Abstract
The two-pore domain K(+) channel, TRESK (TWIK-related spinal cord K(+) channel) is reversibly activated by the calcium/calmodulin-dependent protein phosphatase, calcineurin. In the present study, we report that 14-3-3 proteins directly bind to the intracellular loop of TRESK and control the kinetics of the calcium-dependent regulation of the channel. Coexpression of 14-3-3eta with TRESK blocked, whereas the coexpression of a dominant negative form of 14-3-3eta accelerated the return of the K(+) current to the resting state after the activation mediated by calcineurin in Xenopus oocytes. The direct action of 14-3-3 was spatially restricted to TRESK, since 14-3-3eta was also effective, when it was tethered to the channel by a flexible polyglutamine-containing chain. The effect of both the coexpressed and chained 14-3-3 was alleviated by the microinjection of Ser(P)-Raf259 phosphopeptide that competes with TRESK for binding to 14-3-3. The gamma and eta isoforms of 14-3-3 controlled TRESK regulation, whereas the beta, zeta, epsilon, sigma, and tau isoforms failed to influence the mechanism significantly. Phosphorylation of serine 264 in mouse TRESK was required for the binding of 14-3-3eta. Because 14-3-3 proteins are ubiquitous, they are expected to control the duration of calcineurin-mediated TRESK activation in all the cell types that express the channel, depending on the phosphorylation state of serine 264. This kind of direct control of channel regulation by 14-3-3 is unique within the two-pore domain K(+) channel family.
Highlights
Two-pore domain potassium (2PKϩ) channels give rise to background Kϩ currents that are pivotal regulators of the excitability in neurons and other cell types (6)
Several members of the two-pore domain potassium (2PKϩ) channel family are regulated by calcium-mobilizing hormones and neurotransmitters
The signaling pathway is still debated (the breakdown of membrane phosphoinositides (27, 28), the direct binding of G␣q to the channel (29), the production of diacylglycerol (28), and the phosphorylation by protein kinase C (30, 31) were all suggested as possible mechanisms for the different channels), there is a general consensus that the formation of inositol-1,4,5-trisphosphate and the following calcium signal are not involved in the inhibition of TASK and TREK subfamily members
Summary
Plasmids—The sequences of pXEN and pXEN-pQ108 vectors were deposited to GenBankTM under the accession numbers EU267939 and EU267940, respectively. pXEN-pQ108, coding the flexible, artificial polypeptide chain (LEHQQQQQQQQQ), was obtained by inserting nine orientationally ligated. The proteins were eluted from the resin with G-lysis supplemented with 20 mM glutathione and dialyzed against a solution containing 10 mM HEPES, 100 mM NaCl, 50 mM KCl, 2 mM MgCl2, 1 mM -mercaptoethanol (pH 7.4 with NaOH). TRESKloop-H8 proteins immobilized on 12–25 l of Ni-NTA resin were washed 3 times with EQ solution containing 50 mM HEPES, 50 mM KCl, 1 mM PMSF, 0.1 mM benzamidine, 2 mM -mercaptoethanol (pH 7.2 with NaOH) 20 min before the phosphorylation reaction. The phosphorylation with protein kinase A holoenzyme (protein kinase A (PKA), 1 g/reaction, Sigma P5511, 0.7 units/g) was performed at 37 °C for 30 min in a solution containing 20 mM HEPES, 80 mM KCl, 10 mM MgCl2, 25 mM -glycerol phosphate, 0.5 mM -mercaptoethanol, 0.1 mM sodium orthovanadate, 1 mM cAMP (pH 7.5 with NaOH) supplemented with 50 M Na2ATP and 50 kBq [␥-32P]ATP. NaCl, 50 mM KCl, 1 mM PMSF, 0.1 mM benzamidine, 2 mM -mercaptoethanol, 30 mM imidazole (pH 7.4 with NaOH)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
