The lipid-activated two-pore domain K+ channel TREK-1 is resistant to hypoxia: implication for ischaemic neuroprotection

Abstract

TREK-1 is a member of the two-pore domain potassium (K(2P)) channel family that is mechano-, heat, pH, voltage and lipid sensitive. It is highly expressed in the central nervous system and probably encodes one of the previously described arachidonic acid-activated K(+) channels. Polyunsaturated fatty acids and lysophospholipids protect the brain against global ischaemia. Since both lipids are openers of TREK-1, it has been suggested that this K(2P) channel is directly involved in neuroprotection. Recently, however, this view has been challenged by a report claiming that TREK-1 and its activation by arachidonic acid is inhibited by hypoxia. In the present study, we demonstrate that the bubbling of saline with gases results in the loss of arachidonic acid from solution. Using experimental conditions which obviate this experimental artefact we demonstrate that TREK-1 is resistant to hypoxia and is strongly activated by arachidonic acid even at low P(O(2)) (< 4 Torr). Furthermore, hypoxia fails to affect basal as well as 2,4,6-trinitrophenol- and acid-stimulated TREK-1 currents. These data are supportive for a possible role of TREK-1 in ischaemic neuroprotection and in cell signalling via arachidonic acid.

Figure 1. Effects of bubbling on arachidonic activation of mTREK-1

mTREK-1 currents recorded at room temperature in Hepes buffer. A, effects of 10 μm arachidonic acid on membrane currents in non-aerated and air-sparged solutions. Upper panel, voltage clamp protocol; lower panel, membrane current. B, ramp currents from the experiment in A; a, in control, non-aerated, media; b, media sparged with air; c, media containing arachidonic acid sparged with air; d, in a non-aerated medium containing arachidonic acid. C, mTREK-1 currents recorded in non-aerated Hepes buffer or in buffer sparged with 5% O₂–95% N₂. AA indicates currents recorded in the presence of 10 μm arachidonic acid. D, summary of effects of sparging and arachidonic acid on membrane current at 0 mV (expressed relative to currents recorded in control non-aerated solutions). Black columns are data obtained using air to sparge solutions (n = 5); grey columns are data obtained using 5% O₂–95% N₂(n = 6).

Figure 2. Effects of bubbling on arachidonic acid solutions

A, method used for sparging solutions with air (see Results). B, effects of bubbling on arachidonic acid levels in solution. Data presented are means ± s.e.m.(n = 4) of the activity of [³H]arachidonic acid in samples withdrawn at the indicated time points expressed relative to the mean activity in the three samples withdrawn prior to sparging with air. C, relative activity of [³H]arachidonic acid in solution under control conditions (prior to sparging), after 60 min sparging, and after 60 min sparging followed by addition of 100 μl Triton X-100 and remixing of the solution.

Figure 3. Effects of hypoxia and arachidonic acid on mTREK-1

A, method used for equilibrating solutions with gases (see Results). B, effects of 10 μm arachidonic acid (AA) on mTREK-1 currents in air-equilibrated (Air) and nitrogen-equilibrated (N₂) Hepes buffer at room temperature. The cell was voltage clamped at a holding potential of −70 mV and subject to repetitive 0.5 s voltage ramps from −100 mV to +30 mV at 0.1 Hz. C, mean (± s.e.m.) of mTREK-1 currents at 0 mV expressed relative to control (air equilibrated) in Hepes buffer at room temperature (as in B; n = 6). D, effects of 5 μm arachidonic acid (AA) on mTREK-1 currents in 5% CO₂–95% air-equilibrated (Air) and 5% CO₂–95% N₂-equilibrated (N₂) HCO₃⁻ buffer at 35°C. Voltage clamp protocol as in B. E, mean (± s.e.m.) of membrane currents measured at 0 mV expressed relative to control (5% CO₂–95% air equilibrated) in HCO₃⁻-buffered medium at 35°C (as in D; n = 5).

Figure 4. Effects of hypoxia and arachidonic acid on mTREK-1_long and hTREK-1

Effects of 5 μm arachidonic acid (AA) onTREK-1 currents in bicarbonate buffer at 35°C. Solutions were equilibrated with 5% CO₂–95% air (Air) or 5% CO₂–95% N₂ (N₂). Cells were voltage clamped at −70 mV and subject to repetitive 0.5 s voltage ramps from −100 mV to +30 mV at 0.1 Hz. A, representative recording of mTREK-1_long currents. B, summary of the effects of hypoxia and arachidonic acid on mTREK-1_long. Data are mean (± s.e.m.) of currents at 0 mV expressed relative to control (5% CO₂–95% air equilibrated); n = 5. C, representative recording of hTREK-1 currents. D, summary of the effects of hypoxia and arachidonic acid on hTREK-1. Data are mean (± s.e.m.) of currents at 0 mV expressed relative to control (5% CO₂–95% air equilibrated); n = 6.

Figure 5. Effects of hypoxia and TNP on mTREK-1

mTREK-1 currents recorded at room temperature in Hepes buffer. A, effects of 1 mm TNP on membrane currents in air-equilibrated and nitrogen-equilibrated solutions. Upper panel, voltage clamp protocol; cells were held at −70 mV and subject to a 500 ms voltage from −100 mV to +70 mV followed by four 25 ms depolarizing pulses to −30, 0, 30 and +60 mV at 0.1 Hz. Lower panel, membrane current. B, ramp currents from the experiment in A in air-equilibrated (Air) and nitrogen-equilibrated (N₂) saline in the presence and absence of 1 mm TNP. C, mean (± s.e.m.) of mTREK-1 currents at 0 mV expressed relative to control (air equilibrated) in Hepes buffer at room temperature (n = 6).

Figure 6. Effects of hypoxia and intracellular acidosis on mTREK-1

mTREK-1 currents recorded at room temperature in Hepes buffer. A, effects of 10 mm sodium propionate on membrane currents in air-equilibrated and nitrogen-equilibrated solutions. Upper panel, voltage clamp protocol; cells were held at −70 mV and subject to 500 ms voltage ramps from −100 mV to +70 mV at 0.1 Hz. Lower panel, membrane current. B, ramp currents from the experiment in A in air-equilibrated (Air) and nitrogen-equilibrated (N₂) saline in the presence and absence of 10 mm propionate (Prop). C, mean (± s.e.m.) of mTREK-1 currents at 0 mV expressed relative to control (air equilibrated) in Hepes buffer at room temperature (n = 5).

Figure 7. Effects of hypoxia and acidosis on mTREK-1 channel activity

Inside-out patch recordings of mTREK-1 channel activity at a membrane potential of −70 mV (+70 mV pipette potential). A, continuous recording of mTREK-1 activity in an inside-out patch showing the effects of changing perfusate (intracellular) pH and of hypoxia (N₂-equilibrated P_O2 < 4 Torr). B, 1 s excerpts from above recording shown at higher temporal resolution. Number of open channels is indicated by scale on right of figure. Single channel amplitude was 3.5 pA. C, summary of effects of acidosis and hypoxia upon single channel activity (reported as NP_open calculated for a single channel amplitude of 3.5 pA using a 50% threshold); n = 6 patches.