Extracellular modulation of TREK-2 activity with nanobodies provides insight into the mechanisms of K2P channel regulation

Abstract

Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, we generated camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identified selective binders including several that directly modulate channel activity. Crystal structures of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These tools therefore provide important insights into TREK channel gating and a more selective approach to the modulation of K2P channel activity via their extracellular domains.

Effect of two activators on the gating of a K2Pchannel

“…For instance: The inactivated structure of TREK1 (PDB: 4twk, Figure S6) exhibits co-crystallized water molecules behind SF1 and inside S0 and S1. The presence of hydrophobic moieties in the modulator pocket (i.e., lipids [90, 25], hydrophobic mutations (e.g., G137I mutation [91]), and aromatic residue packing in the pocket [92]) can activate the channel, likely by decreasing hydration levels in the region, similar to those observed in our simulations (Figure S1 D. S9).…”