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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
Author(s) -
Zuzarte Marylou,
Heusser Katja,
Renigunta Vijay,
Schlichthörl Günter,
Rinné Susanne,
Wischmeyer Erhard,
Daut Jürgen,
Schwappach Blanche,
PreisigMüller Regina
Publication year - 2009
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2008.164756
Subject(s) - potassium channel , microbiology and biotechnology , xenopus , fusion protein , biology , lysine , protein targeting , c terminus , chemistry , biophysics , biochemistry , membrane protein , amino acid , gene , recombinant dna , membrane
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.