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Figure 3. State-independent access of MTS-TBAO to the inner pore of TREK-1. (A) TREK-1 currents activated by pH 5 were exposed to repeated applications of 20 µM MTS-TBAO at pH 8 for the times indicated by the arrows in order to test the accessibility of Cys-186 in the closed state. (B) A similar experiment performed in the presence of 1 mM TPenA, which blocks TREK-1 with an IC50 of 13 ± 1 µM.8 The channels were exposed to the blocker during the time indicated by the horizontal bars that resulted in complete inhibition of the current. Twenty µM MTS-TBAO was then applied to the blocked channel at times indicated by the arrows. (C) Time course of the relative current decline from experiments such as those shown in panelsA and B as a function of cumulative reagent exposure (Modification Time x [MTS-TBAO]) resulted in a monoexponential time course of 0.7 ± 0.1 sec (n = 6) in absence and 8.2 ± 0.9 sec (n = 3) in the presence of 1 mM TPenA. Data points represent mean ± SEM. (D) Activation of TREK-1-G186C mutant channels by 10 µM PtdIns(4,5)P2 at pH 8 and subsequent application of MTS-TBAO irreversibly reduced the current. The decay of the current was fitted to a monoexponential function with a Tau (τ) of 1.2 ± 0.1 sec (n = 3). Image published in: Rapedius M et al. (2012) Copyright © 2012 Landes Bioscience. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license Larger Image Printer Friendly View

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