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, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data 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 structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.

CryoEM Structure of the human THIK-1 K2P K+Channel Reveals a Lower ‘Y-gate’ Regulated by Lipids and Anaesthetics

“…The extracellular cap domain above the filter results in a bifurcated exit route for K + . Mutations within these pathways in other K2P channels have been shown to influence channel properties, in particular their sensitivity to blockers that interact with the ‘Keystone Inhibitor Site’ (KIS) immediately above the filter 22 , and obstruction of the pathway by nanobodies has also been shown to inhibit channel activity 23 .…”

“…Two arginine side chains (R92 and R258) are in close proximity to the headgroup of this lipid and F262 on M4 contacts the middle of the lipid ( Figure 4e ). This is of particular interest because in many other K2P channels, F262 is a conserved tryptophan predicted to rotate inwards into this pocket during channel activation 23,28 ( Figure 4f ). Any lipid bound within this site might therefore modulate THIK-1 activity and thus influence microglial function.…”