Fig. 3: Functional characterization of the lower gate in TALK-2 K2P channels.
a Relative current amplitudes from TEVC measurements at pH 8.5 of WT and mutant TALK-2 channels. Currents were elucidated with a voltage protocol ramped from -120 mV to +45 mV within 3.5 s, analyzed at +40 mV and normalized to WT. Inlays showing representative WT TALK-2 (gray traces), TALK-2 L264A, L262A, W255A and V146A mutant channel currents (blue and green traces), respectively and a topology model of a channel protomer highlighting the localization of the g-o-f mutations. b Sequence alignment of the TM2, TM2-TM3 linker, and TM4 regions of the human K2P channels TALK-2 and TASK-1. c Pore homology model of TALK-2 based on the crystal structure of TASK-1 (PDB ID: 6RV3, chains A, B) highlighting the cluster of g-o-f mutations (V146A, L262A and L264A) at the cytosolic pore entrance. d Representative inside-out single channel measurements of WT TALK-2 (gray trace) and TALK-2 L264A mutant channels (blue trace) at -100 mV. e Relative open probability (NPO) and single channel amplitudes (SCA) analyzed from recordings as in d for WT and L264A TALK-2 channels (n = 6). f–h Analysis of the mean channel-open times (g), closed time events (f), and burst behavior (h; see “Methods” section) for WT and TALK-2 L264A mutant channels. i Representative measurement of TALK-2 L264A mutant channels additionally carrying the inner pore mutation L145C (TALK-2 L264A/L145C) at +40 mV showing a fast and irreversible modification and subsequent block upon application of 1.0 mM MTS-ET. The experiment was repeated with similar results (n = 10). j Cartoon illustrating the pore accessibility of MTS-ET in L145C mutant TALK-2 channels with or without carrying an additional g-o-f mutation. k Modification rates of WT, L145C and double mutant TALK-2 channels at +40 mV as indicated. Data shown are the mean ± s.e.m and the number (n) of independent experiments is indicated in the figure and supplementary table 2. Statistical relevance has been evaluated using unpaired, two-sided t-test and exact P values are indicated in the figure. n.d. not determinable.
