title: State-independent intracellular access of quaternary ammonium blockers to the pore of TREK-1.
abstract: 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.
authors.affiliation: Physiological Institute, Christian-Albrechts University, Kiel, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Rapedius
authors.forename: Matthias R
authors.initials: MR
authors.lastname: Schmidt
authors.forename: Chetan
authors.initials: C
authors.lastname: Sharma
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Stansfeld
authors.forename: Mark S P
authors.initials: MS
authors.lastname: Sansom
authors.forename: Thomas
authors.initials: T
authors.lastname: Baukrowitz
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Tucker
mesh_terms: D000818:Animals; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D008958:Models, Molecular; D026902:Potassium Channel Blockers / Q000494:pharmacology; D024683:Potassium Channels, Tandem Pore Domain / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000378:metabolism*; D040681:Structural Homology, Protein; D014981:Xenopus
chemical_list: D026902:Potassium Channel Blockers; D024683:Potassium Channels, Tandem Pore Domain; D000644:Quaternary Ammonium Compounds; C103742:potassium channel protein TREK-1; C027435:tetrapentylammonium