Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Regulation of KCNQ2/KCNQ3 current by G protein cycling: the kinetics of receptor-mediated signaling by Gq. 1517 19

The relationship between receptor-induced membrane phosphatidylinositol-4'5'-bisphosphate (PIP2) hydrolysis and M-current inhibition was assessed in single-dissociated rat sympathetic neurons by simultaneous or parallel recording of membrane current and membrane-to-cytosol translocation of the fluorescent PIP2/inositol 1,4,5-trisphosphate (IP3)-binding peptide green fluorescent protein-tagged pleckstrin homology domain of phospholipase C (GFP-PLCdelta-PH). The muscarinic receptor agonist oxotremorine-M produced parallel time- and concentration-dependent M-current inhibition and GFP-PLCdelta-PH translocation; bradykinin also produced parallel time-dependent inhibition and translocation. Phosphatidylinositol-4-phosphate-5-kinase (PI5-K) overexpression reduced both M-current inhibition and GFP-PLCdelta-PH translocation by both oxotremorine-M and bradykinin. These effects were partly reversed by wortmannin, which inhibits phosphatidylinositol-4-kinase (PI4-K). PI5-K overexpression also reduced the inhibitory action of oxotremorine-M on PIP2-gated G-protein-gated inward rectifier (Kir3.1/3.2) channels; bradykinin did not inhibit these channels. Overexpression of neuronal calcium sensor-1 protein (NCS-1), which increases PI4-K activity, did not affect responses to oxotremorine-M but reduced both fluorescence translocation and M-current inhibition by bradykinin. Using an intracellular IP3 membrane fluorescence-displacement assay, initial mean concentrations of membrane [PIP2] were estimated at 261 microm (95% confidence limit; 192-381 microm), rising to 693 microm (417-1153 microm) in neurons overexpressing PI5-K. Changes in membrane [PIP2] during application of oxotremorine-M were calculated from fluorescence data. The results, taken in conjunction with previous data for KCNQ2/3 (Kv7.2/Kv7.3) channel gating by PIP2 (Zhang et al., 2003), accorded with the hypothesis that the inhibitory action of oxotremorine-M on M current resulted from depletion of PIP2. The effects of bradykinin require additional components of action, which might involve IP3-induced Ca2+ release and consequent M-channel inhibition (as proposed previously) and stimulation of PIP2 synthesis by Ca2+-dependent activation of NCS-1.
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PMID:Relationship between membrane phosphatidylinositol-4,5-bisphosphate and receptor-mediated inhibition of native neuronal M channels. 1580 Jan 95

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.
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PMID:Angiotensin II regulates neuronal excitability via phosphatidylinositol 4,5-bisphosphate-dependent modulation of Kv7 (M-type) K+ channels. 1677 36

M-channels are voltage-gated K+ channels that regulate the excitability of many neurons. They are composed of Kv7 (KCNQ) family subunits, usually Kv7.2 + Kv7.3. Native M-channels and expressed Kv7.2 + 7.3 channels are inhibited by stimulating G(q/11)-coupled receptors - prototypically the M1 muscarinic acetylcholine receptor (M1-mAChR). The channels require membrane phosphatidylinositol-4,5-bisphosphate (PIP(2)) to open and the effects of mAChR stimulation result primarily from the reduction in membrane PIP(2) levels following G(q)/phospholipase C-catalysed PIP(2) hydrolysis. However, in sympathetic neurons, M-current inhibition by bradykinin appears to be mediated through the release and action of intracellular Ca(2)+ by inositol-1,4,5-trisphosphate (IP(3)), a product of PIP(2) hydrolysis, rather than by PIP(2) depletion. We have therefore compared the effects of bradykinin and oxotremorine-M (a muscarinic agonist) on membrane PIP(2) in sympathetic neurons using a fluorescently tagged mutated C-domain of the PIP(2) binding probe, 'tubby'. In concentrations producing equal M-current inhibition, bradykinin produced about one-quarter of the reduction in PIP(2) produced by oxotremorine-M, but equal reduction when PIP(2) synthesis was blocked with wortmannin. Likewise, wortmannin restored bradykinin-induced M-current inhibition when Ca(2)+ release was prevented with thapsigargin. Thus, inhibition by bradykinin can use product (IP(3)/Ca(2)+)-dependent or substrate (PIP(2)) dependent mechanisms, depending on Ca(2)+ availability and PIP(2) synthesis rates.
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PMID:Regulation of M(Kv7.2/7.3) channels in neurons by PIP(2) and products of PIP(2) hydrolysis: significance for receptor-mediated inhibition. 1739 26

KCNQ genes encode five Kv7 K(+) channel subunits (Kv7.1-Kv7.5). Four of these (Kv7.2-Kv7.5) are expressed in the nervous system. Kv7.2 and Kv7.3 are the principal molecular components of the slow voltage-gated M-channel, which widely regulates neuronal excitability, although other subunits may contribute to M-like currents in some locations. M-channels are closed by receptors coupled to Gq such as M1 and M3 muscarinic receptors; this increases neuronal excitability and underlies some forms of cholinergic excitation. Muscarinic closure results from activation of phospholipase C and consequent hydrolysis and depletion of membrane phosphatidylinositol-4,5-bisphosphate, which is required for channel opening. Some effects of M-channel closure, determined from transmitter action, selective blocking drugs (linopirdine and XE991) and KCNQ2 gene disruption or manipulation, are as follows: (i) in sympathetic neurons: facilitation of repetitive discharges and conversion from phasic to tonic firing; (ii) in sensory nociceptive systems: facilitation of A-delta peripheral sensory fibre responses to noxious heat; and (iii) in hippocampal pyramidal neurons: facilitation of repetitive discharges, enhanced after-depolarization and burst-firing, and induction of spontaneous firing through a reduction of action potential threshold at the axon initial segment. Several drugs including flupirtine and retigabine enhance neural Kv7/M-channel activity, principally through a hyperpolarizing shift in their voltage gating. In consequence they reduce neural excitability and can inhibit nociceptive stimulation and transmission. Flupirtine is in use as a central analgesic; retigabine is under clinical trial as a broad-spectrum anticonvulsant and is an effective analgesic in animal models of chronic inflammatory and neuropathic pain.
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PMID:Neural KCNQ (Kv7) channels. 1929 56

G protein-coupled receptors initiate signaling cascades. M(1) muscarinic receptor (M(1)R) activation couples through Galpha(q) to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP(2)). Depletion of PIP(2) closes PIP(2)-requiring Kv7.2/7.3 potassium channels (M current), thereby increasing neuronal excitability. This modulation of M current is relatively slow (6.4 s to reach within 1/e of the steady-state value). To identify the rate-limiting steps, we investigated the kinetics of each step using pairwise optical interactions likely to represent fluorescence resonance energy transfer for M(1)R activation, M(1)R/Gbeta interaction, Galpha(q)/Gbeta separation, Galpha(q)/PLC interaction, and PIP(2) hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M(1)R activation (<100 ms) and M(1)R/Gbeta interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. Galpha(q)/Gbeta separation and Galpha(q)/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP(2) hydrolysis (6.7 s) occurs on the timescale of M current suppression. Overexpression of PLC accelerates the rate of M current suppression threefold (to 2.0 s) to become nearly contemporaneous with Galpha(q)/PLC interaction. Evidently, channel release of PIP(2) and closure are rapid, and the availability of active PLC limits the rate of M current suppression.
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PMID:Fluorescence changes reveal kinetic steps of muscarinic receptor-mediated modulation of phosphoinositides and Kv7.2/7.3 K+ channels. 1933 18

Kv7 K(+)-channel subunits differ in their apparent affinity for PIP(2) and are differentially expressed in nerve, muscle, and epithelia in accord with their physiological roles in those tissues. To investigate how PIP(2) affinity affects the response to physiological stimuli such as receptor stimulation, we exposed homomeric and heteromeric Kv7.2, 7.3, and 7.4 channels to a range of concentrations of the muscarinic receptor agonist oxotremorine-M (oxo-M) in a heterologous expression system. Activation of M(1) receptors by oxo-M leads to PIP(2) depletion through G(q) and phospholipase C (PLC). Chinese hamster ovary cells were transiently transfected with Kv7 subunits and M(1) receptors and studied under perforated-patch voltage clamp. For Kv7.2/7.3 heteromers, the EC(50) for current suppression was 0.44 +/- 0.08 microM, and the maximal inhibition (Inhib(max)) was 74 +/- 3% (n = 5-7). When tonic PIP(2) abundance was increased by overexpression of PIP 5-kinase, the EC(50) was shifted threefold to the right (1.2 +/- 0.1 microM), but without a significant change in Inhib(max) (73 +/- 4%, n = 5). To investigate the muscarinic sensitivity of Kv7.3 homomers, we used the A315T pore mutant (Kv7.3(T)) that increases whole-cell currents by 30-fold without any change in apparent PIP(2) affinity. Kv7.3(T) currents had a slightly right-shifted EC(50) as compared with Kv7.2/7.3 heteromers (1.0 +/- 0.8 microM) and a strongly reduced Inhib(max) (39 +/- 3%). In contrast, the dose-response curve of homomeric Kv7.4 channels was shifted considerably to the left (66 +/- 8 nM), and Inhib(max) was slightly increased (81 +/- 6%, n = 3-4). We then studied several Kv7.2 mutants with altered apparent affinities for PIP(2) by coexpressing them with Kv7.3(T) subunits to boost current amplitudes. For the lower affinity (Kv7.2 (R463Q)/Kv7.3(T)) or higher affinity (Kv7.2 (R463E)/Kv7.3(T)) channels, the EC(50) and Inhib(max) were similar to Kv7.4 or Kv7.3(T) homomers (0.12 +/- 0.08 microM and 79 +/- 6% [n = 3-4] and 0.58 +/- 0.07 microM and 27 +/- 3% [n = 3-4], respectively). The very low-affinity Kv7.2 (R452E, R459E, and R461E) triple mutant was also coexpressed with Kv7.3(T). The resulting heteromer displayed a very low EC(50) for inhibition (32 +/- 8 nM) and a slightly increased Inhib(max) (83 +/- 3%, n = 3-4). We then constructed a cellular model that incorporates PLC activation by oxo-M, PIP(2) hydrolysis, PIP(2) binding to Kv7-channel subunits, and K(+) current through Kv7 tetramers. We were able to fully reproduce our data and extract a consistent set of PIP(2) affinities.
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PMID:Affinity for phosphatidylinositol 4,5-bisphosphate determines muscarinic agonist sensitivity of Kv7 K+ channels. 1985 60

M-type (Kv7, KCNQ) K(+) channels control the resting membrane potential of many neurons, including peripheral nociceptive sensory neurons. Several M channel enhancers were suggested as prospective analgesics, and targeting M channels specifically in peripheral nociceptors is a plausible strategy for peripheral analgesia. However, receptor-induced inhibition of M channels in nociceptors is often observed in inflammation and may contribute to inflammatory pain. Such inhibition is predominantly mediated by phospholipase C. We investigated four M channel enhancers (retigabine, flupirtine, zinc pyrithione and H(2)O(2)) for their ability to overcome M channel inhibition via two phospholipase C-mediated mechanisms, namely depletion of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) and a rise in intracellular Ca(2+) (an action mediated by calmodulin). Data from overexpressed Kv7.2/Kv7.3 heteromers and native M currents in dorsal root ganglion neurons suggest the following conclusions. (i) All enhancers had a dual effect on M channel activity, a negative shift in voltage dependence and an increase of the maximal current at saturating voltages. The enhancers differed in their efficacy to produce these effects. (ii) Both PIP(2) depletion and Ca(2+)/calmodulin strongly reduced the M current amplitude; however, at voltages near the threshold for M channel activation (-60 mV) all enhancers were able to restore M channel activity to a control level or above, while at saturating voltages the effects were more variable. (iii) Receptor-mediated inhibition of M current in nociceptive dorsal root ganglion neurons did not reduce the efficacy of retigabine or flupirtine to hyperpolarize the resting membrane potential. In conclusion, we show that all four M channel enhancers tested could overcome both PIP(2) and Ca(2+)-calmodulin-induced inhibition of Kv7.2/7.3 at voltages close to the threshold for action potential firing (-60 mV) but generally had reduced efficacy at a saturating voltage (0 mV). We suggest that the efficacy of an M channel enhancer to shift the voltage dependence of activation may be most important for rescuing M channel function in sensory neurons innervating inflamed tissue.
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PMID:M channel enhancers and physiological M channel block. 2215 35

ADP and other nucleotides control ion currents in the nervous system via various P2Y receptors. In this respect, Cav2 and Kv7 channels have been investigated most frequently. The fine tuning of neuronal ion channel gating via G protein coupled receptors frequently relies on the formation of higher order protein complexes that are organized by scaffolding proteins and harbor receptors and channels together with interposed signaling components. However, ion channel complexes containing P2Y receptors have not been described. Therefore, the regulation of Cav2.2 and Kv7.2/7.3 channels via P2Y1 and P2Y12 receptors and the coordination of these ion channels and receptors in the plasma membranes of tsA 201 cells have been investigated here. ADP inhibited currents through Cav2.2 channels via both P2Y1 and P2Y12 receptors with phospholipase C and pertussis toxin-sensitive G proteins being involved, respectively. The nucleotide controlled the gating of Kv7 channels only via P2Y1 and phospholipase C. In fluorescence energy transfer assays using conventional as well as total internal reflection (TIRF) microscopy, both P2Y1 and P2Y12 receptors were found juxtaposed to Cav2.2 channels, but only P2Y1, and not P2Y12, was in close proximity to Kv7 channels. Using fluorescence recovery after photobleaching in TIRF microscopy, evidence for a physical interaction was obtained for the pair P2Y12/Cav2.2, but not for any other receptor/channel combination. These results reveal a membrane juxtaposition of P2Y receptors and ion channels in parallel with the control of neuronal ion currents by ADP. This juxtaposition may even result in apparent physical interactions between receptors and channels.
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PMID:Membrane coordination of receptors and channels mediating the inhibition of neuronal ion currents by ADP. 2717 14