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Results for “KCNQ3 Potassium Channel”

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At least 55 records · Page 3Linked to original sources
PubMed2003

PIP(2) activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents.

KCNQ channels belong to a family of potassium ion channels with crucial roles in physiology and disease. Heteromers of KCNQ2/3 subunits constitute the neuronal M channels. Inhibition of M currents, by pathways that stimulate phospholipase C activity, controls excitability throughout the nervous system. Here we show that a common feature of all KCNQ channels is their activation by the signaling membrane phospholipid phosphatidylinositol-bis-phosphate (PIP(2)). We show that wortmannin, at concentrations that prevent recovery from receptor-mediated inhibition of M currents, blocks PIP(2) replenishment to the cell surface. Moreover, we identify a C-terminal histidine residue, immediately proximal to the plasma membrane, mutation of which renders M channels less sensitive to PIP(2) and more sensitive to receptor-mediated inhibition. Finally, native or recombinant channels inhibited by muscarinic agonists can be activated by PIP(2). Our data strongly suggest that PIP(2) acts as a membrane-diffusible second messenger to regulate directly the activity of KCNQ currents.

Androstadienes↗
PubMed2000

Neuronal KCNQ potassium channels: physiology and role in disease.

Humans have over 70 potassium channel genes, but only some of these have been linked to disease. In this respect, the KCNQ family of potassium channels is exceptional: mutations in four out of five KCNQ genes underlie diseases including cardiac arrhythmias, deafness and epilepsy. These disorders illustrate the different physiological functions of KCNQ channels, and provide a model for the study of the 'safety margin' that separates normal from pathological levels of channel expression. In addition, several KCNQ isoforms can associate to form heteromeric channels that underlie the M-current, an important regulator of neuronal excitability.

Animals↗
PubMed2005

Specific enhancement of SK channel activity selectively potentiates the afterhyperpolarizing current I(AHP) and modulates the firing properties of hippocampal pyramidal neurons.

SK channels are Ca2+-activated K+ channels that underlie after hyperpolarizing (AHP) currents and contribute to the shaping of the firing patterns and regulation of Ca2+ influx in a variety of neurons. The elucidation of SK channel function has recently benefited from the discovery of SK channel enhancers, the prototype of which is 1-EBIO. 1-EBIO exerts profound effects on neuronal excitability but displays a low potency and limited selectivity. This study reports the effects of DCEBIO, an intermediate conductance Ca2+-activated K+ channel modulator, and the effects of the recently identified potent SK channel enhancer NS309 on recombinant SK2 channels, neuronal apamin-sensitive AHP currents, and the excitability of CA1 neurons. NS309 and DCEBIO increased the amplitude and duration of the apamin-sensitive afterhyperpolarizing current without affecting the slow afterhyperpolarizing current in contrast to 1-EBIO. The potentiation by DCEBIO and NS309 was reversed by SK channel blockers. In current clamp experiments, NS309 enhanced the medium afterhyperpolarization (but not the slow afterhyperpolarization sAHP) and profoundly affected excitability by facilitating spike frequency adaptation in a frequency-independent manner. The potent and specific effect of NS309 on the excitability of CA1 pyramidal neurons makes this compound an ideal tool to assess the role of SK channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability.

Animals↗
PubMed2003

Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels.

To quantify the modulation of KCNQ2/3 current by [Ca2+]i and to test if calmodulin (CaM) mediates this action, simultaneous whole-cell recording and Ca2+ imaging was performed on CHO cells expressing KCNQ2/3 channels, either alone, or together with wild-type (wt) CaM, or dominant-negative (DN) CaM. We varied [Ca2+]i from <10 to >400 nM with ionomycin (5 microM) added to either a 2 mM Ca2+, or EGTA-buffered Ca2+-free, solution. Coexpression of wt CaM made KCNQ2/3 currents highly sensitive to [Ca2+]i (IC50 70 +/- 20 nM, max inhibition 73%, n = 10). However, coexpression of DN CaM rendered KCNQ2/3 currents largely [Ca2+]i insensitive (max inhibition 8 +/- 3%, n = 10). In cells without cotransfected CaM, the Ca2+ sensitivity was variable but generally weak. [Ca2+]i modulation of M current in superior cervical ganglion (SCG) neurons followed the same pattern as in CHO cells expressed with KCNQ2/3 and wt CaM, suggesting that endogenous M current is also highly sensitive to [Ca2+]i. Coimmunoprecipitations showed binding of CaM to KCNQ2-5 that was similar in the presence of 5 mM Ca2+ or 5 mM EGTA. Gel-shift analyses suggested Ca2+-dependent CaM binding to an "IQ-like" motif present in the carboxy terminus of KCNQ2-5. We tested whether bradykinin modulation of M current in SCG neurons uses CaM. Wt or DN CaM was exogenously expressed in SCG cells using pseudovirions or the biolistic "gene gun." Using both methods, expression of both wt CaM and DN CaM strongly reduced bradykinin inhibition of M current, but for all groups muscarinic inhibition was unaffected. Cells expressed with wt CaM had strongly reduced tonic current amplitudes as well. We observed similar [Ca2+]i rises by bradykinin in all the groups of cells, indicating that CaM did not affect Ca2+ release from stores. We conclude that M-type currents are highly sensitive to [Ca2+]i and that calmodulin acts as their Ca2+ sensor.

Animals↗
PubMed2004

Regulation of KCNQ2/KCNQ3 current by G protein cycling: the kinetics of receptor-mediated signaling by Gq.

Receptor-mediated modulation of KCNQ channels regulates neuronal excitability. This study concerns the kinetics and mechanism of M1 muscarinic receptor-mediated regulation of the cloned neuronal M channel, KCNQ2/KCNQ3 (Kv7.2/Kv7.3). Receptors, channels, various mutated G-protein subunits, and an optical probe for phosphatidylinositol 4,5-bisphosphate (PIP2) were coexpressed by transfection in tsA-201 cells, and the cells were studied by whole-cell patch clamp and by confocal microscopy. Constitutively active forms of Galphaq and Galpha11, but not Galpha13, caused a loss of the plasma membrane PIP2 and a total tonic inhibition of the KCNQ current. There were no further changes upon addition of the muscarinic agonist oxotremorine-M (oxo-M). Expression of the regulator of G-protein signaling, RGS2, blocked PIP2 hydrolysis and current suppression by muscarinic stimulation, confirming that the Gq family of G-proteins is necessary. Dialysis with the competitive inhibitor GDPbetaS (1 mM) lengthened the time constant of inhibition sixfold, decreased the suppression of current, and decreased agonist sensitivity. Removal of intracellular Mg2+ slowed both the development and the recovery from muscarinic suppression. When combined with GDPbetaS, low intracellular Mg2+ nearly eliminated muscarinic inhibition. With nonhydrolyzable GTP analogs, current suppression developed spontaneously and muscarinic inhibition was enhanced. Such spontaneous suppression was antagonized by GDPbetaS or GTP or by expression of RGS2. These observations were successfully described by a kinetic model representing biochemical steps of the signaling cascade using published rate constants where available. The model supports the following sequence of events for this Gq-coupled signaling: A classical G-protein cycle, including competition for nucleotide-free G-protein by all nucleotide forms and an activation step requiring Mg2+, followed by G-protein-stimulated phospholipase C and hydrolysis of PIP2, and finally PIP2 dissociation from binding sites for inositol lipid on the channels so that KCNQ current was suppressed. Further experiments will be needed to refine some untested assumptions.

Cell Membrane↗
PubMed2004

Retigabine stimulates human KCNQ2/Q3 channels in the presence of bupivacaine.

BACKGROUND: Inhibition of KCNQ2/Q3 channels may cause convulsion in humans. The interaction of bupivacaine with these channels is unknown. The anticonvulsant retigabine activates KCNQ2/Q3 channels and may reverse inhibitory actions of bupivacaine. Potassium channel stimulation may thus constitute a novel approach to treat local anesthetic-induced seizures. The aim of this study was to characterize bupivacaine effects on KCNQ2/Q3 channels and to investigate whether retigabine reverses the effects of the local anesthetic. METHODS: KCNQ2/Q3 channels were transiently expressed in Chinese hamster ovary cells. The effects of bupivacaine and retigabine were studied with the patch-clamp technique. RESULTS: Bupivacaine inhibited KCNQ2/Q3 channels in a concentration-dependent and reversible manner. The concentration-response curve was described by a Hill equation (IC50 = 173 +/- 7 microm, Hill coefficient = 1.4 +/- 0.1, mean +/- SEM, n = 37). The inhibitory effect did not differ between bupivacaine and levobupivacaine (42 +/- 4%, n = 7, versus 42 +/- 5%, n = 10; P > 0.05). Ropivacaine was four times less potent than bupivacaine. The inhibition of KCNQ2/Q3 channels by bupivacaine resulted in a significant and reversible depolarization of the membrane potential. Retigabine (300 nm-10 microm) reversed the inhibitory action of bupivacaine on KCNQ2/Q3 channels as well as the depolarization of the membrane potential. CONCLUSIONS: The anticonvulsant retigabine at nanomolar concentrations reverses the inhibitory effect of micromolar concentrations of bupivacaine. Our results allow the hypothesis that activation of KCNQ2/Q3 channels by retigabine may offer a novel therapeutic approach for the treatment of bupivacaine-induced seizures.

Anesthetics, Local↗
PubMed2004

Mice carrying the szt1 mutation exhibit increased seizure susceptibility and altered sensitivity to compounds acting at the m-channel.

PURPOSE: Mutations in the genes that encode subunits of the M-type K+ channel (KCNQ2/KCNQ3) and nicotinic acetylcholine receptor (CHRNA4) cause epilepsy in humans. The purpose of this study was to examine the effects of the Szt1 mutation, which not only deletes most of the C-terminus of mouse Kcnq2, but also renders the Chnra4 and Arfgap-1 genes hemizygous, on seizure susceptibility and sensitivity to drugs that target the M-type K+ channel. METHODS: The proconvulsant effects of the M-channel blocker linopirdine (LPD) and anticonvulsant effects of the M-channel enhancer retigabine (RGB) were assessed by electroconvulsive threshold (ECT) testing in C57BL/6J-Szt1/+ (Szt1) and littermate control C57BL/6J+/+ (B6) mice. The effects of the Szt1 mutation on minimal clonic, minimal tonic hindlimb extension, and partial psychomotor seizures were evaluated by varying stimulation intensity and frequency. RESULTS: Szt1 mouse seizure thresholds were significantly reduced relative to B6 littermates in the minimal clonic, minimal tonic hindlimb extension, and partial psychomotor seizure models. Mice were injected with LPD and RGB and subjected to ECT testing. In the minimal clonic seizure model, Szt1 mice were significantly more sensitive to LPD than were B6 mice [median effective dose (ED50) = 3.4 +/- 1.1 mg/kg and 7.6 +/- 1.0 mg/kg, respectively]; in the partial psychomotor seizure model, Szt1 mice were significantly less sensitive to RGB than were B6 mice (ED50 = 11.6 +/- 1.4 mg/kg and 3.4 +/- 1.3 mg/kg, respectively). CONCLUSIONS: These results suggest that the Szt1 mutation alters baseline seizure susceptibility and pharmacosensitivity in a naturally occurring mouse model.

Animals↗
PubMed2003

Effect of the KCNQ potassium channel opener retigabine on single KCNQ2/3 channels expressed in CHO cells.

KCNQ2/3 potassium channel subunits were co-expressed in Chinese hamster ovary (CHO) cells and currents through single channels recorded using cell-attached patches. Channels had a similar slope conductance in the presence (8.04 +/- 0.02 pS) and absence (7.6 +/- 0.01 pS) of 10 microM retigabine. The mean maximal open probability (Po) for single KCNQ2/3 channels was 0.13 +/- 0.02, with a half-maximal Po potential (Vo) of -28.7 +/- 1.4 mV for control recordings. Retigabine increased mean maximal Po to 0.38 +/- 0.04 and produced a hyperpolarising shift of Vo to -40.1 +/- 3.4 mV. Single KCNQ2/3 channels have multiple voltage-dependent kinetic components in their activity (CL-OS-CM-OL-CS; C = closed, O = open, L = long, S = short, M = medium), giving short, medium and long closed times (tauCS, tauCM, tauCL) and short and long open times (tauOS and tauOL). In the presence of retigabine at 0 mV the combined duration and contributions of the longest closed time tauCL decreased tenfold, while the short and long open times increased fourfold and twofold, respectively. Thus, steady-state kinetics were modified to favour the open channel configuration.

Animals↗
PubMed2006

KCNQ/M-currents contribute to the resting membrane potential in rat visceral sensory neurons.

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.

Aminopyridines↗
PubMed2006

Angiotensin II regulates neuronal excitability via phosphatidylinositol 4,5-bisphosphate-dependent modulation of Kv7 (M-type) K+ channels.

Voltage-gated Kv7 (KCNQ) channels underlie important K+ currents in many different types of cells, including the neuronal M current, which is thought to be modulated by muscarinic stimulation via depletion of membrane phosphatidylinositol 4,5-bisphosphate (PIP2). We studied the role of modulation by angiotensin II (angioII) of M current in controlling discharge properties of superior cervical ganglion (SCG) sympathetic neurons and the mechanism of action of angioII on cloned Kv7 channels in a heterologous expression system. In SCG neurons, which endogenously express angioII AT1 receptors, application of angioII for 2 min produced an increase in neuronal excitability and a decrease in spike-frequency adaptation that partially returned to control values after 10 min of angioII exposure. The increase in excitability could be simulated in a computational model by varying only the amount of M current. Using Chinese hamster ovary (CHO) cells expressing cloned Kv7.2 + 7.3 heteromultimers and AT1 receptors studied under perforated patch clamp, angioII induced a strong suppression of the Kv7.2/7.3 current that returned to near baseline within 10 min of stimulation. The suppression was blocked by the phospholipase C inhibitor edelfosine. Under whole-cell clamp, angioII moderately suppressed the Kv7.2/7.3 current whether or not intracellular Ca2+ was clamped or Ca2+ stores depleted. Co-expression of PI(4)5-kinase in these cells sharply reduced angioII inhibition, but did not augment current amplitudes, whereas co-expression of a PIP2 5'-phosphatase sharply reduced current amplitudes, and also blunted the inhibition. The rebound of the current seen in perforated-patch recordings was blocked by the PI4-kinase inhibitor, wortmannin (50 microM), suggesting that PIP2 re-synthesis is required for current recovery. High-performance liquid chromatographic analysis of anionic phospholipids in CHO cells stably expressing AT1 receptors revealed that PIP2 and phosphatidylinositol 4-phosphate levels are to be strongly depleted after 2 min of stimulation with angioII, with a partial rebound after 10 min. The results of this study establish how angioII modulates M channels, which in turn affects the integrative properties of SCG neurons.

Action Potentials↗
PubMed2000

Retigabine, a novel anti-convulsant, enhances activation of KCNQ2/Q3 potassium channels.

Retigabine [N-(2-amino-4-[fluorobenzylamino]-phenyl) carbamic acid; D-23129] is a novel anticonvulsant, unrelated to currently available antiepileptic agents, with activity in a broad range of seizure models. In the present study, we sought to determine whether retigabine could enhance current through M-like currents in PC12 cells and KCNQ2/Q3 K(+) channels expressed in Chinese hamster ovary cells (CHO-KCNQ2/Q3). In differentiated PC12 cells, retigabine enhanced a linopirdine-sensitive current. The effect of retigabine was associated with a slowing of M-like tail current deactivation in these cells. Retigabine (0.1 to 10 microM) induced a potassium current and hyperpolarized CHO cells expressing KCNQ2/Q3 cells but not in wild-type cells. Retigabine-induced currents in CHO-KCNQ2/Q3 cells were inhibited by 60.6 +/- 11% (n = 4) by the KCNQ2/Q3 blocker, linopirdine (10 microM), and 82.7 +/- 5.4% (n = 4) by BaCl(2) (10 mM). The mechanism by which retigabine enhanced KCNQ2/Q3 currents involved large, drug-induced, leftward shifts in the voltage dependence of channel activation (-33.1 +/- 2.6 mV, n = 4, by 10 microM retigabine). Retigabine shifted the voltage dependence of channel activation with an EC(50) value of 1.6 +/- 0.3 microM (slope factor was 1.2 +/- 0.1, n = 4 to 5 cells per concentration). Retigabine (0.1 to 10 microM) also slowed the rate of channel deactivation, predominantly by increasing the contribution of a slowly deactivating tail current component. Our findings identify KCNQ2/Q3 channels as a molecular target for retigabine and suggest that activation of KCNQ2/Q3 channels may be responsible for at least some of the anticonvulsant activity of this agent.

Animals↗
PubMed2006

Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels.

To resolve the controversy about messengers regulating KCNQ ion channels during phospholipase C-mediated suppression of current, we designed translocatable enzymes that quickly alter the phosphoinositide composition of the plasma membrane after application of a chemical cue. The KCNQ current falls rapidly to zero when phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PI(4,5)P2] is depleted without changing Ca2+, diacylglycerol, or inositol 1,4,5-trisphosphate. Current rises by 30% when PI(4,5)P2 is overproduced and does not change when phosphatidylinositol 3,4,5-trisphosphate is raised. Hence, the depletion of PI(4,5)P2 suffices to suppress current fully, and other second messengers are not needed. Our approach is ideally suited to study biological signaling networks involving membrane phosphoinositides.

Animals↗
PubMed2002

Molecular and functional characterization of ERG, KCNQ, and KCNE subtypes in rat stomach smooth muscle.

Contribution of K(+) channels derived from the expression of ERG, KCNQ, and KCNE subtypes, which are responsible for rapidly and slowly activating delayed rectifier K(+) currents (I(Kr) and I(Ks), respectively) in cardiac myocytes, to membrane currents was examined in stomach circular smooth muscle cells (SMCs). The region-qualified multicell RT-PCR showed that ERG1/KCNE2 transcripts were expressed in rat stomach fundus and antrum SMCs and that KCNQ1/KCNE1 transcripts were expressed in antrum but not fundus. Western blotting and immunocytochemical analyses indicate that ERG1 proteins were substantially expressed in both regions, whereas KCNE1 proteins were faintly expressed in antrum and not in fundus. Both I(Kr)- and I(Ks)-like currents susceptible to E-4031 and indapamide, respectively, were identified in circular SMCs of antrum but only I(Kr)-like current was identified in fundus. It is strongly suggested that I(Kr)- and I(Ks)-like currents functionally identified in rat stomach SMCs are attributable to the expression of ERG1/KCNE2 and KCNQ1/KCNE1, respectively. The membrane depolarization by 1 microM E-4031 indicates the contribution of K(+) channels encoded by ERG1/KCNE2 to the resting membrane potential in stomach SMCs.

Animals↗
PubMed2003

Experiments to test the role of phosphatidylinositol 4,5-bisphosphate in neurotransmitter-induced M-channel closure in bullfrog sympathetic neurons.

Various neurotransmitters excite neurons by suppressing a ubiquitous, voltage-dependent, noninactivating K+ conductance called the M-conductance (gM). In bullfrog sympathetic ganglion neurons the suppression of gM by the P2Y agonist ATP involves phospholipase C (PLC). The present results are consistent with the involvement of the lipid and inositol phosphate cycles in the effects of both P2Y and muscarinic cholinergic agonists on gM. Impairment of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) with the phosphatidylinositol 4-kinase inhibitor wortmannin (10 microm) slowed or blocked the recovery of agonist-induced gM suppression. This effect could not be attributed to an action of wortmannin on myosin light chain kinase or on phosphatidylinositol 3-kinase. Inhibition of PIP2 synthesis at an earlier point in the lipid cycle by the use of R59022 (40 microm) to inhibit diacylglycerol kinase also slowed the rate of recovery of successive ATP responses. This effect required several applications of agonist to deplete levels of various phospholipid intermediates in the lipid cycle. PIP2 antibodies attenuated the suppression of gM by agonists. Intracellular application of 20 microm PIP2 slowed the rundown of KCNQ2/3 currents expressed in COS-1 or tsA-201 cells, and 100 microm PIP2 produced a small potentiation of native M-current bullfrog sympathetic neurons. These are the results that might be expected if agonist-induced activation of PLC and the concomitant depletion of PIP2 contribute to the excitatory action of neurotransmitters that suppress gM.

1-Phosphatidylinositol 4-Kinase↗