XB-ART-48228Channels (Austin) September 18, 2012; 6 (6): 473-8.
State-independent intracellular access of quaternary ammonium blockers to the pore of TREK-1.
We previously reported that TREK-1 gating by internal pH and pressure occurs close to or within the selectivity filter. These conclusions were based upon kinetic measurements of high-affinity block by quaternary ammonium (QA) ions that appeared to exhibit state-independent accessibility to their binding site within the pore. Intriguingly, recent crystal structures of two related K2P potassium channels were also both found to be open at the helix bundle crossing. However, this did not exclude the possibility of gating at the bundle crossing and it was suggested that side-fenestrations within these structures might allow state-independent access of QA ions to their binding site. In this addendum to our original study we demonstrate that even hydrophobic QA ions do not access the TREK-1 pore via these fenestrations. Furthermore, by using a chemically reactive QA ion immobilized within the pore via covalent cysteine modification we provide additional evidence that the QA binding site remains accessible to the cytoplasm in the closed state. These results support models of K2P channel gating which occur close to or within the selectivity filter and do not involve closure at the helix bundle crossing.
PubMed ID: 22991046
PMC ID: PMC3536734
Article link: Channels (Austin)
Genes referenced: kcnk1 kcnk2 mapt tff3.6
Article Images: [+] show captions
|Figure 1. Refined structural models of TREK-1 with TPenA bound. (A) Bottom up view of a structural model of the TREK-1 pore with docked TPenA. The model was based upon the crystal structure of TRAAK (PDB ID: 3UM7). Those residues which interact with TPenA are highlighted as spheres and colored green if they agree with our functional scanning mutagenesis study or red if a false positive. Only residues on one protomer are numbered. (B) Similar model based upon the crystal structure of TWIK-1 (PDB ID: 3UKM). (C) Similar model based upon the crystal structure of KvAP (PDB ID: 1ORQ). (D) Solvent-accessible surface area (SASA) of a TREK-1 model, based on TRAAK, in which the side-fenestrations are still clearly visible. The SASA of the phospholipid head groups is colored green whereas the TREK-1 model is colored gray. For one protomer, the outer (M1 and M3) and inner (M2 and M4) transmembrane helices are highlighted in orange and blue, respectively. The docked TPenA is also visible through the side-fenestration (colored in yellow spheres) and QA ion access to this binding site occurs via the cytoplasmic pore of the channel (indicated by direction of the arrow).|
|Figure 2. MTS-TBAO block and modification of TREK-1. (A) TREK-1 channel currents expressed in Xenopus oocytes measured at -80 mV in inside-out patches, activated from pH 8 by pH 5 and exposed to MTS-TBAO for 120 sec. A small and reversible block [8.2 ± 0.5% at 100 µM; (n = 4)] by the TBAO-moiety was obtained. (B) Concentration-response curves for tetrabutylammonium (TButA) inhibition of wild-type TREK-1 and G186C channels fitted to a standard Hill function with IC50 values and Hill-coefficients of 4.8 ± 0.3 mM and 1.4 ± 0.1 (n = 5) for wt; 0.5 ± 0.1 mM and 1.1 ± 0.1 for G186 (n = 6). Data points represent mean ± SEM (C) Structural model of the P1 subunits in the TREK-1 pore, the M3 and M4 helices from the P2 subunit are removed for clarity. The MTS-TBAO was linked to the introduced cysteines at position 186 with the SH-group in yellow, which is located in close proximity to the predicted QA binding site (green). (D) Application of MTS-TBAO on TREK-1-G186C mutant channels results in an initial fast block [24.2 ± 1% at 20 µM; (n = 5)] followed by a monoexponential time course [τ = 18.6 ± 3.9 sec; (n = 5)] for the modification of this cysteine that irreversibly reduced the current by up to 90%.|
|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).|