title: GsMTx4: Mechanism of Inhibiting Mechanosensitive Ion Channels.
abstract: GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We synthesized six analogs with single lysine-to-glutamate substitutions and tested them against Piezo1 channels in outside-out patches and independently measured lipid binding. Four analogs had ∼20% lower efficacy than the wild-type (WT) peptide. The equilibrium constants calculated from the rates of inhibition and washout did not correlate with the changes in inhibition. The lipid association strength of the WT GsMTx4 and the analogs was determined by tryptophan autofluorescence quenching and isothermal calorimetry with membrane vesicles and showed no significant differences in binding energy. Tryptophan fluorescence-quenching assays showed that both WT and analog peptides bound superficially near the lipid-water interface, although analogs penetrated deeper. Peptide-lipid association, as a function of lipid surface pressure, was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure. Analogs with compromised efficacy had pressure-area isotherms with steeper slopes in this region, suggesting tighter peptide association. The pressure-dependent redistribution of peptide between "deep" and "shallow" binding modes was supported by molecular dynamics (MD) simulations of the peptide-monolayer system under different area constraints. These data suggest a model placing GsMTx4 at the membrane surface, where it is stabilized by the lysines, and occupying a small fraction of the surface area in unstressed membranes. When applied tension reduces lateral pressure in the lipids, the peptides penetrate deeper acting as "area reservoirs" leading to partial relaxation of the outer monolayer, thereby reducing the effective magnitude of stimulus acting on the MSC gate.
authors.affiliation: Department of Physiology and Biophysics, State University of New York, Buffalo, New York.
authors.forename: Radhakrishnan
authors.initials: R
authors.lastname: Gnanasambandam
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas.
authors.forename: Chiranjib
authors.initials: C
authors.lastname: Ghatak
authors.affiliation: Department of Biology, University of Maryland, College Park, Maryland.
authors.forename: Anthony
authors.initials: A
authors.lastname: Yasmann
authors.affiliation: Clinical Laboratory Science, Teikyo University School of Medical Technology, Tokyo, Japan.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Nishizawa
authors.affiliation: Department of Physiology and Biophysics, State University of New York, Buffalo, New York.
authors.forename: Frederick
authors.initials: F
authors.lastname: Sachs
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas.
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Ladokhin
authors.affiliation: Department of Biology, University of Maryland, College Park, Maryland.
authors.forename: Sergei I
authors.initials: SI
authors.lastname: Sukharev
authors.affiliation: Department of Physiology and Biophysics, State University of New York, Buffalo, New York. Electronic address: suchyna@buffalo.edu.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Suchyna
mesh_terms: D001696:Biomechanical Phenomena; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D055595:Mechanical Phenomena; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D011312:Pressure; D013111:Spider Venoms / Q000378:metabolism / Q000494:pharmacology*; D013314:Stress, Mechanical
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; C411249:MTx4 protein, Grammostola spatulata; D010455:Peptides; D013111:Spider Venoms