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The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion., (© 2014 Renigunta et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Two-pore domain potassium (K(2P)) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K(2P) channels. We describe A1899 as a potent and highly selective blocker of the K(2P) channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K(2P) open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K(2P) channels.

The two-pore-domain potassium channels TASK-1 (KCNK3) and TASK-3 (KCNK9) modulate the electrical activity of neurons and many other cell types. We expressed TASK-1, TASK-3 and related reporter constructs in Xenopus oocytes, mammalian cell lines and various yeast strains to study the mechanisms controlling their transport to the surface membrane and the role of 14-3-3 proteins. We measured potassium currents with the voltage-clamp technique and fused N- and C-terminal fragments of the channels to various reporter proteins to study changes in subcellular localisation and surface expression. Mutational analysis showed that binding of 14-3-3 proteins to the extreme C-terminus of TASK-1 and TASK-3 masks a tri-basic motif, KRR, which differs in several important aspects from canonical arginine-based (RxR) or lysine-based (KKxx) retention signals. Pulldown experiments with GST fusion proteins showed that the KRR motif in the C-terminus of TASK-3 channels was able to bind to COPI coatomer. Disabling the binding of 14-3-3, which exposes the KRR motif, caused localisation of the GFP-tagged channel protein mainly to the Golgi complex. TASK-1 and TASK-3 also possess a di-basic N-terminal retention signal, KR, whose function was found to be independent of the binding of 14-3-3. Suppression of channel surface expression with dominant-negative channel mutants revealed that interaction with 14-3-3 has no significant effect on the dimeric assembly of the channels. Our results give a comprehensive description of the mechanisms by which 14-3-3 proteins, together with N- and C-terminal sorting signals, control the intracellular traffic of TASK-1 and TASK-3.

The two-pore-domain potassium channels TASK-1, TASK-3 and TASK-5 possess a conserved C-terminal motif of five amino acids. Truncation of the C-terminus of TASK-1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP-tagged channel proteins. Two-hybrid analysis showed that the C-terminal domain of TASK-1, TASK-3 and TASK-5, but not TASK-4, interacts with isoforms of the adapter protein 14-3-3. A pentapeptide motif at the extreme C-terminus of TASK-1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14-3-3, whereas the last 40 amino acids of TASK-1 were required for strong binding. Deletion of a single amino acid at the C-terminal end of TASK-1 or TASK-3 abolished binding of 14-3-3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK-1 mutants that failed to interact with 14-3-3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK-1 S409A, which interacts with 14-3-3-like wild-type channels, displayed normal macroscopic currents. Co-injection of 14-3-3zeta cRNA increased TASK-1 current in Xenopus oocytes by about 70 %. After co-transfection in HEK293 cells, TASK-1 and 14-3-3zeta (but not TASK-1DeltaC5 and 14-3-3zeta) could be co-immunoprecipitated. Furthermore, TASK-1 and 14-3-3 could be co-immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14-3-3 with TASK-1 or TASK-3 may promote the trafficking of the channels to the surface membrane.