Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 289, Issue 41, Pages 28202-28212Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M114.600437
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Funding
- Fondation pour la Recherche Medicale (Equipe labellisee FRM)
- Agence Nationale de la Recherche (Laboratory of Excellence Ion Channel Science and Therapeutics) [ANR-11-LABX-0015-01]
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Despite a high level of sequence homology, tandem pore domain halothane-inhibited K+ channel 1 (THIK1) produces background K+ currents, whereas THIK2 is silent. This lack of activity is due to a unique combination of intracellular retention and weak basal activity in the plasma membrane. Here, we designed THIK subunits containing dominant negative mutations (THIK1(DN) and THIK2(DN)). THIK2(DN) mutant inhibits THIK1 currents, whereas THIK1(DN) inhibits an activated form of THIK2 (THIK2-A155P-I158D). In situ proximity ligation assays and Forster/fluorescence resonance energy transfer (FRET) experiments support a physical association between THIK1 and THIK2. Next, we expressed covalent tandems of THIK proteins to obtain expression of pure heterodimers. Td-THIK1-THIK2 (where Td indicates tandem) produces K+ currents of amplitude similar to Td-THIK1-THIK1 but with a noticeable difference in the current kinetics. Unlike Td-THIK2-THIK2 that is mainly detected in the endoplasmic reticulum, Td-THIK1-THIK2 distributes at the plasma membrane, indicating that THIK1 can mask the endoplasmic reticulum retention/retrieval motif of THIK2. Kinetics and unitary conductance of Td-THIK1-THIK2 are intermediate between THIK1 and THIK2. Altogether, these results show that THIK1 and THIK2 form active heteromeric channels, further expanding the known repertoire of K+ channels.
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