Spadin selectively antagonises arachidonic acid activation of mTREK-1 channels

We recently revealed the mechano-gated two-pore domain potassium (K2P) channel, TREK-1, to be a major regulator of murine intestinal contractility (Ma et al, 2018). However, verification was complicated by a lack of well-defined K2P modulators and species specific pharmacology. Here, using heterologous expression in Xenopus laevis oocytes and electrophysiological analysis using two-electrode voltage clamp in standard bath solutions, we characterised the pharmacological profile of mouse homologues of TREK-1 and TREK-2 using previously documented human K2P activators; arachidonic acid (AA, 10 µM), BL-1249 (1 µM) and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC, 10 µM) and inhibitors; spadin (1 µM) and barium chloride (Ba2+, 1 mM). Values are stated as mean ± S.E.M. and were statistically compared by ANOVA. Mouse (m) TREK-1 and TREK-2 channel currents were significantly increased by AA, BL-1249 and CDC in a voltage-independent manner. At 0 mV, AA increased mTREK-1 currents by 2.5 ± 0.3-fold (n = 7, p = <0.05) and mTREK-2 by 1.6 ± 0.1-fold (n = 7, p <0.05), BL-1249 increased mTREK-1 currents by 2.9 ± 0.3-fold (n = 9, p = <0.05) and mTREK-2 by 1.5 ± 0.1-fold (n = 9, p = <0.05), and CDC increased mTREK-1 currents by 2.1 ± 0.1-fold (n = 8, p <0.05) and mTREK-2 by 2.3 ± 0.2-fold (n = 6, p = <0.05). Under basal conditions, both mTREK-1 and mTREK-2 currents were insensitive to application of spadin at all activating voltages. In contrast Ba2+ induced a significant, voltage-dependent reduction in outward currents of both channels. At 0 mV, mTREK-1 and mTREK-2 currents were inhibited by 56.1 ± 1.4% (n = 7, p <0.05) and 55.4 ± 2.4% (n = 7, p <0.05) respectively. Furthermore, spadin did not significantly inhibit either mTREK-1 or mTREK-2 currents following pre-activation by either AA, BL-1249 or CDC (n = 8-12, p = >0.05). However, we found pre-exposure to spadin significantly perturbed the activation of mTREK-1 currents by AA, voltage-independently. At 0 mV, activation of mTREK-1 by AA was reduced by 42.5 ± 1.8% (n = 12, p <0.05) compared to AA alone. Activation of mTREK-1 channels by either BL-1249 or CDC was not significantly attenuated by pre-exposure to spadin (n = 8-11, p = >0.05). Similarly, pre-exposure to spadin was unable to antagonise mTREK-2 channel current activation by either AA, BL-1249 or CDC (n = 6-8, p = >0.05). These data demonstrate that pharmacological activation of mouse TREK-1 and TREK-2 channels by AA, BL-1249 and CDC is comparable to that observed in their human orthologues. Additionally, our data suggests spadin is not a pore blocker of mTREK-1 or mTREK-2 channels, like Ba2+, but specifically antagonises the activation of mTREK-1 channels by AA, and not other pharmacological activators. Further studies are required to reveal the precise molecular mechanisms of spadin antagonism.