Tandem Pore Domain Halothane-inhibited K+ Channel Subunits THIK1 and THIK2 Assemble and Form Active Channels

Description

Despite a high level of sequence homology, tandem poredomain halothane-inhibited K channel 1 (THIK1) producesbackground K currents, whereas THIK2 is silent. This lack ofactivity is due to a unique combination of intracellular retentionand weak basal activity in the plasma membrane. Here, wedesigned THIK subunits containing dominant negative mutations (THIK1DN and THIK2DN). THIK2DN mutant inhibitsTHIK1 currents, whereas THIK1DN inhibits an activated formof THIK2 (THIK2-A155P-I158D). In situ proximity ligationassays and Fo¨rster/fluorescence resonance energy transfer(FRET) experiments support a physical association betweenTHIK1 and THIK2. Next, we expressed covalent tandems ofTHIK proteins to obtain expression of pure heterodimers.Td-THIK1-THIK2 (where Td indicates tandem) produces Kcurrents of amplitude similar to Td-THIK1-THIK1 but with anoticeable 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 THIK1and THIK2. Altogether, these results show that THIK1 andTHIK2 form active heteromeric channels, further expandingthe known repertoire of K channels.

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