Cholesterol modulates ion channels via down-regulation of phosphatidylinositol 4,5-bisphosphate

Abstract

Ubiquitously expressed Mg(2+)-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 gamma-(32)P 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.

Fig. 1

Inhibition of endogenous Mg²⁺-inhibitory cation channel (MIC) currents by cholesterol enrichment. (a) Time-dependent activation profile of MIC currents. Currents at –100 mV were plotted in control (n = 6; ○) and cholesterol-treated HEK293 cells (n = 6; ●). Cells were pre-incubated for 1–2 h with 150 μM cholesterol before recordings. MIC currents were elicited by ramp pulses from –100 to +100 mV every 10 s using whole-cell configurations bathed in Ca²⁺-free bath solution. The inset shows the normalized MIC currents from control and cholesterol-treated cells. Currents were normalized relative to the maximum current levels in each condition. (b) Current–voltage relationships of the MIC current. Currents were obtained in response to ramp pulses from –100 to +100 mV during a 200-ms period in control and cholesterol-treated cells. Currents were elicited 500 s after the formation of whole cell-configuration to induce full activation of MIC currents. A dotted line is drawn over the control current trace to show the inward rectification of the MIC current. (c) Recovery of MIC currents by phosphatidylinositol 4,5-bisphosphate (PIP₂) in cholesterol-treated HEK293 cells. In some recordings, 25 μM PIP₂, phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂], phosphatidic acid (PA), or phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] was included in the pipette solution. (d) Effects of cholesterol enrichment on other endogenous currents from HEK293 cells. Ca²⁺ release-activated Ca²⁺ (CRAC) currents and voltage-dependent K⁺ (Kv) currents were measured in control and cholesterol-treated HEK293 cells, and current density values (pA/pF) are presented.

Fig. 2

Inhibition of exogenously expressed ether-a-go-go-related gene (HERG) currents by cholesterol enrichment. (a) Representative HERG K⁺ current traces are shown in control and cholesterol-treated HEK293 cells. Cells were pre-incubated for 1–2 h with 150 μM cholesterol before recordings. Depolarizing steps were applied from a holding potential of –70 mV to between –60 and +50 mV for 4 s, followed by a step to –40 mV to elicit currents. As more depolarized membrane potentials were applied, HERG K⁺ currents were activated more rapidly, as evident in control current traces. (b, c) Averaged current–voltage relations obtained for HERG K⁺ currents (n = 7). Currents were measured at the end of the depolarizing step (I_HERG,step) and for peak tail current amplitude (I_{HERG, tail}) following the step to –40 mV. This pulse protocol was applied at 15 s intervals. In some recordings in cholesterol-treated cells, 25 μM phosphatidylinositol 4,5-bisphosphate (PIP₂) was included in the pipette solution (+PIP₂). In some recordings, cells were treated with cholesterol along with phospholipase C inhibitor, 5 μM edelfosine (+Edel).

Fig. 3

Inhibition of ether-a-go-go-related gene (HERG) currents by acute application of phospholipase C (PLC) activator and cholesterol. (a) The effect of acute application of a PLC activator on the HERG current. Representative current traces show HERG K⁺ current before (0 min) and after (1, 2, 3 and 5 min) the application of the PLC activator, m-3M3FBS (25 μM). Currents were elicited by depolarizing voltage step to +30 mV from a holding potential of –70 mV for 4 s, followed by a step to –40 mV. (b) Steady state currents (I_HERG,step) were measured in different conditions. PLC activator (m-3M3FBS; n = 6) or non-active form (o-3M3FBS; n = 8) was added at the time indicated by the arrow. Vehicle (DMSO) was added for control (n = 6). (c) Steady state currents (I_HERG,step) were measured in different conditions. The effect of 150 μM cholesterol is shown (cholesterol; n = 8). In some cells, the PLC inhibitor edelfosine (10 μM) (cholesterol + Edel; n = 8), were included in whole-cell pipette solutions. Vehicle DMSO was added for control (control; n = 8). Cholesterol was added at the time indicated by the arrow. (d) Comparison of average steady state HERG K⁺ currents expressed as % of control. The data were the same as in (c) except for the PLC inhibitors U73122 (n = 4). The presence of 25 μM PIP₂ in the pipette solution also blocked the inhibitory effect of cholesterol (n = 7).

Fig. 4

Inhibition of KCNQ current by cholesterol enrichment. (a) Inhibition of exogenously expressed KCNQ2/KCNQ3 K⁺ current by cholesterol. Families of K⁺ current elicited by voltage steps from –80 to +40 mV, in 10-mV intervals (see the pulse protocol), with or without incubation of 150 μM cholesterol for 1–2 h (Control vs. Cholesterol in the figure). The holding potential was –70 mV. (b) Current density measured immediately after whole-cell breakthrough in control and cholesterol-treated cells. Cells expressing KCNQ channels were preincubated at 37°C for 2 h in the presence or absence of cholesterol (150 μM). Control, 95.7 ± 14.6 pA/pF; cholesterol, 23.0 ± 6.4 pA/pF, n = 7. *p < 0.01. In some cells, U73122 (2.5 μM) or edelfosine (2.5 μM) was pre-incubated with or without cholesterol for 2 h at 37°C. (c) Time course of KCNQ current modulation by acute application of cholesterol (150 μM). Measurements started 3 min after breaking through to whole-cell recording at 23–25°C. Vertical bars are SEM. Control, n = 3; cholesterol, n = 5. (d) Modulation of KCNQ current by cholesterol and oxotremorine-M (Oxo-M). Whole-cell currents were recorded during the application of cholesterol (150 μM) and Oxo-M (10 μM).

Fig. 5

Down-regulation of phosphatidylinositol 4,5-bisphosphate (PIP₂) by cholesterol. (a) Alteration of PIP₂ metabolism in cholesterol-treated cells. TLC analysis of lipid extracts prepared either from control or cholesterol-treated (150 μM for 2 h) HEK293 cells. PIP, phosphatidylinositol 4-monophosphate. (b) Quantification of TLC data.