Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The M-type potassium current (I(M)) plays a dominant role in regulating membrane excitability and is modulated by many neurotransmitters. However, except in the case of bradykinin, the signal transduction pathways involved in M-channel modulation have not been fully elucidated. The channels underlying I(M) are produced by the coassembly of KCNQ2 and KCNQ3 channel subunits and can be expressed in heterologous systems where they can be modulated by several neurotransmitter receptors including histamine H(1) receptors. In HEK293T cells, histamine acting via transiently expressed H(1)R produced a strong inhibition of recombinant M-channels but had no overt effects on the voltage dependence or voltage range of I(M) activation. In addition, the modulation of I(M) by histamine was not voltage sensitive, whereas channel gating, particularly deactivation, was accelerated by histamine. Non-hydrolysable guanine nucleotide analogues (GDP-beta-S and GTP-gamma-S) and pertussis toxin (PTX) treatment demonstrated the involvement of a PTX-insensitive G protein in the signal transduction pathway mediating histamine-induced I(M) modulation. Abrogation of the histamine-induced modulation of I(M) by expression of a C-terminal construct of phospholipase C (PLC-beta1-ct), which buffers activated Galpha(q/11) subunits, implicates this G protein alpha subunit in the modulatory pathway. On the other hand, abrogation of the histamine-induced modulation of I(M) by expression of two constructs which buffer free betagamma subunits, transducin (Galphat) and a C-terminal construct of a G protein receptor kinase (MAS-GRK2-ct), implicates betagamma dimers in the modulatory pathway. These findings demonstrate that histamine modulates recombinant M-channels in HEK293T cells via a PTX-insensitive G protein, probably Galpha(q/11), in a similar manner to a number of other G protein-coupled receptors. However, histamine-induced I(M) modulation in HEK293T cells is novel in that betagamma subunits in addition to Galpha(q/11) subunits appear to be involved in the modulation of KCNQ2/3 channel currents.
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PMID:Activation of a PTX-insensitive G protein is involved in histamine-induced recombinant M-channel modulation. 1248 85

A variety of G-protein-coupled receptors regulate membrane excitability via M-type K(+) current (M-current) modulation. Muscarinic m1 and m3 acetylcholine receptors have both been implicated in the modulation of M-current. The muscarinic m5 receptor, like muscarinic m1 and m3 receptors, couples to phospholipase C via a pertussis toxin-insensitive G protein. Since a number of other receptors which activate phospholipase C also modulate M-current, we investigated if muscarinic m5 receptors could modulate recombinant M-type (KCNQ2/KCNQ3) K(+) channels after heterologous expression in human embryonic kidney (HEK) 293T cells. Application of Oxo-tremorine M to HEK293T cells expressing muscarinic m1, m3, or m5 receptors produced a similar robust inhibition of M-current, whereas muscarinic m2 and m4 receptor stimulation was without effect. Muscarinic m1, m3, or m5 receptor stimulation decreased the deactivation time constants of M-current at -50 mV. The inhibition of M-current by stimulation of muscarinic m1, m3, or m5 receptors was insensitive to overnight treatment with pertussis toxin or cholera toxin, which interfere with G(i/o) and G(s) G-protein signaling. These data suggest that muscarinic m1, m3, and m5 receptors inhibit M-channels via the activation of a common G protein.
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PMID:Activation of muscarinic m5 receptors inhibits recombinant KCNQ2/KCNQ3 K+ channels expressed in HEK293T cells. 1259 Oct 92

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.
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PMID:PIP(2) activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents. 1267 Apr 25

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.
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PMID:Experiments to test the role of phosphatidylinositol 4,5-bisphosphate in neurotransmitter-induced M-channel closure in bullfrog sympathetic neurons. 1283 15

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

Some ion channels are regulated by inositol phospholipids and by the products of cleavage by phospholipase C (PLC). KCNQ channels (Kv7) require membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) and are turned off when muscarinic receptors stimulate cleavage of PIP(2) by PLC. We test whether diacylglycerols are also important in the regulation of KCNQ2/KCNQ3 channels using electrophysiology and fluorescent translocation probes as indicators for PIP(2) and diacylglycerol in tsA cells. The cells are transfected with M(1) muscarinic receptors, channel subunits, and translocation probes. Although they cause translocation of a fluorescent probe with a diacylglycerol-binding C1 domain, exogenously applied diacylglycerol (oleoyl-acetyl-glycerol and dioctanoyl glycerol) and phorbol ester do not mimic or occlude the suppression of KCNQ current by muscarinic agonist. Blocking the metabolism of endogenous diacylglycerol by inhibiting diacylglycerol kinase with R59022 or R59949 slows the decay of diacylglycerol twofold but does not mimic or occlude muscarinic regulation and recovery of current. Blocking diacylglycerol lipase with RHC-80267 also does not occlude muscarinic modulation of current. We conclude that the diacylglycerol produced during activation of PLC, any activation of protein kinase C that it may stimulate, and downstream products of its metabolism are not essential players in the acute muscarinic modulation of KCNQ channels.
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PMID:Does diacylglycerol regulate KCNQ channels? 1672 10

The recently identified Mas-related gene (Mrg) family of G-protein-coupled receptors is expressed almost exclusively in dorsal root ganglion (DRG) neurons. The expression of one family member, MrgD, is even further confined to IB4+, nonpeptidergic, small-diameter nociceptors. Although the functional consequences of MrgD activation are not known, this expression profile provides intriguing potential for a role in pain sensation or modulation. In a recombinant cell line, we first assessed the functional significance of MrgD activation by coexpressing MrgD with the KCNQ2/3 potassium channel, a channel implicated in pain. Whole-cell voltage-clamp recordings revealed that bath application of the ligand for MrgD, beta-alanine, resulted in robust inhibition of KCNQ2/3 activity. Pharmacological blockade of G(i/o) and phospholipase C signaling revealed a partial and complete block of the response, respectively. We extended these observations to dissociated DRG neuron cultures by examining MrgD modulation of M-currents (carried primarily by KCNQ2/3). Here too, beta-alanine-induced activation of endogenous MrgD inhibited M-currents, but primarily via a pertussis toxin-sensitive pathway. Finally, we assessed the consequence of beta-alanine-induced activation of MrgD in phasic neurons. Phasic neurons that fired a single action potential (AP) before beta-alanine application fired multiple APs during beta-alanine exposure. In sum, we provide evidence for a novel interaction between MrgD and KCNQ/M-type potassium channels that contributes to an increase in excitability of DRG neurons and thus may enhance the signaling of primary afferent nociceptive neurons.
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PMID:MrgD activation inhibits KCNQ/M-currents and contributes to enhanced neuronal excitability. 1744 34

Voltage-evoked signals play critical roles in neural activities, muscle contraction and exocytosis. Ciona voltage-sensor containing phosphatase (Ci-VSP) consists of the transmembrane voltage sensor domain (VSD) and a cytoplasmic domain of phosphoinositide phosphatase, homologous to phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Previous experiments utilizing potassium channels as the sensor for phosphoinositides have demonstrated that phosphatase activities of Ci-VSP are voltage dependent. However, it still remained unclear whether enzyme activity is activated by depolarization or hyperpolarization. Further, a large gap in voltage dependency was found between the charge movement of the VSD and potassium channel-reporting phosphatase activities. In this study, voltage-dependent dynamics of phosphoinositides mediated by Ci-VSP were examined by confocal imaging and electrical measurements in Xenopus oocytes. Imaging of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) using green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains from phospholipase C delta subunit (PLC-delta) showed that PtdIns(4,5)P(2) concentration is reduced during depolarization. In the presence of Ci-VSP, IRK1 channels with higher sensitivity to phosphoinositide than GIRK2 channels decreased their magnitude during depolarization over 0 mV, indicating that the PtdIns(4,5)P(2) level is reduced upon depolarization. KCNQ2/3 channels coexpressed with Ci-VSP exhibited voltage-dependent decay of the outward current that became sharper with higher depolarization in a voltage range up to 100 mV. These results indicate that Ci-VSP has an activity that depletes PtdIns(4,5)P(2) unlike PTEN and that depolarization-activated voltage sensor movement is translated into activation of phosphatase activity.
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PMID:Depolarization activates the phosphoinositide phosphatase Ci-VSP, as detected in Xenopus oocytes coexpressing sensors of PIP2. 1761 6

The first-generation antihistamines are widely prescribed medications that relieve allergic reactions and urticaria by blocking the peripheral histamine H(1) receptor. Overdose of these drugs often results in serious neuronal toxic effects, including seizures, convulsions and worsening of epileptic symptoms. The KCNQ/M K(+) channel plays a crucial role in controlling neuron excitability. Here, we demonstrate that mepyramine and diphenhydramine, two structurally related first-generation antihistamines, can act as potent KCNQ/M channel blockers. Extracellular application of these drugs quickly and reversibly reduced KCNQ2/Q3 currents heterologously expressed in HEK293 cells. The current inhibition was concentration and voltage dependent. The estimated IC(50) (12.5 and 48.1 microM, respectively) is within the range of drug concentrations detected in poisoned patients (30-300 microM). Both drugs shifted the I-V curve of KCNQ2/Q3 channel to more depolarized potentials and altered channel gating properties by prolonging activation and shortening deactivation kinetics. Mepyramine also inhibited the individual homomeric KCNQ1-4 and heteromeric KCNQ3/Q5 currents. Moreover, mepyramine inhibited KCNQ2/Q3 current in an outside-out patch excised from HEK293 cells and the inhibitory effect was neither observed when it was applied intracellularly nor affected by blocking phospholipase C (PLC) activity, indicating an extracellular and direct channel blocking mechanism. Finally, in cultured rat superior cervical ganglion (SCG) neurons, mepyramine reduced the M type K(+) current in a concentration-dependent manner and led to marked membrane potential depolarization. It is likely that these effects may be involved in the adverse neuroexcitatory effects observed in patients experiencing an overdose of antihistamines.
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PMID:Antihistamine mepyramine directly inhibits KCNQ/M channel and depolarizes rat superior cervical ganglion neurons. 1822 95


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