Question:

Two-pore-domain potassium channels (K_2P) mediate K⁺ background currents that modulate the membrane potential of excitable cells. K_2P18.1 (TRESK, TWIK-related spinal cord K⁺ channel) mediates hyperpolarizing background currents in neurons. Recently a dominant negative loss-of-function mutation in K_2P18.1 has been implicated in migraine, and activation of K_2P18.1 channels was proposed as therapeutic strategy. Here we elucidate molecular mechanisms underlying protein kinase-dependent activation of K_2P18.1 currents.

Methods:

Human K_2P18.1 channels were heterologously expressed in Xenopus laevis oocytes, and currents were recorded using the two-electrode voltage clamp technique.

Results:

Stimulation of protein kinase C (PKC) with phorbol 12-myristate-13-acetate (PMA) activated hK_2P18.1 current by 3.1-fold in concentration-dependent fashion (EC₅₀ = 17.9 nM). The inactive analog 4α-PMA did not alter channel activity. Activation of K_2P18.1 required protein kinase C, as specific PKC-inhibitors (bisindolylmaleimide I, Ro-32-0432, chelerythrine) reduced PMA-induced channel activation. Selective stimulation of conventional PKC isoforms with thymeleatoxin (100 nM) did not reproduce K_2P18.1 channel activation, and signaling crosstalk with protein kinase A or calcineurin was excluded. Mutation of putative PKC phosphorylation sites did not prevent PMA-induced K_2P18.1 channel activation. Finally, PMA-induced activation was blocked by the diacylglycerol kinase (DGK)-Inhibitor R59949 (30 µM), identifying DGK as critical signaling factor in K_2P18.1 activation.

Conclusions:

We report PKC- and DGK-dependent modulation of K_2P18.1 background K⁺ currents. Activation of K_2P18.1 through PKC and DGK may serve as novel molecular target for migraine treatment.