title: Involvement of intracellular transport in TREK-1c current run-up in 293T cells.
abstract: The TREK-1 channel, the TWIK-1-related potassium (K+) channel, is a member of a family of 2-pore-domain K+(K2P) channels, through which background or leak K+currents occur. An interesting feature of the TREK-1 channel is the run-up of current: i.e. the current through TREK-1 channels spontaneously increases within several minutes of the formation of the whole-cell configuration. To investigate whether intracellular transport is involved in the run-up, we established 293T cell lines stably expressing the TREK-1c channel (K2P2.1) and examined the effects of inhibitors of membrane protein transport, N-methylmaleimide (NEM), brefeldin-A, and an endocytosis inhibitor, pitstop2, on the run-up. The results showing that NEM and brefeldin-A inhibited and pitstop2 facilitated the run-up suggest the involvement of intracellular protein transport. Correspondingly, in cells stably expressing the mCherry-TREK-1 fusion protein, NEM decreased and pitstop2 increased the cell surface localization of the fusion protein. Furthermore, the run-up was inhibited by the intracellular application of a peptide of the C-terminal fragment TREK335-360, corresponding to the interaction site with microtubule-associated protein 2 (Mtap2). This peptide also inhibited the co-immunoprecipitation of Mtap2 with anti-mCherry antibody. The extracellular application of an ezrin inhibitor (NSC668394) also suppressed the run-up and surface localization of the fusion protein. The co-application of these inhibitors abolished the TREK-1c current, suggesting that the additive effects of ezrin and Mtap2 enhance the surface expression of TREK-1c channels and the run-up. These findings clearly showed the involvement of intracellular transport in TREK-1c current run-up and its mechanism.
authors.affiliation: a Department of Physiology , Kansai Medical University , Hirakata , Osaka , Japan.
authors.forename: Naaz
authors.initials: N
authors.lastname: Andharia
authors.affiliation: a Department of Physiology , Kansai Medical University , Hirakata , Osaka , Japan.
authors.forename: Ancy
authors.initials: A
authors.lastname: Joseph
authors.affiliation: a Department of Physiology , Kansai Medical University , Hirakata , Osaka , Japan.
authors.forename: Mikio
authors.initials: M
authors.lastname: Hayashi
authors.affiliation: a Department of Physiology , Kansai Medical University , Hirakata , Osaka , Japan.|b Department of Medical Life Science, College of Life Science , Kurashiki University of Science and the Arts , Kurashiki , Okayama , Japan.
authors.forename: Masayoshi
authors.identifier: http://orcid.org/0000-0001-8515-7143
authors.initials: M
authors.lastname: Okada
authors.affiliation: a Department of Physiology , Kansai Medical University , Hirakata , Osaka , Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Matsuda
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D055724:Electrophysiological Phenomena; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D024683:Potassium Channels, Tandem Pore Domain / Q000378:metabolism*; D055550:Protein Stability; D021381:Protein Transport; D000099149:Ezrin
chemical_list: D003598:Cytoskeletal Proteins; D008869:Microtubule-Associated Proteins; D024683:Potassium Channels, Tandem Pore Domain; D000099149:Ezrin; C103742:potassium channel protein TREK-1
keywords: 293T cell; TREK-1; ezrin; intracellular transport; microtubule-associated protein 2; run-up; surface localization; two-pore-domain K+ channel