Analyses of gating thermodynamics and effects of deletions in the mechanosensitive channel TREK-1: comparisons with structural models.
TREK-1, a mechanosensitive K channel from the two-pore family (K(2)P), is involved in protective regulation of the resting potential in CNS neurons and other tissues. The structure of TREK-1 and the basis of its sensitivity to stretch and variety of lipid-soluble factors remain unknown. Using existing K channel structures as modeling templates, TREK-1 was envisioned as a two-fold symmetrical complex with the gate formed primarily by the centrally positioned TM2b helices of the second homologous repeat. Opening was modeled as a conical expansion of the barrel separating TM2b's accompanied by extension of TM2a helices with the cytoplasmic TM2a-TM1b connector. Seeking first experimental support to the models we have accomplished thermodynamic analysis of mouse TREK-1 gating and functional testing of several deletion mutants. The predicted increase of the channel in-plane area (ΔA) of ~5 nm(2) in models was supported by the experimental ΔA of ~4 nm(2) derived from the slope of open probability versus membrane tension in HEK-293T cells and their cytoskeleton-depleted blebs. In response to steps of suction, wild-type channel produced transient currents in cell-attached patches and mostly sustained currents upon patch excision. TREK-1 motifs not present in canonical K channels include divergent cytoplasmic N- and C-termini, and a characteristic 50-residue extracellular loop in the first homologous repeat. Deletion of the extracellular loop (Δ76-124) reduced the average current density in patches, increased spontaneous activity and generated a larger sub-population of high-conductance channels, while activation by tension augmented by arachidonic acid was fully retained. Further deletion of the C-terminal end (Δ76-124/Δ334-411) removed voltage dependency but otherwise produced no additional effect. In an attempt to generate a cysteine-free version of the channel, we mutated two remaining cysteines 159 and 219 in the transmembrane region. C219A did not compromise channel activity, whereas the C159A/S mutants were essentially inactive. Treatment with β-mercaptoethanol suggested that none of these cysteines form functionally-important disulfides.
Department of Biology, University of Maryland, College Park, USA.
Grigory
G
Maksaev
Adina
A
Milac
Andriy
A
Anishkin
H Robert
HR
Guy
Sergei
S
Sukharev
D000818:Animals; D003545:Cysteine; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D040542:Mechanotransduction, Cellular; D008564:Membrane Potentials; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D024683:Potassium Channels, Tandem Pore Domain / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011312:Pressure; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014162:Transfection
D024683:Potassium Channels, Tandem Pore Domain; C103742:potassium channel protein TREK-1; D008623:Mercaptoethanol; D003545:Cysteine