Molecular Mechanism For Modulation Of PIP₂Metabolism By Membrane Potential

PtdIns(4,5)P2(PIP₂) is a membrane phospholipid that regulates manyimportant cellular processes, including the attachment of the cytoskeleton tothe plasma membrane, exocytosis, endocytosis, membrane trafficking, and theactivation of enzymes. At the same time, PIP₂is the phospholipid precursor ofthree second messengers, inositol trisphosphate (IP3), diacylglycerol (DAG),and phosphatidylinositol3,4,5-trisphosphate (PIP3). In addition, it is now clearthat phosphoinositides are not only precursors, but also they can act as secondmessengers themselves, and have been implicated to play a role in differentimportant nuclear signaling events such as cell cycle progression, apoptosis,chromatin remodeling, transcriptional regulation and mRNA processing.Finally, PIP₂modulates the function of ion transporters and ion channels,including potassium channel (inwardly rectifying K+channels, M/KCNQchannels, two-pore channels, EAG/ERG channels), voltage-gated calciumchannels (P/Q-and N-type, L-type channels), transient receptor potentialchannels (TRPC, TRPM, TRPV1, TRPA1). PIP₂hydrolysis by PLC, initiatedby activation of Gq-coupled receptor contributed to the mechanism ofreceptor-mediated inhibition of M/KCNQ potassium currents.Apart from PLC-induced, events-related cleavage of membrane PIP₂, thesteady state PIP₂level in the cellular membrane is dynamically balanced bythe activity of specific phosphoinositides kinases and specific lipidphosphatases. The dominant pathway of PIP₂synthesis is probably thesequential phosphorylation in the sarcolemma of phosphatidylinositol (PI) atthe4-and then the5-positions of inositol. PI4P, the PI(4,5)P2precursor, issynthesized from PI through phosphorylation by the PI4-kinase family; PIP isfurther catalyzed by PIP kinases to phosphatidylinositol4,5-bisphosphate(PI4,5P2). Type I PIP kinases use PI4P as a substrate to create PI(4,5)P2, and type II kinases prefer PI5P as a substrate. Alteration of activity of thesekinases will inevitably change PIP₂level and subsequently the PIP₂-dependentprotein or cell functions. The membrane PIP₂level can be manifested by thefunction of a membrane ion channel whose activity depends on the PIP₂. Thusblockage of PI4kinase by wortmannin or phenylarsine oxide blocks there-synthesis of PIP₂and thus the reactivation of M/KCNQ currents afterinhibition induced by PIP₂hydrolysis; when membrane PIP₂abundance waselevated by overexpression of PI(4)P5K, the channel activity of KCNQ2andKCNQ2/3dramatically increased; a similar maneuver greatly blunts the extentof M/KCNQ current inhibition by Gq/11-coupled receptor stimulation.Every cell in our body contains an aqueous cytosol enwrapped by thehydrophobic plasma membrane perforated by the specific permeabilitypathways for water (water channels) and different inorganic and organic ions(ion channels, transporters and pumps). Transmembrane potential is affectedby changes in gradients of various ion concentrations due to the downstreamactivity of membrane channels and gradients in ion concentrations across themembrane. It is well established that transmembrane potential can modulateand even control functioning of various membrane proteins such as voltagegated ion channels, enzymes, ligand gated channels, ion transporters and veryrecently GPCRs.This project started following finding that membrane depolarizationinduced enhancement of KCNQ2/Q3currents in Xenopus oocytes throughincreased PIP₂synthesis. In this thesis, we systematically studied themolecular mechanism for modulation of PIP₂metabolism by the membranepotential, and have the following major findings:(1) The depolarizationselectively activated PKC II isoform and enhanced its interaction with PI4kinase β, which is responsible for the enhanced PIP₂synthesis;(2) G protein(coupled receptors) was possibly involved in the membranedepolarization-induced enhancement of PIP₂synthesis;(3) Mechanismunderlying the effect of hypertonic solutions on cellular function abolished themembrane depolarization-induced enhancement of PIP₂synthesis;(4) The depolarization-induced synthesis of PIP₂is blocked by the PLC blocker thusthe PLC was indicated in the enhanced synthesis of PIP₂;(5) Edelfosinepotentiated the KCNQ2/Q3currents independent of PIP₂.Part1Membrane depolarization increases membrane PtdIns(4,5)P2levelthrough PKC beta II and PI4kinase mechanismsObjective: In our previous study, we described a novel mechanism formembrane PIP₂modulation. In this case, the membrane depolarizationelevated membrane PIP₂level and enhanced PIP₂-dependent KCNQ2/Q3currents expressed in Xenopus oocytes, and the enhanced PIP₂synthesis waspossibly due to increasing activity of PI4kinase. Here, we investigate how theactivity of KI4kinase was enhanced and to study whether protein kinase C(PKC) is responsibly for the activation of PI4kinase.Methods:(1) Transcription of the KCNQ2and KCNQ3genes in vitro.All cRNAs were transcribed using RibomaxTMLarge Scale RNA ProductionSystems T7Kit.(2) Currents were measured from oocytes2-3days aftercRNA injection under two-electrode voltage clamp using0.5-1.0M microelectrodes filled with3M KCl (pH7.2) with a Geneclamp500Bamplifier (Axon Instruments).(3) The proteins were resolved by SDS-PAGEand transferred to polyvinylidene difluoride membranes. Standard Westernblots, using Tris-buffered saline (TBS) and5%non-fat milk powder wereperformed.(4) Immunoprecipitation to detect the interaction between PKCβand PI4Kβ was performed.(5) Immunocytochemistry was used to study thecellular locations of PKCβ and PI4Kβ in oocytes and cardiomyocytes. Thecells were scanned using the laser scanning confocal microscopy.(6) RT-PCR.Total RNA was extracted from different groups of oocytes and concentrationswere adjusted to800ng/l. RNA was reversely transcribed into DNA and thelatter was used as a template to amplify PI4Ks with PI4K-specific primer, andthe resulted PCR product indicates the mRNA level of PI4Ks.(7) RNAi.Double strands RNA (dsRNA) or siRNA interference (RNAi) againstendogenous PI4kianse and PKC of Xenopus oocytes (PI4K) was used todepress the enzyme.(8) Measurement of PKC activity. PKC activity of Xenopus oocyte was measured by kit of PepTag Assay for Non-RadioactiveDetection of Protein Kinase C.Results:(1) Results from RT-PCR and western blot indicated that PI4Kβ,but not PI4Kα expressed in Xenopus oocytes.(2) PMA affected function ofKCNQ2/Q3expressed in Xenopus oocytes in a similar way to thedepolarization. PMA (50nM), when applied for10min, increased KCNQ2/Q3by1.98±0.21folds (n=8).(3) Depolarization (ND96-K) increasedexpression of PI4Kβ. Similarly, PMA treatment also induced an increase inPI4Kβ expression by88±23%. PMA-induced enhancement of KCNQ2/Q3currents was abolished by PI4Kβ knock down. TLC results showed PMAtreatment increased PIP and PIP2level of oocytes membrane by87±33%and48±23%, respectively.(4) Both the depolarization and PMA treatmentincreased the activity of PKC by31±3%(n=7) and37±6%(n=7),respectively.(5) Among5isozymes of PKC, only PKC βII in the membranewas increased by30±5%(n=5) by the depolarization, whereas all5isozymes were increased by PMA treatment. There existed a low level of basalinteractions between both PKCα and PKCβII with PI4Kβ. PMA (50nM)enhanced the both PKCα and PKCβII interactions with PI4K, whereas themembrane depolarization only enhanced the PKCβII interaction with PI4K.(6)Enzastaurin (LY317615), a selective inhibitor of PKCβ significantly decreasedthe membrane depolarization-induced enhancement of KCNQ2/Q3currents.The depolarization-induced enhancement of KCNQ currents was also greatlyreduced in siRNA injected oocytes.(7) The interaction between PI4K andPKCs was also found in DRG neuron and in cardiomyocytes of rats.Conclusion: The depolarization-induced elevation of membrane PIP2isthrough activation of PKCβ and subsequently increased activity of PI4kinaseβ.Part2Role of GPCRs in the membrane depolarization–induced synthesisof membrane PIP2levelObjective: To investigate the possible role of GPCRs in the membranepotential-mediated effects on phosphatidylinositol metabolism. Methods:(1) Transcription of the KCNQ2and KCNQ3genes in vitro.All cRNAs were transcribed using RibomaxTMLarge Scale RNA ProductionSystems T7Kit.(2) Currents were measured from oocytes2-3days aftercRNA injection under two-electrode voltage clamp using0.5-1.0M microelectrodes filled with3M KCl (pH7.2) with a Geneclamp500Bamplifier (Axon Instruments).(3) Antisense oligonucleotides against Gαsubunits of the G proteins were microinjection into oocytes to inhibit theexpressions Gα subunits.(4) RNAi. Double strands RNA (dsRNA) or siRNAinterference (RNAi) against endogenous PI4kianse (PI4K) and PKC ofXenopus oocytes was used to depress the enzyme.(5) Immunoprecipitationwas used to detect the interaction between Gα and Gβ subunits.Results:(1) QX-314abolished membrane depolarization-inducedpotentiation of KCNQ2/Q3currents. However, QX-314did not affect theeffects of RTG and QO-58, two KCNQ openers, on KCNQ2/Q3currents,neither did it affect the effect of XE991, a KCNQ channel blocker.(2)Another local anesthetics ropicacine inhibited the KCNQ2/Q3currentsexpressed in Xenopus oocytes, but had no effect on the depolarization-inducedcurrent increase.(3) PTX and PMT, which depress the activity of Gα subunits,did not affect the depolarization-induced enhancement of KCNQ2/Q3currents.Antisense oligonucleotides against Gα subunits also did not affect thedepolarization-induced enhancement of KCNQ2/Q3currents.(4) Theinteraction between Gαq and Gβ or Gαs and Gβ subunits was regulateddifferentially by membrane depolarization or PMA.Conclusion: G protein (coupled receptors) may be involved in thedepolarization-induced enhancement of PIP₂synthesis.Part3Mechanism underlying the effect of hypertonic solutions oncellular function abolished the membrane depolarization-inducedenhancement of PIP₂synthesisObjective: We aim to study the effects of different osmotic pressure onKCNQ2/Q3currents and the currents enhancement induced by the membranedepolarization and then to explore mechanisms for the effect of osmotic pressure on PIP₂metabolism.Methods:(1) Transcription of the KCNQ2and KCNQ3genes in vitro.All cRNAs were transcribed using RibomaxTMLarge Scale RNA ProductionSystems T7Kit.(2) Currents were measured from oocytes2-3days aftercRNA injection under two-electrode voltage clamp using0.5-1.0M microelectrodes filled with3M KCl (pH7.2) with a Geneclamp500Bamplifier (Axon Instruments).(3) Western blots was used to detecttranslocations of PKC.(4) Measurement of PKC activity. PKC activity ofXenopus oocyte was measured by kit of PepTag Assay for Non-RadioactiveDetection of Protein Kinase C.Results:(1) Extracellular hypo-osmotic pressure did not affect themembrane depolarization-and PMA (50nM)-induced enhancement ofKCNQ2/Q3currents in Xenopus oocytes.(2) Hypertonic extracellularsolutions abolished the depolarization-and PMA-induced currents increased.The effect of PMA (50nM) on the KCNQ2/Q3currents was reversed byhypertonic solutions (sucrose, glucose and mannitol) from potentiation toinhibition.(3) The hypertonic solutions increased the activity of PKCβⅡ andPKCα.(4) Measurement of PKC activity. PKC activity of Xenopus oocyte wasmeasured by kit of PepTag Assay for Non-Radioactive Detection of ProteinKinase C.Conclusion: The membrane depolarization-induced enhancements ofKCNQ2/Q3currents through PIP₂synthesis may share the similar mechanismwith the effects of hypertonic solutions on cellular function.Part4Phospholipase C is involved in the membrane depolarization-induced KCNQ2/Q3currents enhancement in Xenopus oocytesObjective: To study the role of PLC plays in the membranedepolarization in modulation of PIP₂synthesis in Xenopus oocytes.Methods:(1) Transcription of the KCNQ2and KCNQ3genes in vitro.All cRNAs were transcribed using RibomaxTMLarge Scale RNA ProductionSystems T7Kit.(2) Currents were measured from oocytes2-3days aftercRNA injection under two-electrode voltage clamp using0.5-1.0M microelectrodes filled with3M KCl (pH7.2) with a Geneclamp500Bamplifier (Axon Instruments).(3) The proteins were resolved by SDS-PAGEand transferred to polyvinylidene difluoride membranes. Standard Westernblots using Tris-buffered saline (TBS) and5%non-fat milk powder wereperformed.(4) Immunoprecipitation was used to detect the interactionbetween Gβ and PLCβ and the tyrosine-phophorylated PLCγ1.(5) RNAi.Double strands RNA (dsRNA) against endogenous PLCβ and PLCγ1ofXenopus oocytes were used to depress the enzymes.(6) The association ofPI(3,4,5)P3with PLCγ1was determined by immunoprecipitation method.Results:(1) U73122but not its inactive analogue U73343stronglyinhibited the KCNQ2/Q3currents increase induced by the membranedepolarization and PMA.(2) U73122also abolished the membranedepolarization-induced PIP and PIP₂increase.(3) PLCγ1was the dominantform of PLC localized on the membrane of oocytes. The membranedepolarization and PMA significantly increased the activity of PLCγ1.(4)Oocytes were treated with ND96-K for15min or PMA for20min. Oocyteslysates were isolated. PLCγ1and PI3K-p85in the total cell lysates weredetermined by western analysis. Association of PI(3,4,5)P3with PLCγ1wasdetermined by immunoprecipitation with PLCγ1antibody followed byincubation of the immunoprecipitate-spotted (the spotting of chloroformextract were2,6,4μl for each line) membrane with PI(3,4,5)P3antibody.(5)The depolarization-induced enhancement of KCNQ currents was greatlyreduced in oocytes injected with dsRNA for PLCγ1but not with dsRNA forPLCβ3.(6) The depolarization increased the amount of PIP3bound to PLCγ1;the depolarization induced phosphorylation of Akt.Conclusion: PLCγ1is necessary for the depolarization-inducedmodulation of PIP₂metabolism.Part5Edelfosine potentiates the currents of KCNQ2/3channelsexpressed in Xenopus oocytes and CHO cellsObjective: To examined the effects of edelfosine on recombinantKCNQ2and KCNQ3potassium currents, expressed either alone or in combination in Xenopus oocytes or CHO cells.Methods:(1) Transcription of the KCNQ2and KCNQ3genes in vitro.All cRNAs were transcribed using RibomaxTMLarge Scale RNA ProductionSystems T7Kit.(2) Currents were measured from oocytes2-3days aftercRNA injection under two-electrode voltage clamp using0.5-1.0M microelectrodes filled with3M KCl (pH7.2) with a Geneclamp500Bamplifier (Axon Instruments).(3) The proteins were resolved by SDS-PAGEand transferred to polyvinylidene difluoride membranes. Standard Westernblots using Tris-buffered saline (TBS) and5%non-fat milk powder wereperformed.Results:(1) KCNQ2/Q3currents were increased by1.35±0.14(n=10)folds after10min application of10μM edelfosine. Theconcentration-response relationship for edelfosine was established. Edelfosineincreased KCNQ2/Q3currents with EC50of1.35±0.51μM.(2) Edelfosinedid not affect the activation kinetics of KCNQ2/Q3currents, but significantlyslowed channel deactivation in a voltage-dependent manner at membranepotentials from–140to–90mV.(3) KCNQ2(H328C)/Q3responded toedelfosine similarly as KCNQ2/Q3(edelfosine-induced increase: KCNQ2/Q3,1.35±0.31folds, n=6; KCNQ2(H328C)/KCNQ3,1.11±0.44, n=6).Edelfosine (10μM) did not increase PIP2or PIP levels.(4) Edelfosine (10μM)markedly enhanced KCNQ2currents (1.44±0.34folds increase, n=7); it alsoincreased KCNQ1(1.36±0.44folds increase, n=8) and KCNQ1/KCNE1(0.85±0.28folds increase, n=6) currents.(5) Edelfosine treatment did notchange the membrane level of the KCNQ2/Q3protein in either Xenopusoocytes or CHO cells.(6) Pretreatment with M-β-CD (2.5mM,30mins)partly abolished the edelfosine-induced enhancement of KCNQ2/Q3currents(1.35±0.14vs0.57±0.14).Conclusion: Edelfosine potentiates KCNQ currents, and the effectdepends on the integrity of the cell membrane structure (lipids raft).