Introduction

Two-pore-domain potassium (K2P) channels are a novel family of potassium channels with four transmembrane segments and two pore-forming domains located in tandem1, 2. These channels control neuronal excitability through their influence on resting membrane potential (RMP). Thus, they are classified as background potassium channels or leak potassium channels3, 4. To date, 17 human K2P channel subunits have been identified according to their amino acid sequence identity and regulatory mechanisms. They can be divided into six subfamilies: TWIK, THIK, TASK, TALK, TREK, and TRESK5, 6.

TREK-1 is one of the most important members of the K2P channel family and is expressed throughout the central nervous system (CNS)4, 7. In addition to its unusual gating properties, such as background channel activity and sensitivity to membrane stretch, the TREK-1 channel can be modulated by many different intracellular and extracellular chemical agents. For example, TREK-1 is activated by increased temperature, membrane stretch and internal acidosis and is also sensitive to the presence of some polyunsaturated fatty acids [such as arachidonic acid (AA)] and gaseous general anesthetics (such as halothane and nitrous oxide) 8, 9, 10, 11. It has been recently reported that the TREK-1 channel is also modulated by neuroprotective agents such as riluzole and plays an important role in neuroprotection12, 13. In our previous studies, we showed that the expression of TREK-1 mRNA and protein significantly increased after acute and chronic cerebral ischemia, suggesting that the TREK-1 channel may be closely linked to pathological conditions such as cerebral ischemia14, 15.

3-n-Butylphthalide (NBP) is a potent neuroprotectant that was approved by the State Food and Drug Administration (SFDA) of China at the end of 2002 as a new drug for the treatment of ischemic stroke16. Pre-clinical and clinical studies have demonstrated that racemic NBP (dl-NBP) is a promising drug for the treatment of ischemic stroke. This neuroprotectant influences several pathophysiological processes such as improving rat brain microcirculation, inhibiting platelet aggregation, preventing oxidative damage from ischemia and reducing neuronal apoptosis Full size image

l-NBP inhibited TREK-1 channel currents in a dose-dependent manner

We elicited TREK-1 currents with depolarizing voltage steps from a holding potential of -80 mV (Figure 3A). l-NBP-mediated inhibition of TREK-1 currents was partially reversed upon washout. This inhibition was concentration dependent over the range of 0.01 to 10 μmol/L. The maximum inhibition of TREK-1 current by l-NBP (70.0%±2.0%, n=8) was observed at a concentration of 10 μmol/L (Figure 3B). This dose-dependent response was well fitted to the Hill equation, with an IC50 of 0.06±0.03 μmol/L and a Hill coefficient of 0.54±0.13. Figure 3C shows the current-voltage relationship (I–V) curve for the inhibition of TREK-1 channels by 0.3 μmol/L l-NBP. The inhibition was gradual and usually reached a peak 3–5 min after l-NBP exposure. Whole-cell current density was normalized to control currents, and the voltage dependence of the blockade by 10 μmol/L l-NBP was calculated (Figure 3D). The inhibition did not change substantially between -80 mV and +80 mV, indicating a lack of voltage dependence for the effect of l-NBP. This effect was partially reversed upon washout.

Figure 3
figure 3

l-NBP inhibited TREK-1 channel currents in a concentration-dependent manner. (A) The inhibition of TREK-1 currents by l-NBP. Representative current evoked by 300-ms voltage pulses from -80 mV to +80 mV in 40 mV increments. (a) Currents in TREK-1/CHO cells. (b) Inhibition of TREK-1 currents by 10 μmol/L l-NBP. (c) The TREK-1 currents returned to near the control level after washout. (B) Concentration-response curve for the inhibition of TREK-1 channels by l-NBP measured at +80 mV from the holding potential -80 mV at the end of a 300-ms pulse. Data are expressed as means±SEM from at least six cells. The IC50 was calculated as 0.06±0.03 μmol/L. (C) The I–V curve for the inhibition of TREK-1 channels by 0.3 μmol/L l-NBP was measured at +80 mV from the holding potential of -80 mV at the end of a 300-ms pulse. Data are expressed as means±SEM. (D) Voltage-independent inhibition of TREK-1 currents by l-NBP (10 μmol/L). Whole-cell current densities were normalized to control currents (lcontrol). The normalized current density for l-NBP-treated cells did not change significantly.

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Effects of l-NBP on the membrane potential of TREK-1/CHO cells

Inhibition of TREK-1 channels has been reported to depolarize the cell membrane7, 12. Therefore, we compared the effects of l-NBP on the RMPs of Wt/CHO and TREK-1/CHO cells in current-clamp mode. The results show that 10 μmol/L l-NBP shifted the RMP from -55.3±2.4 mV to -42.9±2.1 mV (n=25, Figure 4, P<0.05) in TREK-1/CHO cells but not in Wt/CHO cells (n=7), confirming the role of this channel in the maintenance of the RMP.

Figure 4
figure 4

l-NBP depolarized the membrane potential of TREK-1/CHO cells but not Wt/CHO cells. (A) Effects of 10 μmol/L l-NBP on the RMPs of Wt/CHO cells and TREK-1/CHO cells. Under a current clamp, the RMPs of the two cell lines were measured similarly every 20 s for 30 min. The RMPs for control cells and cells treated with 10 μmol/L l-NBP were monitored at 10 and 20 min, respectively. (B) Summary the RMP changes in Wt/CHO and TREK-1/CHO cells before and after exposure to 10 μmol/L l-NBP. Bar a shows the control RMPs of Wt/CHO cells and TREK-1/CHO cells. The effects of 10 μmol/L l-NBP on the RMP of the two cell lines are shown in bar b. The results are presented in pA/pF as means±SEM (Wt/CHO, n=7; TREK-1/CHO, n=25); eP<0.05 vs control.

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