The Effect Of M-β-CD And Role Of Lipid Raft Play In The Metabolism Of Membrane PIP₂

Cell membrane is important in exchanging material and informationacross the cells. When stimulated, numerous signal pathways on cellmembrane are activated at the same time, with the help of localized structureson the membrane. Lipid raft is one of these localized structures for membranesignal transduction. Lipid rafts are membrane domains that are rich incholesterol and sphingomyelin microstructure, are closely related with signaltransduction and membrane localization of proteins. One of the lipid structure,caveolea, is a detergent insoluble membrane area, characterized by caveolinprotein, and is involved in cellular cholesterol transport, cell membranesynthesis, signal transduction and tumor formation. There are three types ofcaveolin: caveolin-1(including alpha and beta variants), caveolin-2andcaveolin-3. Studies have shown that caveolin-1has the ability of bindingwith cholesterol, and participates in the maintenance of intracellularcholesterol homeostasis and process of cholesterol transportation. Methyl-β-cyclodextrin (M-β-CD) is a type of detergent, and can extract cholesterol frommembrane, disassociating the integrity of the cell membrane.Transmembrane potential is established due to the gradients of variousion concentrations cross the membrane, and selective permeation of certainions by the ion channels localized on the cell membrane. The membranepotential can be sensed not only by membrane ion channels, but also can besensed by other membrane proteins such as some enzymes.Previous studies shown that, the cell membrane depolarization inXenopus oocytes can increase membrane PIP2levels, depending on theactivity of enzymes PKC, PI4K and PLC. This increased metabolism ofphosphatidyl inositol maintain the membrane PIP2at a high level, which result in a PIP2dependent increase of activity of KCNQ2/Q3potassiumchannels expressed in oocytes.This project was performed following the previous findings thatextracellular hypertonic solution can block the membranedepolarization-induced increase of membrane PIP2and enhancement ofKCNQ2/Q3currents. We hypothesize that membrane depolarization-inducedincrease of PIP2synthesis and enhancement of KCNQ2/Q3currents may berelated to the physical properties of the cell membrane, and more specificallythe localized structure of the membrane. Therefore, we aim to study the role ofstructural integrity of cell membrane plays in the modulation of PIP2metabolism in Xenopus oocytes.Objective: We aim to study the effects and the mechanism of M-β-CD onthe membrane PIP2metabolism indicated by the currents of recombinantKCNQ2and KCNQ3potassium channels expressed in oocytes；to study therole of lipid raft of cell membrane plays in modulation of PIP2metabolism inXenopus oocytes.Methods:(1) Transcription of KCNQ2and KCNQ3RNA in vitro. AllcRNAs were transcribed by RibomaxTMLarge Scale RNA Production SystemsT7Kit.(2) KCNQ2/Q3currents were measured from oocytes usingtwo-electrode voltage clamp technique.(3) The proteins were detected bywestern blot.Results:(1) KCNQ2/Q3currents were increased by application of5mMM-β-CD, andα-CD had no effect on KCNQ2/Q3currents;(2) PLC inhibitorU73122, PI4K inhibitor PIK93, PKC inhibitor Bis all inhibited theM-β-CD-induced enhancement of KCNQ2/Q3currents；(3) M-β-CDabolished the membrane depolarization-induced and PMA-inducedenhancement of KCNQ2/Q3currents；(4) Hypertonic extracellular solutionsabolished the M-β-CD-induced and PMA-induced increase of KCNQ2/Q3currents；(5) Cholesterol abolished the M-β-CD-induced increase ofKCNQ2/Q3, and inhibited KCNQ2/Q3currents；(6) M-β-CD anddepolarization increased the activity of PI4K, and M-β-CD reduced the expression of caveolin-1.Conclusion: The membrane depolarization-induced enhancement of PIP2synthesis and increased activity of KCNQ2/Q3currents may share the similarmechanism with the effects of M-β-CD, which involve a disassociation ofmembrane lipid raft.