Fig. 4: Selective extracellular activation of TREK-2 by Nb-Activator-76.

A Dose-response relationships showing activation of TREK-2 channel activity by Nb-Activator-76 (Nb76). Whole-cell TREK-2 currents were recorded at +40 mV and perfused with different concentrations of Nb76. The effect of mutating different residues in Nb76, which interact with TREK-2, are shown. The curves shown for the R53A and W98A mutant nanobodies were fitted by hand. (Error bars represent mean ± S.D; n ≥ 5). B Crystal structure of TREK-2 in complex with Nb76 bound to both sides of the Cap and the P2-M4 loop. C Interactions with the side of the Cap. W98 on CDR3 inserts between Cap helices of different TREK-2 subunits to form an interaction with E128 on EH2 of TREK-2, whilst R53 on Nb76 interacts with Q106 on EH1 of a different subunit of TREK-2. Mutation of either W98 or R53 on Nb76 reduces its activatory effect. D Expanded view of the interaction of N56 on CDR3 with N292 on the P2-M4 loop. Mutation of N56 also reduces activation by Nb76. E Alignment with TREK-2 Up-state (4BW5 in Grey) highlights displacement of the top of M4 and the inward rotation of W306. This is accompanied by the outward rotation of F164 on Pore-Helix-1 (PH1). F The right-hand panel rotates this view through 90° to better visualise the relative reorientation of W306 and F164 and the outward displacement of M4. This expands the K2P modulator site behind the selectivity filter (red asterisk).