|Home | About | Journals | Submit | Contact Us | Français|
Ubiquitously expressed Mg2+-inhibitory cation (MIC) channels are permeable to Ca2+ and Mg2+ and are essential for cell viability. When membrane cholesterol level was increased by pre-incubating cells with a water-soluble form of cholesterol, the endogenous MIC current in HEK293 cells was negatively regulated. The application of phosphatidylinositol 4,5-bisphosphate (PIP2) recovered MIC current from cholesterol effect. As PIP2 is the direct modulator for MIC channels, high cholesterol content may cause down-regulation of PIP2. To test this possibility, we examined the effect of cholesterol on two exogenously expressed PIP2-sensitive K+ channels: human Ether-a-go-go related gene (HERG) and KCNQ. Enrichment with cholesterol inhibited HERG currents, while inclusion of PIP2 in the pipette solution blocked the cholesterol effect. KCNQ channel was also inhibited by cholesterol. The effects of cholesterol on these channels were blocked by pre-incubating cells with inhibitors for phospholipase C, which may indicate that cholesterol enrichment induces the depletion of PIP2 via phospholipase C activation. Lipid analysis showed that cholesterol enrichment reduced γ-32P incorporation into PIP2 by approximately 35%. Our results suggest that cholesterol may modulate ion channels by changing the levels of PIP2. Thus, an important cross-talk exists among two plasma membrane-enriched lipids, cholesterol and PIP2.
Cholesterol constitutes a key component of the mammalian plasma membrane and is capable of influencing its structural and physical properties such as fluidity, curvature and stiffness. Therefore, cholesterol levels may affect the function of various ion channels and receptors in the plasma membrane. Cholesterol is known to be essential for the functionality of the nicotinic acetylcholine receptor because it is necessary for maintaining proper lipid–protein interaction (Fong and McNamee 1986). Manipulation of cholesterol content affects the equilibrium between the closed and open states of volume-regulated anion channels (Levitan et al. 2000). Similarly, cholesterol content affects the kinetic and steady-state parameters of activation and inactivation of voltage-dependent K+ (Kv1.3) channels in activated T lymphocytes (Hajdu et al. 2003), as well as structural coupling between L-type Ca2+ channels and adjacent regulatory proteins in ventricular myocytes (Tsujikawa et al. 2008). Cholesterol content has been shown to affect the gating of Ca2+-activated K+ channels in vascular smooth muscle cells (Chang et al. 1995). In some cases, cholesterol content regulates the formation of lipid raft micro-domain structures, thereby regulating the targeting of K+ channels (Romanenko et al. 2004; Abi-Char et al. 2007), the activity of large-conductance Ca2+-activated K+ channels from colonic epithelia (Lam et al. 2004), and the surface expression of TRPC3 channels (Graziani et al. 2006). Collectively, these studies imply that cholesterol content may directly affect lipid–channel protein interactions, change channel gating kinetics and participate in targeting channels to specific micro-domains.
The various effects of cholesterol on ion channels are thought to occur through direct physical interactions with other lipids or target proteins. Alternatively, cholesterol may modulate ion channel activities by changing the signaling properties of the plasma membrane. For example, phosphoinositides (i.e. phosphorylated derivatives of phosphatidylinositol) are major signaling molecules found in the cell membrane. Among them, phosphatidylinositol 4,5-bisphosphate (PIP2) plays a fundamental role in the plasma membrane, where it regulates signal transduction, exocytosis/endocytosis, actin dynamics, and ion channel and transporter function (Suh and Hille 2005; Di Paolo and De Camilli 2006). For instance, a large number of ion channels have been shown to be inhibited when PIP2 is hydrolyzed by phospholipase C (PLC) (Suh and Hille 2005). In this study, we showed that increased cholesterol content decreases PIP2 levels and inhibits PIP2-sensitive channels, including the endogenous Mg2+-inhibitory cation (MIC) channel and the exogenously expressed HERG K+ and KCNQ K+ channels. Cholesterol also acutely inhibited the HERG current. The inclusion of PIP2 or PLC inhibitors in the pipette solution prevented acute inhibition by cholesterol. Also, pre-incubating cells with PLC inhibitors prevented cholesterol effect on these channels, indicating that increasing cholesterol levels activate the PLC pathway. Taken together, our results suggest that one of novel function of cholesterol is to modulate ion channels via regulation of PIP2 level.
All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), except for U73122 (Research Biochemicals, Natick, MA, USA), m-3M3 fetal bovine serum (FBS), o-3M3FBS (Tocris, Ellisville, MO, USA) and edelfosine (Calbiochem, Gibbstown, NJ, USA). Phosphatidylinositol phosphates including DiC8-PIP2 and phosphatidic acid (PA) were dissolved in pipette solutions for whole-cell recordings and sonicated for 10 min at ice-cold temperature just before use.
Human embryonic kidney (HEK)293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated FBS and penicillin/streptomycin at 37°C in a 5% CO2 incubator. To enrich the cells with cholesterol, we exposed them to DMEM culture medium containing cholesterol water-solubilized with methyl-β-cyclodextrin (MβCD-cholesterol) for 1–2 h. During the incubation, cells were maintained in a humidified CO2 incubator at 37°C. HERG cDNA was cloned into BamHI/EcoRI sites of the plasmid pCDNA3.0 (Invitrogen, Carlsbad, CA, USA). DNA sequencing was used to verify the integrity of the HERG. HEK293 cells were transiently co-transfected with HERG-pCDNA3.0 and a green fluorescent protein cDNA plasmid (Clontech Laboratories, Mountain View, CA, USA) using the lipofectamine method (Invitrogen). Cells with green fluorescence were used for a patch clamp 24–48 h after transfection. For electrophysiologic analysis, cells were harvested with trypsinization, washed with phosphate-buffered saline (PBS), and studied within 8 h of harvest. Human embryonic kidney tsA-201 (tsA) cells were cultured and transiently transfected using lipofectamine with various cDNAs: M1-muscarinic receptor and the channel subunits KCNQ2 and KCNQ3. TsA cells were maintained in DMEM supplemented with 10% FBS and 0.2% penicillin/streptomycin and used 24–48 h after transfection on poly-l-lysine-coated coverslips.
Cells grown in 100 mm plates were assayed in duplicate for cholesterol using the Amplex red cholesterol assay kit (Molecular Probes, Eugene, OR, USA). First, cells were incubated for 2 h with MβCD-cholesterol, washed twice with ice-cold PBS and homogenized with hypotonic buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM EGTA, 25 mM beta-glycerol phosphate, 5 mM sodium fluoride, 2 mM sodium pyrophosphate and 1 mM sodium orthovandate) using a 22-gauge needle. The samples were then centrifuged at 1000 g for 10 min to remove nuclei and cell debris. Membranes were pelleted from the post-nuclear supernatants by centrifugation for 1 h at 100 000 g, then assayed according to the supplier's instructions.
Cell viability was assessed by using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl-tetrazolium bromide assay. Briefly, around 2 × 104 cells per well were seeded in 96-well microtiter plates with 100 μL of medium. After incubating cells in different conditions, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl-tetrazolium bromide (5 mg/mL stock in PBS) was added to each well and subsequently incubated for 1 h at 37°C. Absorption readings were performed at 540 nm with reference at 690 nm.
Patch pipettes were fabricated from borosilicate glass (TW150-4; World Precision Instrument, Sarasota, FL, USA) using a Flaming/Brown micropipette puller (P-97; Sutter Instrument, Novato, CA, USA). Patch-clamp experiments were conducted in a standard whole-cell recording configuration at 23–25°C. For MIC current, extracellular Na+-based divalent cation-free recording solution contained: 130 mM sodium methanesulfonate, 5 mM NaCl, 10 mM HEPES and 12 mM N-hydroxyethyl-ethylenediamine-triacetic acid. pH was adjusted to 7.2 with NaOH. In the absence of divalent cations, MIC channels conduct large Na+ inward current (Aarts et al. 2003). To prevent activation of Ca2+ release-activated Ca2+ (CRAC), the internal-free [Ca2+] was buffered at 100 nM, calculated using Maxchelator software (Stanford University). The fully developed MIC current was almost completely blocked by 2-aminoethoxydiphenyl borate (Jeong et al. 2006), and the conductance decreased significantly by the presence of Ca2+ in extracellular solution as shown previously (Aarts et al. 2003). For the recording of CRAC current separately from MIC current, extracellular Ca2+-containing bath solution was used, which contained: 135 mM sodium methanesulfonate, 3 mM CaCl2, 0.5 mM EDTA and 10 mM HEPES. pH was adjusted to 7.2 with NaOH. Pipette solution contained: 135 mM Cs methanesulfonate, 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N, N′,N′-tetraacetic acid, 10 mM HEPES and 5 mM MgCl2. Intracellular Mg2+ prevents the activation of MIC current (Hermosura et al. 2002). pH was adjusted to 7.2 with CsOH. Thapsigargin (1 μM) and inositol trisphosphate (IP3;1 μM) were also included in pipette solution to deplete the intracellular Ca2+ store. For Kv current recordings, the bath solution contained: 140 mM NaCl, 3 mM MgCl2, 10 mM HEPES, 5 mM KCl, 10 mM glucose (pH adjusted to 7.4 with NaOH). Pipette solution contained: 140 mM KCl, 5 mM NaCl, 1 mM MgCl2, 10 mM HEPES, 5 mM EGTA, 2 mM Na-ATP and 1 mM Na-GTP (pH adjusted to 7.4 with KOH). Junction potentials were zeroed with the electrode in the standard bath solution.
Currents were amplified using an Axopatch 1D patch-clamp amplifier (Axon Instruments, Foster City, CA, USA). For KCNQ current and HERG current, transfected TsA cells and HEK cells were whole-cell clamped at 23–25°C, 24–48 h after transfection, as described previously (Bian et al. 2001; Suh et al. 2006). Data were analyzed using pClamp7 (Axon Instruments) and the Origin 6.0 program (OriginLab, Northampton, MA, USA). For all experiments with individual cells, at least three independent batches of cells were used in different experimental days.
Anion-exchange high-performance liquid chromatography analysis of phosphoinositides and phosphoinositide radiolabeling using liposomes and [γ32P]ATP were performed as described (Berman et al. 2008). Radioactivity was quantified using a phosphorimager. In some experiments, cytosols were pre-incubated for 5 min at 37°C with 1 mM GDP or 0.2 mM GTP-γS. ‘P2’ subcellular fractions were prepared for metabolic labeling as follows: HEK cell was homogenized in 0.4 mL of 25 mM HEPES, pH 7.4, and 0.32 M sucrose (HEPES-based buffer), and centrifuged at 10 000 ×g in a microfuge. The resulting supernatant was diluted in 4 mL HEPES-based buffer and centrifuged at 45 000 ×g for 15 min at 4°C in an SS34 Sorvall centrifuge rotor. The pellet was re-suspended in 4 mL of 130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES (pH 7.33) and 30 mM glucose (control buffer) and re-centrifuged and re-suspended in the same buffer at a final protein concentration of 2 mg/mL. Aliquots of 100 μL were incubated with [32P]orthophosphate at 2 mCi/mL for 30 min at 37°C. At the end of this incubation, samples were subjected to lipid extraction. Counts in the PIP2 spot were normalized to those in the PA spot, which were not affected by the various genotypes. Lipids were extracted using chloroform/methanol (2 : 1) supplemented with 20 mg/mL cold phosphoinositides and processed for TLC.
The amount of PIP2 extracted from HEK293 cells were measured by using PIP2 Mass ELISA kit (Echelon Biosciences Inc., Salt Lake City, UT, USA). PIP2 was extracted from control or cholesterol-treated cell for 1 h at 150 μM. Cellular PIP2 quantities were estimated by comparing the values from standard curve, which showed linear relationship at the range from 0.5 to 1000 pM concentrations.
The transient receptor potential melastatin 7 (TRPM7)-associated MIC channel has been implicated in the control of cellular proliferation and viability by transporting various trace metal ions, in the regulation of Mg2+ and Ca2+-homeostasis, and in anoxic neuronal death (Nadler et al. 2001; Aarts et al. 2003; Monteilh-Zoller et al. 2003; Schmitz et al. 2003). We measured endogenous MIC currents from HEK293 cells using the whole-cell patch clamp method in divalent cation-free bath solution. In agreement with a previous report (Jeong et al. 2006), MIC current recorded from control cells showed time-dependent activation, reaching a steady-state level approximately 400 s after obtaining whole-cell configuration (Fig. 1a). To determine if increasing the cholesterol level might regulate the activity of the MIC channel, we pre-incubated cells with 150 μM cholesterol water-solubilized with MβCD-cholesterol for 1–2 h prior to recordings. Under these conditions, membranous cholesterol levels increased by more than twofold, while no cell death was observed (data not shown). MIC currents were down-regulated by over 90% with this treatment, as shown in Fig. 1(a). However, the normalized current traces from control and cholesterol-enriched cells showed similar time-dependent activation, indicating that the activation rate of MIC currents was not changed by cholesterol enrichment (inset of Fig. 1a). Current-voltage relationships were obtained by applying ramp voltage pulses (Fig. 1b). Control cells showed slightly inward rectification, which is typical of MIC current. Cholesterol enrichment did not alter the shape of this curve, but reduced the currents at all voltages. These results suggest that cholesterol enrichment decreases the number of activated MIC channels. When cells were pre-incubated with MβCD alone for 1–2 h to deplete cholesterol, MIC currents were increased by twofold, which was opposite to the effect of MβCD-cholesterol (data not shown). MβCD did not change the time-dependent activation rate of MIC currents. These results verified that the effects of MβCD and MβCD-cholesterol on MIC currents were specific for cholesterol.
It has been shown that PIP2, but not its metabolites such as diacylglycerol and IP3, directly modulates TRPM7 channels (Runnels et al. 2002). Specifically, the finding that PIP2 depletion inhibits TRPM7 channels led us to hypothesize that down-regulation of endogenous MIC currents by cholesterol enrichment may be due to decreased levels of PIP2. To test this possibility, we determined if increasing PIP2 levels could restore normal MIC currents in cholesterol-enriched cells. When water soluble DiC8-PIP2 (25 μM) was directly applied to the cytoplasmic side of cells in the whole-cell configuration, MIC current was partially recovered as shown in Fig. 1(c). In contrast, application of the stereoisomer phosphatidylinositol 3,4-bisphosphate, PA, or phosphatidylinositol 3,4,5-trisphosphate was not as effective at rescuing MIC currents as PIP2 at the same concentration (Fig. 1c). Next, the effect of cholesterol enrichment on other endogenous currents was tested in HEK293 cells. Neither CRAC current nor Kv current was inhibited by cholesterol enrichment (Fig. 1d). Thus, cholesterol enrichment inhibited MIC channels, but spared CRAC and Kv channels. Pre-incubating cells with MβCD alone for 1–2 h was without effect on CRAC current or Kv current (data not shown).
To further test whether enhanced cholesterol levels down-regulate MIC currents by reducing PIP2 levels, we investigated the effect of cholesterol-enrichment on other PIP2-sensitive channels. K+ currents were measured in HEK293 cells over-expressing the HERG K+ channel, which has been implicated in the control of repolarization rate during the cardiac action potential (Bian et al. 2001). Exogenously expressed HERG K+ channels gave rise to very slow activation behavior (Fig. 2a), as reported previously (Bian et al. 2001). Similar to the recordings for MIC currents, testing for the effect of cholesterol enrichment on the HERG K+ current was carried out by pre-incubating cells with 150 μM water-soluble cholesterol for 1–2 h before recordings. Cholesterol enrichment inhibited the HERG K+ current as shown in Fig. 2(a) for typical recordings. Increased cholesterol levels were inhibitory at all voltages tested, either when maximum currents were measured (Fig. 2b) or when tail currents were measured (Fig. 2c). The inclusion of 25 μM PIP2 in the whole-cell pipette solution partially recovered HERG K+ current from cholesterol-enriched cells (Fig. 2b and c). These results are consistent with a previous report showing that HERG K+ current is under the regulation of PIP2 (Bian et al. 2004), and they strongly suggest that increased cholesterol induces depletion of PIP2.
A pharmacological approach was used to determine if the acute manipulation of endogenous PIP2 levels could regulate HERG K+ current. A major catabolic pathway for PIP2 involves PLC-mediated hydrolysis of PIP2 to generate diacylglycerol and IP3 (Horowitz et al. 2005). We therefore used a PLC activator to down-regulate cellular PIP2 levels. Figure 3(a) shows HERG K+ current traces measured by step voltage pulses before and after the application of 25 μM m-3M3FBS, the active-form of PLC activator (Bae et al. 2003). The time-dependent change of current levels is shown in Fig. 3(b). Without drug treatment, control HERG K+ currents showed little run-down during the recordings. In contrast, application of the PLC activator induced very rapid inhibition of the current, while the inactive-form, o-3M3FBS, was without effect. Based on these results, we confirmed that acute depletion of PIP2 inhibits HERG K+ current. We then tested the effect of acutely applied cholesterol on HERG K+ currents. As shown in Fig. 3(c), current inhibition started approximately 5 min after the application of water-soluble cholesterol. About 20% of current inhibition was achieved within 15 min. Thus, even though cholesterol enrichment was less effective and required more time than the PLC activator did, both treatments induced similar acute inhibition of HERG K+ current. These results suggest that the inhibitory effect of cholesterol enrichment on HERG K+ currents may occur via the activation of PLC. To test this hypothesis, we included PLC inhibitors in the whole-cell pipette solution. As shown in Fig. 3(c), the presence of PLC inhibitor, 10 μM edelfosine, in the pipette solution partially prevented the acute inhibitory effect of cholesterol. The acute effect of cholesterol was also prevented by the presence of structurally different PLC inhibitor, 5 μM U73122 (Fig. 3d). Inclusion of PIP2 in the whole-cell pipette solution also prevented cholesterol effect (Fig. 3d), which may suggest that the acute depletion of PIP2 suffices to suppress PIP2-sensitive HERG channels. However, the effect of PIP2 on the acute cholesterol inhibition was different from that of PIP2 on cholesterol effect from whole-cell patches in Fig. 2(b).
It is possible that PIP2-induced increase in HERG current from cholesterol-treated cell is completely independent of the inhibitory effect of cholesterol. Thus, the recovery of HERG current by PIP2 could occur by the overlap between cholesterol-induced inhibition and PIP2-induced facilitation. To test this possibility, we pre-incubated cells with PLC inhibitor edelfosine (5 μM) along with cholesterol for 1–2 h before recordings. As shown in Fig. 2(b) and (c), the addition of edelfosine prevented most of inhibitory effect of cholesterol on HERG current. Edelfosine alone was without effect on HERG current (data not shown). We also tested the effect of PLC inhibitors on the endogenous MIC currents. When 5 μM edelfosine or 1 μM U73122 were pre-incubated along with cholesterol, MIC current densities were 64.7 ± 6.0 pA/pF (n = 9) and 68.7 ± 7.5 pA/pF (n = 8), respectively. These values were similar to MIC current density from control cells (Fig. 1c). Therefore, the addition of PLC blockers prevented the inhibitory effect of cholesterol on MIC current as well. These results suggest that cholesterol enrichment inhibits either endogenous MIC current or exogenous HERG current via the membrane PIP2 depletion by PLC activation.
Many cellular proteins, including ion channels and transporters, are inhibited when PIP2 is hydrolyzed by PLC (Suh and Hille 2005). Although we showed that cholesterol enrichment depletes PIP2, causing the down-regulation of PIP2-sensitive MIC and HERG channels via the activation of PLC, we could not rule out other signaling molecules downstream of PLC or changes in other phospholipids. Recently, the depletion of PIP2 has been shown to be sufficient to suppress KCNQ K+ current without the need for other second messengers (Suh et al. 2006). Thus, we utilized HEK293-derived cell line tsA-201 cells expressing KCNQ channels to determine if cholesterol enrichment suppresses exogenously expressed KCNQ K+ current. Families of current were elicited using voltage steps from –80 to +40 mV, in 10 mV intervals, in control and cholesterol-enriched cells (Fig. 4a). When voltage dependences of tail currents were compared, cholesterol-enriched cells showed a small rightward shift (data not shown). Deactivation and activation time constants were also increased by cholesterol enrichment. However, the most prominent effect of cholesterol enrichment on KCNQ K+ channels was on current densities. When currents were measured immediately after whole-cell breakthrough, cholesterol-enriched cells showed only 27% of the current density level compared with control cells (Fig. 4b).
We tested the effects of PLC inhibitors on KCNQ K+ currents. Pre-incubating cells with 2.5 μM U73122 or 2.5 μM edelfosine alone showed inhibitory effects on KCNQ K+ currents as shown in Fig. 4(b). However, the presence of these blockers prevented the inhibitory effect of cholesterol on KCNQ K+ currents (Fig. 4b). These results also suggest that cholesterol enrichment induces the depletion of PIP2 via the activation of PLC. We also tested the acute effect of cholesterol on KCNQ K+ currents. They showed slight rundown to 79% of the initial current at 20 min when measured at –20 mV (Fig. 4c). The acute application of cholesterol induced the inhibition of the current up to 53% of the initial current. In cells expressing M1 muscarinic receptors, the application of muscarinic agonist oxotremorine-M following the application of cholesterol rapidly suppressed the current (Fig. 4d), confirming that depletion of PIP2 inhibits KCNQ K+ currents.
We analyzed phosphoinositide turnover using a cell-free radiolabeling assay in the presence of [γ-32P] (Di Paolo et al. 2004), and tested the effect of cholesterol enrichment on PIP2 levels. We analyzed extracts prepared from control and cholesterol-enriched HEK293 cells using TLC (Fig. 5a) and found a 35% reduction in γ-32P incorporation into PIP2 in cholesterol-enriched cells, relative to control cells (n = 4; Fig. 5b). A considerable decrease in PA labeling was also observed in cholesterol-enriched cells, while there was no change in the labeling of phosphatidylinositol phosphates. Incorporation of radioactive phosphate into phospholipids could occur through lipid kinase-mediated phosphorylation, as well as through contributions from phosphatases and phospholipases. PIP2 levels at steady state were also measured by using PIP2 ELISA kit from HEK293 cells. Compared with control cells, cholesterol-enriched HEK293 cells showed 18.1 ± 0.4% (n = 6) reduction in PIP2 level. Thus, our lipid analysis indicates that cholesterol enrichment depletes PIP2 by either diminishing synthesis or enhancing breakdown of PIP2.
In this study, we show that cholesterol enrichment inhibits PIP2-sensitive channels by down-regulating PIP2 levels. We tested the effects of cholesterol on one of the endogenous non-selective channels (MIC) and two different exogenously expressed K+ channels (HERG and KCNQ). All of these channels are known to be positively regulated by PIP2.Asa growing number of channels are known to be under the regulation of PIP2 (Suh et al. 2006), the activities of all these channels may be affected by plasma membrane cholesterol. However, the extent of modulation may not only be dependent on the expression of these ion channels in specific cells, but also on the sensitivity of a specific channel to PIP2, because the regulation of ion channels by PIP2 is dependent on the apparent affinity of the channel protein for PIP2 (Gamper and Shapiro 2007).
Increasing PIP2 levels restored MIC (Fig. 1c) and HERG currents (Fig. 2b and c) in cholesterol-enriched cells. In addition, addition of PLC inhibitors or PIP2 to the pipette solution prevented the acute inhibitory effect of cholesterol on HERG K+ current (Fig. 3d). Also, we confirmed that the presence of PLC inhibitors prevented most of cholesterol effect on all of three channels tested. Thus, these results suggest that depletion of PIP2 by cholesterol enrichment may occur through the activation of PLC. Activation of PLC by cholesterol was previously suggested to explain the mitogen-like action of cholesterol on ascites tumor cell growth and macromolecule synthesis (Haeffner and Wittmann 1999). Cholesterol enrichment may induce activation of PLC through several mechanisms. First, cholesterol may induce translocation of some PLC isoforms from the cytosol to the plasma membrane, where breakdown of PIP2 may occur. Intracellular translocation of phosphoinositide-specific PLC-δ isoform to the cell surface is known to be cell cycle-dependent (Yagisawa et al. 2006). Second, cholesterol may activate PLC by promoting its phosphorylation (Kim et al. 2000). Third, cholesterol may increase the expression levels of PLC. We are currently investigating these possibilities.
Although PIP2 is a minor component in the plasma membrane, it plays important regulatory roles in a variety of cell functions, such as rearrangement of the cytoskeleton and membrane trafficking (Di Paolo and De Camilli 2006). To explain the multiple roles of PIP2, the concept of spatially confined PIP2 pools was proposed (Janmey and Lindberg 2004). Cholesterol- and sphingolipid-rich rafts may serve to confine PIP2 within the plasma membrane. Thus, the confinement of PIP2 to rafts may allow PIP2 hydrolysis to occur locally and restrict signaling mechanisms to the site of activation (Pike and Miller 1998; Hur et al. 2004). However, this hypothesis, termed ‘raft-delimited PIP2 signaling,’ has been disputed recently (van Rheenen et al. 2005). It is possible that the steady-state level of PIP2 is determined by the concerted action of phosphoinositide kinases and phosphatases. Our results may suggest another way of regulating PIP2 levels within specific micro-domains. Cholesterol content in a specific micro-domain may regulate PIP2 levels via PLC activity. Interestingly, PLCb1 is shown to localize in detergent-resistant membrane microdomain prepared from synaptic plasma membrane fraction of rat brain (Taguchi et al. 2007).
It has been suggested that cholesterol plays a crucial role in the development and maintenance of neuronal plasticity and function (Pfrieger 2003). Failure of cholesterol homeostasis has been suggested as the unifying cause of synaptic degeneration (Koudinov and Koudinova 2005). For example, there is evidence suggesting that cholesterol levels are closely connected to neurological disorders such as Alzheimer's disease (Puglielli et al. 2003). When cholesterol homeostasis is compromised in such neurodegenerative conditions, perturbation of PIP2 levels may follow, according to our results. Consequently, the activities of PIP2-sensitive ion channels change, leading to modifications not only in passive ionic permeability, but also in the active properties of the neurons, such as their action potentials. Interestingly, we found that the activation of MIC channels is chronically suppressed by the presence of familial Alzheimer's disease-associated mutant presenilin (Landman et al. 2006). The down-regulation of PIP2 contributes to the observed channel deficits, as well as to the generation of amyloidogenic β-amyloid peptide, Aβ42. Alternatively, a recent report has shown that the oligomeric form of Aβ decreases the level of PIP2, resulting in synaptic dysfunction (Berman et al. 2008). These results suggest that PIP2 may serve as the molecule linking cholesterol metabolism to the pathogenesis of Alzheimer's disease. Thus, membrane cholesterol content may share the same molecular mechanism with Alzheimer's disease-causing presenilin mutations (i.e. down-regulation of PIP2).
This work was also supported by Samsung Biomedical Research Grant and Korea Research Foundation Grant funded by NIH grant R01 NS056049 (G.D.P.) and the Korean Government (KRF-2006-E00009) to S.C.