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.
