Spatiotemporal Dynamics Of Membrane PtdIns(4,5)P₂ Metabolism And The Modulation Of Kir2.0 Subfamily By Membrane Lipids

With the development of research for relationship between ion channels and lipids environment, there is a growing consensus that lipids not only provide the environment for ion channels settleing, but also take part in the regulation of ion channels. Membrane lipids consist of phospholipids, glycolipid and cholesterol, all of which have important role in the ion channels modulation. Phospholipids include glycerophopholipid and sphingomyelin. After twentys'study, it is known that phospholipase can both decrease the content of phospholipids and produce many activated hydrolysate. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂,PIP₂) is one of these lipids components. PIP2 is a precursor of important second messengers, such as the diffusible inositol 1,4,5-triphosphate (InsP₃), which regulates Ca²⁺ release from intracellular Ca²⁺ stores, and the protein kinase C activator, diacylglycerol (DAG). On the other hand, PIP₂ itself is a regulator of a great variety of target molecules, including ion channels and several proteins that regulate actin polymerization and the cytoskeleton, providing a link between the plasma membrane and the cortical cytoskeleton. PIP₂ also has been implicated in several forms of membrane remodeling events, including the fusion of secretory vesicles with the plasma membrane, clathrin-mediated endocytosis, and membrane recovery by endocytosis during neurotransmitter release. Such diverse functions rely upon interaction of the lipid with a large number of regulator molecules. It is critical to understand the underlying mechanism of these interactions. At present, most data are come from studies using artifical lipids membrane, which is far away from normal cell physiology enviroment. So it is important to investigate the interaction in intact cells. The starting point for the research is to build up a good method to visualize the PIP₂ metablisim in intact cells. The first and the second parts of this thesis are designed to visualize the dynamics of PIP2 hydrolysis and resynthesis during activation of GPCR and RTK, and to observe modulation of these processes by pharmacological agents. A fusion construct of green fluorescent protein(GFP) with the PH domain of phospholipase C_δ1 (PLC_δ1PH)(PLC_δ1PH-GFP)was used to visualize PIP₂. PLC_δ1PH is known to bind PIP₂ specifically, and laser-scanning confocal microscopy was used to trace PIP₂ translocation. The lipid raft hypothesis was formulated more than ten years ago, and it is now becoming clear that lipid microenvironments containing special lipid and protein on the cell surface, known as lipid rafts or microdomains, also take part in the interaction between ion channel and lipid. Membrane lipid raft mainly consists of dynamic assemblies of cholesterol and sphingolipids. PIP₂ and arachdonic acid (AA) are two other important components of lipid raft. Inwardly rectifying potassium potassium (Kir) channels comprise a super family composed of seven subfamilies (Kir 1-7) containing about 20 members in mammals. Kir channels are expressed in many tissues. Kir2.0 subfamily is predominantly expressed in heart, skeletal muscle and nervous system. Kir2.0 plays a role in controlling the excitability of heart and brain. Recently, some experiments showed that both arachdonic acid and cholesterol directly or indirectly affected on Kir channels. However, it is not known whether or not the action of cholesterol and AA is specific, to some of Kir2.0 subfamily members, and whether ot not PIP2 is involved in the actions of cholesterol and AA. The third and the fourth parts of this thesis are designed to answer these questions. We expressed different kinds of Kir2.0 subfamily in Xenopus oocytes and CHO cells to investigate both the specific effects of cholesterol or AA on the channels and the relationship with PIP₂. 1. Spatiotemporal dynamics of pharmacological modulation of membrane PIP₂ metabolism by different agents Aim: To visualize the dynamics of PIP₂ hydrolysis and resynthesis during activation of G protein couple receptor (GPCR) and receptor tyrosine kinase(RTK), and to observe the modulation of these processes by pharmacological agents wortmannin, LiCl, U73122 and neomycin. Methods: By using a fusion construct of green fluorescent protein(GFP) with the PH domain of phospholipase C_δ1(PLC_δ1PH)(PLC_δ1PH-GFP)that is known to bind PIP₂ specifically, and by using laser-scanning confocal microscopy to trace PIP₂ translocation. Results: Stimulation of endogenous P2Y receptors by ATP in CHO cells or stimulation of endogenous RTK receptor by insulin in COS-7 cells induced a reversible PLC_δ1PH-GFP translocation, indicating PIP₂ hydrolysis through the receptor-mediated phospholipase C (PLC) activation. Wortmannin and LiCl did not affect the translocation of PLC_δ1PH-GFP from plasma membrane to cytosol but blocked the recovery after the translocation. The transient translocation from plasma membrane was blocked by the PLC inhibitor U73122 but was not affected by another PLC inhibitor neomycin. The potence of insulin hydrolyzing to PIP₂ is weaker than that of ATP's effect. Insulin can induce the fluorescence of GRP1PH-GFP translocate from cytosolic to plasma membtrane, indicating the course of PtdIns(3,4,5)P₃ (PIP₃) produced from PIP₂. Wortmannin (100nM) is able to block PIP₃ production from PIP₂. Conclusion: PLC_δ1PH-GFP can be used as a valuable fluorescence probe to visualize the dynamic change of PIP₂ in living cells. Wortmannin, LiCl, U73122 and neomycin have distinct modulation effects on PIP₂ metabolism. PLC_δ1PH,when bound to PIP₂, prevents neomycin from inhibiting PLC hydrolyzing PIP₂. Insulin is less potent than ATP in inducing hydrolysis of PIP₂. The course of PIP₃ produced from PIP₂ induced by insulin receptor activation can be viewed by he fluorescence of GRP1PH-GFP translocate from cytosolic to plasma membtrane. 2. Binding of PLC_δ1PH-GFP to PtdIns(4,5)P₂ prevents neomyocin inhibition of PLC hydrolysis of PtdIns(4,5)P₂ Aim: To investigate the effects of pleckstrin homology (PH) domains of phospholipase C_δ1 (PLC_δ1PH) on neomycin inhibition of PLC hydrolysis of Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂).Methods: By using a fusion construct of green fluorescent protein(GFP) with the PLC_δ1PH (PLC_δ1PH-GFP) that is known to bind PtdIns(4,5)P₂ specifically, and by using laser-scanning confocal microscopy to trace PtdIns(4,5)P₂ translocation. Results: Stimulation of type I Muscarinic (M₁) receptor and Type II Brandykin (BK₂) receptors induced a reversible PLC_δ1PH-GFP translocation from the membrane to cytosol in COS-7 cell, indicating PtdIns(4,5)P₂ hydrolysis through the receptor-mediated PLC activation. Phospholipase C (PLC) inhibitor U73122 blocked the translocation; Wortmannin, a known PtdIns kinase inhibitor, did not affect the translocation induced by ACh, but blocked the recovery after translocation; Neomyocin, a often used phospholipase C blocker, failed to block the receptor-induced PLC_δ1PH-GFP translocation, indicating the inability of neomycin to block PLC-mediated PtdIns(4,5)P₂ hydrolysis. However, in the absence of PLC_δ1PH-GFP expression, neomycin indeed abolished the receptor-induced PLC hydrolysis of PtdIns(4,5)P₂, assessed by the observation of rising intracellular calcium. We also observed the similar effects of neomycin on ATP activation of endogenesis P2Y receptor. Conclusion: Although PLC_δ1PH and neomycin bind to PtdIns(4,5)P₂ in a similar nature of interaction, they have distinct effects on receptor-mediated activation of PLC and PtdIns(4,5)P₂ hydrolysis. 3. The modulation of Kir2.1,Kir2.3, Kir2.3(I213L)currents by arachidonic acid Aim: To investigate the effects of arachdonic acid (AA) on Kir2.1,Kir2.3 and Kir2.3(I213L)currents in the absence and presence of M1 receptor activation Methods: The mRNA of Kir2.1,Kir2.3 å’ŒKir2.3(I213L)channels synthesized by using in vitro transcription technique were injected into Xenopus oocytes to express the channels. The two electrodes voltage clamp technique was used to observe Kir currents. Results: â‘ AA reversibly and concentration-dependently increasedKir2.3 currents. The maximal effects of Kir2.3 currents by AA (10μM) were 230±30.9% of control (in ND96, P<0.01) and 121±11.2% (in ND96K, P<0.01) of control, with an EC50 value of 1.08μM at -100mV. â‘¡A A didn't affect Kir2.1 currents (98±6%, vs control, P>0.05) and slightly increased Kir2.3 (I213L) currents (115±10%, vs control, P<0.05). â‘¢AA significantly reduced the inhibition of Kir2.3 currents by M1 activation (P<0.01), but had no effects on Kir2.1 and Kir2.3(I213L) currents modulation by M₁ receptor activation (P>0.05). Conclusion: AA selectively potentiated Kir2.3 currents, and didn't affect Kir2.1 currents. This specific modulation of Kir2.3 currents by AA might related to PIP2, since Kir2.3 (I213L), a mutation of Kir2.3 that enhances Kir2.3-PIP2 interaction reduced greatly the modulation of Kir2.3 currents by AA, and Kir2.1 that is known to interact with PIP2 stronger than Kir2.3, was not affected by AA. 4.Modulation of Kir2.1 and Kir2.3 currentsand PIP2 by the level of cellular cholesterol Aim: To investigate the modulation of Kir2.1, Kir2.3 currents, and fluidity or content of membrane PIP₂ by the level of cellular cholesterol. Methods: (1) Elephysiological technique: Kir2.1and Kir2.3 channels were expressed in CHO cells by DNA transfection using lipofectamine transfectin reagen. Whole-cell patch clamp technique was used to record the effects of cholesterol on Kir2.1and Kir2.3 currents. (2) Confocal technique: PIP2 in the membrane was visualized to examine the effects of cholesterol on fluidity and content of PIP₂. Results:(1)Modulation of Kir2.1 currents by cholesterol. In high K⁺ extracellular solution, current densities measured at -90mV were -419±35.6 pA/pF, -325±26.8pA/pF and -260±24.3pA/pF in cholesterol-depleted cells, control cells and in cholesterol-enriched cells, respectively. In low K⁺ extracellular solution, current densities measured at -130mV were -16.8±2.23 pA/pF, -11.3±1.78pA/pF and -7.83±1.44 pA/pF in cholesterol-depleted cells, control cells and in cholesterol-enriched cells, respectively. Thus in both highand low K⁺ solutions, cholesterol depletion and enrichment affected Kir2.1 currents (p<0.01) (2)Kir2.3 currents were not affected by the level of cellular cholesterol. In high K⁺ extracellular solution, current densities of Kir2.3 measured at -90mV were -309±22.9pA/pF, -318±24.6pA/pF and -324±26.3 pA/pF in cholesterol-depleted cells, control cells and cholesterol-enriched cells (P>0.05, vs control), respectively. (3)Modulation of fluidity of membrane PIP2 by the level of cellular cholesterol. FR (fluorescence recovery rate) were 78.8±3.6, 68.6±3.6 and 40.8±2.7 in cholesterol-depleted cells, control cells and cholesterol-enriched cells, respectively (P<0.01, vs control). t_1/2 were 2.6±0.3 S, 7.8±0.5 S and 14.2±1.3 S in cholesterol-depleted cells, control cells and cholesterol-enriched cells, respectively (P<0.01, vs control). (4) Modulation of fluorescence intensity of GFP attached to membrane PIP2 by the level of cellular cholesterol. Relative fluorescence intensity and Fm/Fc of membrane PIP₂ were decreased from 58.4±6.2 to 41.4±4.3 (P<0.01) and from 5.27±0.5 to 2.73±0.3 (P<0.01) in cholesterol-depleted cells, respectively. Neither relative fluorescence intensity nor Fm/Fc of membrane PIP₂ was affected in cholesterol-enriched cells. Conclusion: The decrease of cellular cholesterol results in 1) a significant elevation in Kir2.1 current density, but not in Kir2.3 current density; 2) a significant increase in membrane fluidity of PIP2; 3) a decrease in PIP2 contant of membrane. These results indicate that the specific modulation of Kir2.1 current density by cholesterol may relate to membrane PIP2.