Insights into the stimulatory mechanism of 2-aminoethoxydiphenyl borate on TREK-2 potassium channel

2-Aminoethoxydiphenyl borate (2-APB) has been recently identified as a common agonist of TWIK-related K+ channel (TREK)/TRAAK channels, a subfamily of two-pore domain K+ (K2P) channels. TREK-2 displays much higher sensitivity to 2-APB compared with TREK-1, despite that these two channels share the highest homology among K2P members. However, the structural basis for their difference in response to 2-APB still remains unknown. Here we identified that the cytosolic C-terminus (Ct) domain plays a dominant role in controlling the stimulatory effects of 2-APB on TREK-2 channel. The distal Ct region negatively regulates the effect of 2-APB, while the proximal Ct is sufficient to evoke the full 2-APB activation of the channel. Further mapping within the proximal Ct revealed that His368 is required for 2-APB activation, and the cooperation of the other non-conserved residues is also necessary. We also identified a secondary active site for 2-APB, which is located at the bottom of the transmembrane segment M2. Finally, we demonstrated that key residues or domains required for 2-APB activation are not involved in the gating mechanism of the selectivity filter. In summary, we reveal a unique modulatory model of TREK-2-Ct that distinguishes it from TREK-1 in high sensitivity to 2-APB. The cooperation of the non-conserved residues within the proximal Ct of TREK-2 plays a dominant role in the 2-APB-induced channel opening, whereas the distal Ct negatively regulates the process.