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The Effect of G_q Protein-coupled-receptor Activation on KCNQ/M Currents and Signaling Mechanism

Author: LiuLi
Tutor: ZhangHaiLin
School: Hebei Medical University
Course: Pharmacology
Keywords: M currents PIP2 Calcium PLC G protein -coupled receptors
CLC: R96
Type: Master's thesis
Year: 2008
Downloads: 74
Quote: 0
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G protein-coupled receptors (G protein-coupled-receptors, GPCRs) found the cell surface receptor superfamily have important physiological significance of pathology and pharmacology. Its unique structural features and important role in signal transduction as good drug targets. GPCRs specific agonists and blockers have good prospects for drug development. The fact is that more than half of the drugs on the market are based on GPCRs as targets. Different GPCR through and the G protein (G protein)-coupled can respond to a variety of extracellular stimulation signal neurotransmission in the cells, taste, smell, visual and cellular metabolism, differentiation, proliferation, secretion, etc. series of physiological effects. G protein more types, but all types of G protein composed of three different sub-unit, i.e., α, β, and γ. The α-subunit has the specificity of the GTP binding site and GTP enzyme activity. According to the structure of the α-subunit of the G protein can be divided into four subfamilies: G s , the G i / o , of G q G 12 . M currents at the earliest in 1980 by Brown and Admas found in bullfrog superior cervical sympathetic ganglion is a slowly activating, non-inactivating voltage-dependent outward K + current, its muscarinic receptors can be activated after (M receptors) inhibited the name. M current suppression will lead to increased excitability of the nervous system. The M channel dysfunction is closely related with benign familial neonatal convulsions disease (BFNCs), Alzheimer's disease (Alzheimer), epilepsy and other diseases. The molecular basis of the M channel is a the KCNQ potassium ion channel, has been found in five KCNQ subtypes: KCNQ1, which KCNQ2-5, especially KCNQ2/Q3 participate in constituting the M current. M current regulation mechanisms already found some G q -protein-coupled receptor activation can cause inhibition of M-current. G q protein coupled receptor-mediated phospholipase C (PLC) signal transduction pathway is believed to be the principal means of its inhibition of the M-current. Has proved that activation of the G protein αq First activate PLC β , and thus the hydrolysis membrane PIP 2 the PIP 2 hydrolysis generates two second messengers IP 3 and DAG, IP 3 can trigger the release of endoplasmic reticulum calcium stores, while DAG further activation of PKC. PIP 2 hydrolysis, PKC and intracellular calcium may M-current regulation GPCRs play a role. However, it is still unclear whether all G q -protein coupled receptors activate KCNQ / M current regulation using the same mechanism. Objective: To observe the expression HEK293 cells exogenous histamine (H1), angiotensin II (AT1) purine (P2Y1 and P2Y2) receptor inhibition of the co-expression of KCNQ2 / 3 channel currents to the cell membrane PIP 2 and intracellular calcium release as the main observation point to study the mechanism of inhibition of signaling pathways, and lay the foundation for future experiments. Methods: The whole cell patch clamp recording current change: a laser scanning confocal microscope (LSCM) observation cell membrane PIP 2 -sensitive fluorescent probe (PLC-PH-GFP) translocation between the cell membrane and cytoplasm , and then understand the PIP 2 hydrolysis; determination of changes in the concentration of intracellular calcium calcium imaging technology using LSCM and dual-wavelength ratio method. Results: (1) using the whole cell patch clamp recording to HEK293 cells expressing exogenous KCNQ2 / 3 current can be co-expressed H1, AT1, P2Y1 and P2Y2 receptor activation inhibited, H1, AT1, P2Y1 and P2Y2 activation of KCNQ2 / 3 current inhibition rate were 87.7% ± 6.9% (n = 7), 55.0% ± 4.9% (n = 4), 57.7% ± 5.1% (n = 6) and 62.0% ± 3.1 % (n = 6). (2) LSCM observation HEK293 cell membrane PIP 2 hydrolysis: more than four receptor agonist can obviously see the fluorescent probe (PLC-PH-GFP) from the membrane at the cytoplasm at The translocation, wash receptors inflammatory drugs, the fluorescent probe again by the cytoplasmic translocation to the cell membrane (reset), showing a reversible change. PI4 kinase inhibitor wortmannin persistent cases, the fluorescent probes from the cytoplasm to the reset of the membrane was suppressed, indicating the reset of the fluorescent probe is PI4 kinase mediated PIP 2 resynthesis results. After pre-incubation of the cells with the PLC blocker U73122, receptor activation caused by the membrane to the cytoplasm fluorescence translocation not happen again, receptor activation causes translocation of fluorescent probes PLC-mediated PIP 2 a result of hydrolysis. (3) changes in intracellular calcium ion concentration was measured with LSCM and the proportion of dual-wavelength method calcium imaging technology: more than four receptor agonist, whether with or without outside the presence of calcium, can cause intracellular calcium increase, this effect can be U73122 blocked. (4) excited histamine H1 receptor inhibit KCNQ2 / 3 current mechanism: whole cell patch clamp recording HEK293 cells expressing KCNQ2 / 3 current observe the H1 receptor to activate the expression of KCNQ2 / 3 current inhibition and observation PLC blocker U-73122 (PI4) kinase blocking agent wortmannin and calcium store depleting agent thapsigargin on the role, and thereby infer PIP 2 and intracellular calcium in H1 receptor activation inhibit KCNQ2 The role / 3 current. PLC blocker U73122 allows current suppression rate from 74.5 ± 0.6% (n = 5) was significantly decreased to 7.1 ± 0.6% (n = 5, p <0.01), basically canceled H1 receptor activation current inhibition, Description H1 receptor activation of PLC KCNQ2 / 3 current inhibition; P14 kinase inhibitor wortmannin recovery rate from the current is suppressed to 88.4 ± 6.2% (n = 7) decreased significantly to 10.5 ± 3.4% (n = 8, p <0.01), the PIP 2 resynthesis is a necessary condition for the current recovery by histamine H1 receptor inhibits KCNQ2 / 3 current; the current inhibition rates before and after the application of internal calcium store depletion agent thapsigargin were 90.0 ± 1.6% (n = 6), 88.6 ± 3.9% (n = 6, p> 0.05), no significant difference was not involved in the release of intracellular calcium histamine H1 receptor inhibits KCNQ2 / 3 current process. Conclusion: The results showed that in HEK293 cells, H1, AT1, P2Y1 and P2Y2 receptors can inhibit KCNQ2 / 3 M current the above receptor agonist by activating the PLC can hydrolyze cell membrane PIP 2 and can cause elevated intracellular calcium. H1 receptors and other GPCRs activated mainly through activation of PLC hydrolysis of membrane PIP 2 instead of intracellular calcium to inhibit KCNQ2 / 3 current.

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