Abstract
TRESK (TWIK-related spinal cord K+ channel, KCNK18) is a major background K+ channel of sensory neurons. Dominant-negative mutation of TRESK is linked to familial migraine. This important two-pore domain K+ channel is uniquely activated by calcineurin. The calcium/calmodulin-dependent protein phosphatase directly binds to the channel and activates TRESK current several-fold in Xenopus oocytes and HEK293 cells. We have recently shown that the kinase, which is responsible for the basal inhibition of the K+ current, is sensitive to the adaptor protein 14-3-3. Therefore we have examined the effect of the 14-3-3-inhibited PAR-1/MARK, microtubule-associated-protein/microtubule affinity-regulating kinase on TRESK in the Xenopus oocyte expression system. MARK1, MARK2 and MARK3 accelerated the return of TRESK current to the resting state after the calcium-dependent activation. Several other serine-threonine kinase types, generally involved in the modulation of other ion channels, failed to influence TRESK current recovery. MARK2 phosphorylated the primary determinant of regulation, the cluster of three adjacent serine residues (S274, 276 and 279) in the intracellular loop of mouse TRESK. In contrast, serine 264, the 14-3-3-binding site of TRESK, was not phosphorylated by the kinase. Thus MARK2 selectively inhibits TRESK activity via the S274/276/279 cluster, but does not affect the direct recruitment of 14-3-3 to the channel. TRESK is the first example of an ion channel phosphorylated by the dynamically membrane-localized MARK kinases, also known as general determinants of cellular polarity. These results raise the possibility that microtubule dynamics is coupled to the regulation of excitability in the neurons, which express TRESK background potassium channel.
Highlights
TRESK is abundantly expressed in dorsal root ganglion (DRG) neurons and has been suggested to play an important role in pain disorders [1,2,3,4,5]
TRESK is activated by the elevation of cytoplasmic calcium concentration via calcineurin in HEK293 cells
TRESK current in HEK293 cells was measured under wholecell patch clamp conditions with unusual calcium-free pipette and bath solutions containing EGTA at low (50 mM) concentration
Summary
TRESK is abundantly expressed in dorsal root ganglion (DRG) neurons and has been suggested to play an important role in pain disorders [1,2,3,4,5]. The channel has recently attracted particular attention, because its dominant-negative mutation was reported to be linked to familial migraine with aura [8]. These findings indicate the importance of TRESK in pain control and points to the need for better understanding of the regulatory properties of the channel. Stimulation of Gq protein-coupled receptors activated TRESK by 40–80% in COS-7 cells under whole-cell patch clamp conditions [10,11]. In the absence of specific inhibitors, separation of native whole-cell TRESK current from the other endogenous background K+
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