Members of the 2P (KCNK) channel subfamily are composed of two subunits, each possessing four transmembrane segments and two pore-domains. They express background/leak current, accordingly, they are open in a wide range of the membrane potential. A growing body of evidence indicates their important role in different cellular processes. Their activity can be regulated by extracellular factors i.e. by pH, temperature, hypoxia, membrane stretch etc., and by intracellular signalling mechanisms. The last member of the 2P potassium channel family, TRESK, was cloned from human spinal cord mRNA in 2003(1), and subsequently from mouse cerebellum (2). While TRESK shows the characteristic structural features of 2P channel (two pore domains, and four transmembrane segments), it has an unusually long intracellular loop between the second and third TMS and a short C terminal intracellular tail. TRESK is regulated, like many other 2P channels; however, the mechanism of its regulation is unique in the 2P potassium channel family. In order to study the electrophysiological properties and regulation of TRESK, we subcloned its cDNA into a Xenopus expression vector and expressed the channel in oocytes. TRESK current was measured by two electrode voltage clamp, and single channel patch clamping. The intracellular loop of the channel was expressed as a GST-fusion protein for experiments addressing protein-protein interactions. TRESK was found to express a background potassium current, which was activated by calcium mobilizing agonists. Stimulation of the oocytes by coexpessed M1 muscarinic receptor increased the K⁺ current 10.4±1.4-fold,(n =5). This effect could be mimicked by ionomycin and also by injection of saturated Ca²⁺/EGTA buffer into the oocytes (6.2±0.4-fold (n=57) and 8.4±1.3-fold (n=4) stimulation, respectively). Under single channel conditions, however, application of high calcium to inside-out membrane patches failed to affect the channel activity. On the other hand, inhibitors of the calcium-activated protein phosphatase 2B (calcineurin, CN), cyclosporin or FK506 prevented the calcium induced activation. Coexpression of a truncated, constitutively active CN with TRESK in the oocytes elevated the basal current 3.3±1.2-fold (n=16), while the degree of stimulation in response to ionomycine was reduced from 6.3±0.4 (n=17) to 2.2±0.3 (n=16)-fold, indicating that the phosphatase activated the channel even in the absence of elevated calcium. Injection of a calcineurin inhibitory peptide into the oocytes, coexpressing consitutively active calcineurin with the channel, reduced the (elevated) basal current of TRESK, indicating that the catalytic activity of calcineurin is necessary for the activation of the channel. As calcineurin is a serine/threonine phosphatase we mutated all of the intracellular Ser and Thr residues of the channel to alanine in order to localize the potential site of action of the phosphatase. The S276A mutant (of the intracellular loop), which mimics the dephosphorylated state, showed higher-than-normal basal activity (4.2±0.7-fold (n=11)), whereas the stimulation by calcium was diminished (only 1.22±0.04-fold activation (n=11)). On the other hand, the S276E mutant, mimicking the phosphorylated state, showed low basal activity, and at the same time its activation by ionomycin was also reduced. A distinguished substrate of calcineurin is NFAT (nuclear factor of activated T cells). The dephosphorylation of NFAT, necessary for the activation and nuclear import of this transcription factor, requires the direct binding of calcineurin to a defined peptide motif of NFAT, which is distinct from the dephosphorylated site. The consensus sequence for this binding had been determined as PXIXIT. A similar sequence, PQIVID, was recognized in the intracellular loop of TRESK. Mutation of this sequence of the channel to PQIVIA, PQIVAD and PQAVAD progressively abolished the stimulation by ionomycin from the 7.5±0.6 fold value of the wild type to 4.1±0.3, 1.6±0.1 and 1.1±0.1-fold, respectively. The injection of a competing peptide “VIVIT” into the oocytes similarly reduced the activation of the current. These results suggest that calcineurin binds to the NFAT-like motif of TRESK and this interaction is necessary for the activation. The binding was confirmed also under in vitro conditions, using the purified GST/TRESK-loop protein. This protein, which was attached to glutathion agarose affinity matrix, bound constitutively active calcineurin. The interaction could be prevented also in this in vitro system by VIVIT peptide. The wild type calcineurin bound to the fusion protein with higher affinity in the presence of Ca²⁺/calmodulin, suggesting that the interaction of the two proteins is accelerated by the activation of the phosphatese in vivo.