Previous studies have suggested that certain native members of the tandem P domain K+ (K2P) channel family are involved in cellular responses to reduced PO2 (Hartness et al. 2001; Plant et al. 2002) and that such responses are maintained in recombinant expression systems (Lewis et al. 2001). Here, we report the effects of hypoxia on the human recombinant arachidonic acid- and membrane stretch-sensitive K2P channel, hTREK-1 (Meadows et al. 2000).
The full-length hTREK-1 was cloned and stably expressed in human embryonic kidney (HEK 293) cells as previously described (Meadows et al. 2000). Whole-cell currents were recorded by the patch-clamp technique using solutions and voltage-clamp protocols described by Hartness et al. (2001). All data reported herein were taken from currents recorded at a 200 ms test potential of +60 mV from a holding potential of -70 mV. Acute hypoxia (~20 mmHg) was achieved in the recording chamber by perfusing with extracellular solution pre-equilibrated with 100 % N2.
In normoxia (150 mmHg), K+ current density was 111 ± 17 pA pF-1 (mean ± S.E.M., n = 10 cells). Hypoxia caused reversible inhibition of K+ currents by 38.5 ± 3.7 %, to 68.0 ± 10.7 pA pF-1 (n = 10, P < 0.001, Student’s paired t test). Application of arachidonic acid (AA) caused rapid and reversible enhancement of K+ current density in a concentration-dependent manner, with maximal enhancement (corresponding to a 2.18 ± 0.26-fold increase (n = 6, P < 0.01) in current density observed when applying 10 mM AA. In the presence of AA (10 mM), exposure to hypoxia reduced K+ current density to 53.5 ± 17.2 pA pF-1 (n = 6, P < 0.01), a value not significantly different from that seen under hypoxic conditions in the absence of AA. Furthermore, negative pipette pressure of more than 30 mmH2O significantly increased K+ current (to 133.2 ± 5.6 % of control; n = 12) and this increase was reversed by hypoxia, decreasing currents to 62.5 ± 8.5 % (n = 4) of control; a degree of hypoxic K+ current inhibition normally observed in the absence of membrane stretch.
In conclusion, hTREK-1 is O2 sensitive when stably expressed in HEK 293 cells. Furthermore, the activation of this channel by either AA or membrane stretch is prevented by hypoxia suggesting that the structural requirements for regulation by fatty acids, stretch and oxygen may co-localise.
This work was supported by The Wellcome Trust and the British Heart Foundation.