resemble those of M currents, and it is likely that the function of the KCNQ4 channel is to regulate the subthreshold electrical activity of excitable cells.

Topics: Animals; Anthracenes; Bepridil; Calcium Channel Blockers; Cells, Cultured; Electrophysiology; Humans; Indoles; Ion Channel Gating; KCNQ Potassium Channels; Kidney; Mammals; Membrane Potentials; Oocytes; Potassium Channels; Potassium Channels, Voltage-Gated; Pyridines; Transfection; Xenopus

Linopirdine and XE991, selective blockers of K(+) channels belonging to the KCNQ family, were applied to hair cells isolated from gerbil vestibular system and to hair cells in slices of pigeon crista. In type II hair cells, both compounds inhibited a slowly activating, slowly inactivating component of the macroscopic current recruited at potentials above -60 mV. The dissociation constants for linopirdine and XE991 block were <5 microM. A similar component of the current was also blocked by 50 microM capsaicin in gerbil type II hair cells. All three drugs blocked a current component that showed steady-state inactivation and a biexponential inactivation with time constants of approximately 300 ms and 4 s. Linopirdine (10 microM) reduced inward currents through the low-voltage-activated K(+) current in type I hair cells, but concentrations up to 200 microM had little effect on steady-state outward K(+) current in these cells. These results suggest that KCNQ channels may be present in amniote vestibular hair cells.

Topics: Animals; Anthracenes; Capsaicin; Columbidae; Electric Conductivity; Gerbillinae; Hair Cells, Vestibular; Indoles; Potassium; Potassium Channel Blockers; Pyridines

In an effort to improve the pharmacokinetic and pharmacodynamic properties of the cognition-enhancer linopirdine (DuP 996), a number of core structure analogues were prepared in which the 4-pyridyl pendant group was systematically replaced with 2-fluoro-4-pyridyl. This strategy resulted in the discovery of several compounds with improved activity in acetylcholine (ACh) release-enhancing assays, in vitro and in vivo. The most effective compound resulting from these studies, 10, 10-bis[(2-fluoro-4-pyridinyl)methyl]-9(10H)-anthracenone (9), is between 10 and 20 times more potent than linopirdine in increasing extracellular hippocampal ACh levels in the rat with a minimum effective dose of 1 mg/kg. In addition to superior potency, 9 possesses an improved pharmacokinetic profile compared to that of linopirdine. The half-life of 9 (2 h) in rats is 4-fold greater than that of linopirdine (0.5 h), and it showed a 6-fold improvement in brain-plasma distribution over linopirdine. On the basis of its pharmacologic, pharmacokinetic, absorption, and distribution properties, 9 (DMP543) has been advanced for clinical evaluation as a potential palliative therapeutic for treatment of Alzheimer's disease.

Topics: Acetylcholine; Alzheimer Disease; Animals; Anthracenes; Drug Evaluation, Preclinical; Hippocampus; In Vitro Techniques; Indoles; Male; Microdialysis; Nootropic Agents; Pyridines; Rats; Structure-Activity Relationship

The M-current regulates the subthreshold electrical excitability of many neurons, determining their firing properties and responsiveness to synaptic input. To date, however, the genes that encode subunits of this important channel have not been identified. The biophysical properties, sensitivity to pharmacological blockade, and expression pattern of the KCNQ2 and KCNQ3 potassium channels were determined. It is concluded that both these subunits contribute to the native M-current.

Topics: Adult; Animals; Anthracenes; Brain; Ganglia, Sympathetic; Gene Expression; Humans; Indoles; KCNQ2 Potassium Channel; KCNQ3 Potassium Channel; Kinetics; Neurons; Oocytes; Patch-Clamp Techniques; Potassium; Potassium Channels; Potassium Channels, Voltage-Gated; Pyridines; Rats; Sympathetic Nervous System; Tetraethylammonium; Xenopus

Linopirdine (3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one, DUP996) is an extensively studied representative of a class of cognition enhancing compounds that increase the evoked release of neurotransmitters. Recent studies suggest that these agents act through the blockade of specific K+ channels. We have recently identified more potent anthracenone analogs of linopirdine: 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE991) and 10,10-bis(2-fluoro-4-pyridinylmethyl)-9(10H)-anthracenone (DMP 543). Although linopirdine possesses an EC50 of 4.2 microM for enhancement of [3H]ACh release from rat brain slices, XE991 and DMP 543 have EC50S of 490 and 700 nM, respectively. In addition to greater in vitro potency relative to linopirdine, both compounds show greater in vivo potency and duration of action. Although 5 mg/kg (p.o.) linopirdine does not lead to statistically significant increases in hippocampal extracellular acetylcholine levels, 5 mg/kg (p.o.) XE991 leads to increases (maximal effect > 90% over baseline) which are sustained for 60 min. Moreover, DMP 543 at 1 mg/kg causes more than a 100% increase in acetylcholine levels with the effect lasting more than 3 hr. At doses relevant to their release-enhancing properties, the only overt symptom consistently observed was tremor, possible via a cholinergic mechanism. These results suggest that XE991 and DMP 543 may prove to be superior to linopirdine as Alzheimer's disease therapeutics. In addition, these agents should be useful pharmacological tools for probing the importance of particular ion channels in the control of neurotransmitter release.

Topics: Acetylcholine; Alzheimer Disease; Animals; Anthracenes; Dose-Response Relationship, Drug; Indoles; Male; Potassium Channel Blockers; Pyridines; Rats; Rats, Sprague-Dawley; Rats, Wistar