TRESK Background K+ Channel Is Inhibited by Phosphorylation via Two Distinct Pathways

Abstract

The two-pore domain K ؉ channel, TRESK (TWIK-related spinal cord K ؉ channel, KCNK18) is directly regulated by the calcium/calmodulin-dependent phosphatase calcineurin and 14-3-3 adaptor proteins. The calcium signal robustly activates the channel via calcineurin, whereas the anchoring of 14-3-3 interferes with the return of the current to the resting state after the activation in Xenopus oocytes. In the present study, we report that the phosphorylation of TRESK at two distinct regulatory regions, the 14-3-3 binding site (Ser-264) and the cluster of three adjacent serine residues (Ser-274, Ser-276, and Ser-279), are responsible for channel inhibition. The phosphorylation of Ser-264 by protein kinase A accelerated the return of the current of S276E mutant TRESK to the resting state after the calcineurin-dependent activation. In the presence of 14-3-3, the basal current of the S276E mutant was reduced, and its calcineurin-dependent activation was augmented, suggesting that the direct binding of the adaptor protein to TRESK contributed to the basal inhibition of the channel under resting conditions. Unexpectedly, we found that 14-3-3 impeded the recovery of the current of S264E mutant TRESK to the resting state after the calcineurin-dependent activation, despite of the mutated 14-3-3 binding site. This suggests that 14-3-3 inhibited the kinase phosphorylating the regulatory cluster of Ser-274, Ser-276, and Ser-279, independently of the direct interaction between TRESK and 14-3-3. In conclusion, two distinct inhibitory kinase pathways converge on TRESK, and their effect on the calcineurin-dependent regulation is differentially modulated by the functional availability of 14-3-3.The two-pore domain K ϩ channel, TRESK 3 (TWIK-related spinal cord K ϩ channel, KCNK18) is a major background K ϩ channel of pseudounipolar sensory neurons (1-3). Abundant expression of TRESK mRNA and protein has been detected in dorsal root ganglia (DRG) (1, 2). TRESK cDNA was originally cloned from human spinal cord (4), and subsequently from mouse cerebellum (5) and testis (6). The distribution of the immunolocalization of TRESK in different central nervous system regions has also been recently reported (7). In DRG neurons, TRESK current was reliably identified at the single channel level (1). It was found to be the most prominent determinant of the resting K ϩ conductance of the plasma membrane together with another two-pore domain K ϩ (K2P) channel, TREK-2 (1, 2). TRESK has recently attracted particular attention, because of its efficient inhibition by the paresthesia-inducing pungent agents of the medicinal herb Szechuan pepper (8) and because of its probable role in nociception (9 -11).Although TRESK is abundant in DRG neurons, its coexpression with other two-pore domain K ϩ (K2P) channels (against which no selective inhibitors exist at present) impedes its examination as a whole cell current. Therefore we embarked on the investigation of this important K ϩ channel in a heterologous system, in Xenopus laevis oocytes. We demonstrated that...

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