Ion occupancy of the selectivity filter controls opening of a cytoplasmic gate in the K2P channel TALK-2

Two-pore domain K+ (K2P) channel activity was previously thought to be controlled primarily via a selectivity filter (SF) gate. However, recent crystal structures of TASK-1 and TASK-2 revealed a lower gate at the cytoplasmic pore entrance. Here, we report functional evidence of such a lower gate in the K2P channel K2P17.1 (TALK-2, TASK-4). We identified compounds (drugs and lipids) and mutations that opened the lower gate allowing the fast modification of pore cysteine residues. Surprisingly, stimuli that exclusively target the SF gate (i.e., pHe., Rb+ permeation, membrane depolarization) also opened the cytoplasmic gate suggesting that the SF can induce global structural changes in TALK-2. Reciprocally, opening of the lower gate reduced the electrical work required to force ions into the SF to induce its opening as apparent in large shifts of the conductance-voltage (G-V) curves. These shifts, thereby, represent the mechanical work done by the SF to induce a global structural re-arrangement that opened the lower gate. In conclusion, it appears that the SF is so rigidly locked into the TALK-2 protein structure that changes in ion occupancy can pry open a distant lower gate. Vice versa, we show that opening of the lower gate concurrently forces the SF gate to open. This concept might extent to other K+ channels that contain two gates (e.g., voltage-gated K+ channels) for which such a positive gate coupling has been suggested, but so far not directly demonstrated. SynopsisO_LITALK-2 channels, like most K2P channels, possess a functional gate in the selectivity filter (SF; the upper gate) that is opened by rising extracellular pH and voltage-dependent ion binding (voltage gating). C_LIO_LIA second (lower) permeation gate in TALK-2 at the cytoplasmic end of TM4 is identified using cysteine modification, scanning mutagenesis and structural modelling. This gate can be opened by anionic lipids (LC-CoA) as well as pharmacological ligands (e.g., 2-APB). C_LIO_LIThe modification reactivity of a cysteine introduced between the two gates reveal that stimuli targeting the SF gate also open the lower gate. Furthermore, stimuli that open the lower gate reduce the voltage (i.e., electrical work or mechanical load) required to open the SF gate. These findings demonstrate a tight positive coupling between the two gates. C_LIO_LIThe concept of strong positive gate coupling might extend to other K+ channels with two gates (e.g., voltage-gated K+ channels) for which positive gate coupling has been suggested but so far not directly demonstrated. C_LI