Intracellular traffic of the K+ channels TASK-1 and TASK-3: role of N- and C-terminal sorting signals and interaction with 14-3-3 proteins.
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.
Zuzarte
Marylou
M
Institute of Physiology, Marburg University, Deutschhausstrasse 2, 35037 Marburg, Germany.
Heusser
Katja
K
Renigunta
Vijay
V
Schlichthörl
Günter
G
Rinné
Susanne
S
Wischmeyer
Erhard
E
Daut
Jürgen
J
Schwappach
Blanche
B
Preisig-Müller
Regina
R
D048948:14-3-3 Proteins / Q000378:metabolism / Q000502:physiology*; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D005260:Female; D006801:Humans; D042541:Intracellular Space / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D024683:Potassium Channels, Tandem Pore Domain / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport / Q000235:genetics; D014982:Xenopus laevis
D048948:14-3-3 Proteins; C498655:KCNK9 protein, human; D009419:Nerve Tissue Proteins; D024683:Potassium Channels, Tandem Pore Domain; D021382:Protein Sorting Signals; C420132:potassium channel subfamily K member 3