Fig. 4: Direct stimulation of the SF produces a state-dependent cysteine modification in the pore of TALK-2.
a TALK-2 current responses to voltage step families as indicated using symmetrical K+ concentrations (120 mM [K+]ex./120 mM [K+]int.) at pH 7.4 on both sides (black traces), or at extracellular pH 9.5 (brown traces). b Cartoon illustrating a simplified TALK-2 channel gating model and pore accessibility to MTS-ET by alterations of the pHe that directly affects the SF. c Representative modification and subsequent irreversible inhibition with 1.0 mM MTS-ET of TALK-2 L145C channels pre-activated by extracellular alkalinization (pHe 9.5). d TALK-2 channel currents with intracellular Rb+ (120 mM [K+]ex./120 mM [Rb+]int.) at pH 7.4 for different potentials as indicated showing a maximum PO reached for potentials positive to ~+135 mV (Vmax), as further depolarizations do not increase the tail current amplitudes. e Voltage activation (conductance-voltage (G–V) curves) with V1/2 values of 72 ± 2 mV and 66 ± 3 mV of WT TALK-2 (n = 15) and L145C mutant channels (n = 10), respectively. The highlighted voltages (orange) represent the voltage activation levels for MTS-ET modification experiments shown in i. f Cartoon of a simplified gating model with Rb+ as an amplifier for voltage activation targeting the SF and subsequently the lower gate in TALK-2 channels. g, h Representative measurements at +40 mV of WT (g) and L145C mutant TALK-2 channels (h) showing a non-modifiable state or an almost complete modification/inhibition with 1.0 mM MTS-ET within 60 s in intracellular Rb+, respectively. i Correlation between the fold change of tail current amplitudes (black squares) of TALK-2 L145C channels and the incidental rate of MTS-ET modification (1/τ) (orange squares) with intracellular Rb+ at different potentials as indicated. 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 and supplementary tables 1–3.
