The human tandem-P domain K+ channel, hTREK1, is O2 sensitive and hypoxia occludes its modulation by arachidonic acid (AA), membrane deformation (Miller et al. 2003a) or intracellular acidosis (Miller et al. 2003b). Although these findings question the neuroprotective role of hTREK1 during the acidosis/anoxia of central ischaemia, its role during physiological alkalosis has not been investigated. Here we report the effects of increased intracellular pH on the ability of hypoxia to inhibit, and AA to activate, this channel.
Experiments employed whole-cell or cell-attached patch clamp recordings from HEK293 cells stably transfected with hTREK1. Data reported are derived from currents recorded at a pipette potential of either +60 mV (whole-cell) or -130 mV (cell-attached) from a holding potential of -70 mV or 0 mV, respectively (see Miller et al. 2003a for details).
Following intracellular alkalinization with 20 mM trimethylamine (TMA), whole-cell current density was significantly reduced from 456 ± 50 to 223 ± 39 pA pF-1 (n = 6; mean ± S.E.M.). Further significant reduction was observed when TMA was co-applied with hypoxia such that current density was decreased to 110 ± 20 pA pF-1 (P < 0.01; Student’s paired t test for each manoeuvre). These effects were mirrored by alkalinization with ammonium chloride in the absence and presence of hypoxia (n = 6). Furthermore, the order of application of TMA and hypoxia did not affect the ability of each to inhibit currents. Thus, hypoxia reduced current density from 435 ± 64 to 326 ± 44 pA pF-1 whilst co-application of TMA reduced further the current density to 122 ± 23 pA pF-1 (n = 9; P < 0.02 for each manoeuvre). Although alkalosis was a potent inhibitor of hTREK1 current, it was without effect on AA action. Thus, following inhibition with TMA, 10 µM AA evoked a robust activation from 236 ± 35 to 567 ± 70 pA pF-1 (n = 9); this was completely occluded by hypoxia. In contrast, AA was without effect when hypoxia was pre-applied (in the presence of TMA; n = 11). The inability of TMA to block activation by AA was conserved in cell-attached recordings where TMA evoked mild but significant inhibition of currents from 21.3 ± 3.2 to 17.2 ± 3.2 pA and co-application of AA caused robust activation to 86.7 ± 31.5 pA (n = 7; P < 0.05); hypoxia completely reversed this effect.
In conclusion, although hypoxia occludes the activation of hTREK1 by either acidosis or arachidonic acid, it is without effect on the inhibitory effect of alkalosis, suggesting that regulation by oxygen/arachidonic acid and alkalosis are mechanistically distinct.
Funded by GlaxoSmithKline, British Heart Foundation and Wellcome Trust.