Silencing of the Tandem Pore Domain Halothane-inhibited K+ Channel 2 (THIK2) Relies on Combined Intracellular Retention and Low Intrinsic Activity at the Plasma Membrane

• Published in: The Journal of biological chemistry 2013-12, Vol.288 (49), p.35081-35092
• Main Authors: Chatelain, Franck C., Bichet, Delphine, Feliciangeli, Sylvain, Larroque, Marie-Madeleine, Braud, Véronique M., Douguet, Dominique, Lesage, Florian
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Animals; Bioinformatics; Cell Membrane - metabolism; Chemical Sciences; Cheminformatics; Computer Science; Dogs; Electrophysiology; Endoplasmic Reticulum (ER); Endoplasmic Reticulum - metabolism; Female; Gating; Gene Silencing; Humans; Intracellular Space - metabolism; Life Sciences; Madin Darby Canine Kidney Cells; Medicinal Chemistry; Membrane Biology; Membrane Potentials; Molecular Sequence Data; Mutagenesis, Site-Directed; Oocytes - metabolism; Pharmaceutical sciences; Phosphorylation; Plasma Membrane; Potassium Channels; Potassium Channels, Tandem Pore Domain - chemistry; Potassium Channels, Tandem Pore Domain - genetics; Potassium Channels, Tandem Pore Domain - metabolism; Protein Phosphorylation; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Trafficking; Xenopus laevis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.503318

The tandem pore domain halothane-inhibited K+ channel 1 (THIK1) produces background K+ currents. Despite 62% amino acid identity with THIK1, THIK2 is not active upon heterologous expression. Here, we show that this apparent lack of activity is due to a unique combination of retention in the endoplasmic reticulum and low intrinsic channel activity at the plasma membrane. A THIK2 mutant containing a proline residue (THIK2-A155P) in its second inner helix (M2) produces K+-selective currents with properties similar to THIK1, including inhibition by halothane and insensitivity to extracellular pH variations. Another mutation in the M2 helix (I158D) further increases channel activity and affects current kinetics. We also show that the cytoplasmic amino-terminal region of THIK2 (Nt-THIK2) contains an arginine-rich motif (RRSRRR) that acts as a retention/retrieval signal. Mutation of this motif in THIK2 induces a relocation of the channel to the plasma membrane, resulting in measurable currents, even in the absence of mutations in the M2 helix. Cell surface delivery of a Nt-THIK2-CD161 chimera is increased by mutating the arginines of the retention motif but also by converting the serine embedded in this motif to aspartate, suggesting a phosphorylation-dependent regulation of THIK2 trafficking. Background: THIK2 does not generate macroscopic currents in heterologous systems. Results: THIK2 produces K+ currents when mutated in its pore region and/or in its cytoplasmic amino terminus. Conclusion: Silencing of THIK2 is due to low intrinsic activity and intracellular retention. Significance: THIK2 is a functional but silent channel.