Fig. 5: Open channel blocker show state-dependent pore accessibility and slowing of deactivation kinetics in TALK-2.

a Current responses of WT TALK-2 channels activated with indicated voltage steps under symmetrical ion conditions with either intracellular K⁺ (black trace, basal state) or Rb⁺ (red trace, activated state) and with 1.0 mM TPenA in Rb⁺ (orange trace). Note, the presence of TPenA shows slowing of deactivation resulting in a tail current cross-over. Cartoon depicting a simple model for TALK-2 channel gating, whereby Rb⁺ activation of the SF enables blocker (e.g., TPenA) binding in the pore and unbinding facilitates lower and SF gate closure at −80 mV. b Same recording as in (a) with TREK-2 channels showing inhibition with 50 µM TPenA without tail current cross-over. c Representative current responses of WT TALK-2 channels to voltage steps as indicated in the absence (black) and presence of 1.0 mM TPenA (orang) applied to the intracellular membrane side. d Representative measurement of TALK-2 channel currents at +40 mV showing dose-dependent TPenA inhibition in the pre-activated state with 1.0 mM 2-APB. e Dose-response curves of TPenA inhibition from measurements as in d for TALK-2 in unstimulated conditions (black, n = 16) and pre-activated states with 2-APB (blue) with altering apparent affinities for TPenA (IC₅₀ (0.2 mM 2-APB, n = 7) = 778 ± 116, IC₅₀ (0.5 mM 2-APB, n = 5) = 215 ± 28, IC₅₀ (1.0 mM 2-APB, n = 16) = 54 ± 10). f Residual currents of WT and L264A mutant TALK-2 channels at +40 mV upon 1.0 mM TPenA block at indicated conditions. g Residual currents of unstimulated (black), 2-APB pre-activated WT (blue) and L264A mutant (gray) TALK-2 channels after inhibition with the indicated blocker. h Simplified gating scheme indicating that blocker interact with the open state of TALK-2 to produce inhibition. Data shown are the mean ± s.e.m and the number (n) of independent experiments and repeats of representative measurements with similar results is indicated in the figure. Statistical relevance has been evaluated using unpaired, two-sided t-test and exact P values are indicated in the figure.